Effect of Salicylic Acid Spraying on Physiological and Enzymatic Characteristics of three Broad bean Cultivars under Saline Soil Conditions

A field experiment was conducted during the winter season (2022-2023) in the field of the Department of Crops / Faculty of Agriculture - Al-Qasim Green University, to find out the effect of spraying salicylic acid on the physiological and enzymatic characteristics of three Cultivars of beans under saline soil conditions using the design of complete random sectors in the order of splinter panels, as the main panels included Cultivars (Spanish, Turkish, Dutch), while the secondary panels included foliar spraying of salicylic acid (without spraying, spraying at a concentration of 100 mg L -1, spraying at a concentration of 200 mg L -1).The results of the analysis of variance indicate a significant difference at the level of probability of 5% for all the studied traits, as the Spanish Cultivar outperformed in the total chlorophyll content 23. (mg100 g soft weight) and proline 1.284(mcg g-1 dry matter), while Dutch Cultivar outperformed in the enzyme POD 20.44 (units of absorption g-1 soft weight). Also, foliar spraying of salicylic acid achieved a positive effect at a concentration of 200 mg/L in the leaf content of total chlorophyll pigments 24.239 (mg100 g soft weight)and enzyme CAT 10.94 (units of absorption g-1 soft weight)and enzyme POD 22.251 (units of absorption g-1 soft weight) and seed yield 2.40 tons ha-1 and harvest indx 49.23%.


Introduction
Broad bean Vicia faba .L is one of the most important leguminous crops and the most widespread in the world, due to its high nutritional importance and is grown in various countries with the aim of obtaining juicy green pods and dry seeds that are used as a basic food after grass crops for humans and as animal fodder, as well as beans are a basic food for the population of poor countries, especially North African countries and the countries of the Middle East for its high content of proteins 25-35% [1].The productivity of the bean crop can be increased by using some chemical compounds such as salicylic acid, as salicylic acid is one of the plant hormones that has important physiological roles in encouraging plant growth, increasing the efficiency of photosynthesis and flowering, plant tolerance to various stresses [2] and increasing the activity of antioxidant enzymes [3].It was stated [4] that the main function of salicylic acid lies in protecting cell membranes as well as protein vector bonds (H+-ATP-as membrane pumps) and repairing their structure and action against poisoning and the decomposition effect of ROS active oxygen radicals during the stress period as well as absorbing and transporting the largest number of elements.He also noted [5] that treatment with acetylsalicylic acid (SA) plays a role in activating photosynthesis and increasing the concentrations of essential pigments for this process.He also pointed out [6] that spraying bean plants with a concentration of 200ppm of salicylic acid led to a significant increase in the effectiveness of antioxidant enzymes Catalase and Super oxidase dismutase in the plant, which amounted to 55.41 and 205.70% compared to the comparison plants, which gave the lowest rate of these traits of 36.91 and 66.33% respectively.[7] In an experiment involving two Cultivars of beans (Giza 843 and Giza 3) under the influence of salt stress, it was found that there were significant differences in the content of proline, CAT and POX enzymes in plant leaves The Giza843 Cultivar excelled by giving it the highest average proline and CAT content of 112.1 and (17.1 mmol.g -1 respectively), and therefore the study aimed to reduce the effect of soil salinity on the physiological and enzymatic properties of pea Cultivars by foliar spraying of salicylic acid.It was also found [7] that spraying (0.7) mmol of salicylic acid that spraying this concentration on the vegetative total of the bean plant, achieved significant superiority in the characteristics of the studied yield Total seed yield and biological yield amounted to (34.06 g plant -1 and 63.94 Plant -1 g) sequentially compared to the comparison plants that gave the lowest average of (8.20.14 g plant -1 and 42.08 g plant -1) respectively.Accordingly, the importance of this study came to know the role of salicylic acid in regulating physiological and enzymatic processes and their reflection on the yield of pean Cultivars under conditions of salt stress.

Materials and Methods
A field experiment of the winter season (2022-2023) was carried out in the field of the Department of Crops / Faculty of Agriculture -Al-Qasim Green University, to find out the effect of spraying salicylic acid on the physiological and enzymatic characteristics of three Cultivars of beans under saline soil conditions using the design of complete random sectors in the order of splinter panels, as the main panels included Cultivars (Spanish, Turkish, Dutch), while the secondary panels included foliar spraying of salicylic acid (without spraying, spraying at a concentration of 100 mg L -1 , spraying at a concentration of 200 mg L -1 ).The area of the experimental unit is 3 m×2 m Each experimental unit included four Maroz distance of 70 cm between Marz and another and 20 cm between one joura and another, the process of spraying transactions according to the stages of growth (at the beginning of flowering and the second spray after 15 days the date of the first spray) in the early morning for each of the stages mentioned using a dorsal sprinkler (capacity of 16 Ls) with the use of diffuser material (Al-Zahi) to reduce the surface tension of the water (until complete wetness), planting was done with saline soil with EC=7ds Soil and crop service operations were carried out from patching and loosening as needed, and a week after the completion of the spraying operations, the soft and dry leaves were taken for the purpose of conducting laboratory analyzes of the studied qualities, After collecting the results, they were statistically classified and analyzed using the Genstat variance analysis method, and the least significant difference (LSD) test was used, with a probability level of 0.05 to compare the arithmetic averages [9].

Chemical Indicators
The fourth paper was taken near the growing apex of several plants from each experimental unit [10] (10 days after the last spray), on which the following estimates were made:

Total Chlorophyll Content of the Leaves (mg 100 g -1 Soft Weight)
The total chlorophyll pigment in the leaves of the Broad bean plant and for five paper samples was randomly estimated from each experimental unit according to the method [11]and the leaf content of total chlorophyll was calculated according to the following equations:

Concentration of the Amino Acid Free Proline in Leaves (μg g -1 Dry Matter)
Estimating proline according by method [12].

Estimation of Peroxidase Enzyme (Peroxidase -POX g -1 Soft Weight Absorption Unit)
Estimation of Enzyme Activity by [13].

Estimation of the Activity of the Enzyme (CAT) (g -1 Adsorption Unit by Soft Weight)
The activity of the CAT enzyme was estimated by spectrophotomete spectrophotomete) according to the [14].

Characteristics Yield
Measurements and indicators of the yield and its components were taken until signs of maturity appeared and the pods reached the stage of physiological maturity as a dry yield for each experimental unit as follows.

Total Chlorophyll Content of the Leaves (mg 100 g -1 Soft Weight)
The results in Table (1) that salicylic acid has a significant effect on the total chlorophyll dye content, as the plants sprayed with a concentration of 200 mg/L of salicylic acid achieved the highest average total chlorophyll content of 24.23 (mg100 g soft weight), while the comparison treatment achieved the lowest average total chlorophyll content of 18.036 (mg100 g soft weight).The reason for the concentration exceeding 200 mg/L may be due to the ability of Salicylic acid to improve the growth manifestations of plants prone to salt stress through its role in stimulating photosynthesis by maintaining the enzymes involved in this process, the permeability of plasma membranes and increasing photosynthetic pigments [15], As for the Cultivars , the Spanish Cultivar achieved the highest average total chlorophyll content of 23.01 (mg100 g soft weight)compared to the Turkish Cultivar, which gave the lowest average for this trait amounting to 19.83 (mg100 g soft weight).This indicates that the Spanish Cultivar is highly efficient in exploiting the growth factors available to serve photosynthesis and increase the pigment chlorophyll [16].

Concentration of the Amino Acid Free Proline in Leaves (μg g -1 dry matter)
The results in Table (2) indicated that there are significant differences between the concentrations of salicylic acid in the content of proline in the leaves, meaning that by increasing the concentration of salicylic acid, the proline content decreases, as the comparison treatment outperformed the rest of the treatments by giving it the highest average for this trait of 1.284 ( mcg g -1 dry matter) compared to while the treatment at a concentration of 200 mg L gave the lowest average of 0.747 ( mcg g -1 dry matter and did not differ significantly from the treatment at a concentration of 100 mg L -1 .Perhaps the reason for the decrease in the content of the leaves of proline when treated with acid Salicylic is due to its ability to increase the protein content of the cell by increasing the building of proteins and activating the enzyme nitrate reduction NR as it works to protect enzymes from oxidation, so it reverses the damage caused to the plant by salt tension, the plant resorts to getting rid of the accumulation of ammonia and toxic nitrates by forming proline acid due to oxidation and reducing the activity of protein-forming enzymes in the cell, while Salicylic acid works to activate antioxidants and activate Protein-building enzymes in the BA cell thus reduce the production of free amino acids such as proline [17] .The results in the same table also indicated that there are significant differences between the Cultivars , as the Spanish Cultivar achieved the highest average of 1.28 compared to the Dutch Cultivar, which achieved the lowest average of 0.47 ( mcg g -1 dry matter), and this is due to the difference in the genetic characteristics of the Cultivars and the extent to which they are affected by the surrounding conditions and the difference in the efficiency of photosynthesis in the Cultivars and their content of internal hormones [18] and this is consistent with what he found [19].

Estimation of the Activity of the Enzyme (CAT) (g -1 adsorption unit by soft weight)
The results of Table (3) showed a significant effect of salicylic acid on the effectiveness of the CAT enzyme in the plant and the Cultivars did not differ significantly among themselves, as the effectiveness of the enzyme increased after adding 200 mg/L of acid amounted to 10.94 (units of absorption g-1 soft weight) , compared to the comparison treatment that achieved the lowest rate of 7.37 (units of absorption g -1 soft weight), where the same behavior of the enzyme was observed under the influence of this acid, but the important thing is that the high concentrations of them led to an increase in the effectiveness of the CAT enzyme [20].
Table 3.Effect of salicylic acid on Enzyme (CAT) (g -1 adsorption unit by soft weight)of faba bean Cultivars.

Estimation of Peroxidase Enzyme (Peroxidase -POX g -1 Soft Weight Absorption Unit):
The results of Table (4) showed that there are significant differences between the concentrations of salicylic acid in the average enzyme peroxide POD, as the plants sprayed with a concentration of 200 mg/L of salicylic acid achieved a significant superiority by giving them the highest average of 22.25 (units of absorption g -1 soft weight) compared to the comparison plants (SA0), which gave the lowest average for this characteristic amounted to 15.46 (units of absorption g -1 soft weight), as for the significant increase in the effectiveness of the enzyme when treated with Salicylic acid, it may be due to the ability of this acid to stimulate the formation of Antioxidants for ROS such as Peroxidase [20], Salicylic acid increases the production of Peroxidase and Ascorbate to reduce ROS damage which increases due to the plant's exposure to stress conditions [21] .The results of Table ( 4) also showed that the Dutch Cultivar has significantly outperformed by giving it the highest average POD enzyme, which amounted to 20.44 (units of absorption g -1 soft weight) compared to the Spanish Cultivar, which achieved the lowest average of this enzyme of 18.97 (units of absorption g -1 soft weight), perhaps the reason for the superiority of the Dutch Cultivar is attributed and this indicates that genetically different Cultivars also differ in their biochemical content of antioxidant enzymes [22].

Seed Yield (ton ha -1 )
It is clear from Table (5) that there is a significant increase in the yield of seeds per unit area with an increase in the concentration of spraying with salicylic acid, as the concentration of 200 mg/L achieved the highest average characteristic of 2.40 (ton ha -1 ) compared to the comparison treatment that gave the lowest yield of 1.56 (ton ha -1 ).This is due to the role of salicylic acid, which regulates growth by increasing enzymatic activity (Table 3 and 4), as well as the enzyme alpha-amlase and reductase nitrate, which leads to the accumulation of sugars transported from the leaves to storage centers [23] All these factors were positively reflected in improving the yield characteristics, including increasing the seed yield per unit area.It is also shown from Table (5) that the Spanish Cultivar has achieved the highest average of the seed yield of 2.49 (ton ha -1 ) and significantly superior to the Dutch Cultivar, which gave the lowest average of the characteristic of 1.47 (ton ha -1 ), that the moral superiority of the Spanish Cultivar in the components of the yield must be reflected positively in the increase of seed yield per unit area [24[.Table 5.Effect of salicylic acid on seed yield (ton ha -1 ) of braod bean cultivars.

Biological Yield (ton ha -1 )
Table (6) shows that there is a significant increase in the biological yield with the increase in the concentration of spraying with salicylic acid, as the concentration of 200 mg/L gave the highest average of 4.88 (ton ha -1 ) compared to the comparison plants that gave the lowest average of 3.52 (ton ha -1 ).The reason for this is that salicylic acid has a physiological role in increasing the levels of the plant hormones auxin, gibberellin and cytokinin [25], which have a positive role in increasing the IOP Publishing doi:10.1088/1755-1315/1259/1/0120996 overall growth and yield of the plant, including the number of leaves, the number of branches in the plant and the number of pods.In this area, a number of researchers found a significant effect of salicylic acid in increasing biological yield in leguminous crops [26 and 27].The results of the table also showed the superiority of the Turkish Cultivar significantly over the rest of the Cultivars , as it achieved the highest yield of 4.81 tons h -1 , while the Dutch Cultivar achieved the lowest yield of 3.23 (ton ha -1 ), and this is due to the difference in the genetic characteristics of the Cultivars and the extent to which they are affected by the surrounding conditions, which was reflected in the characteristics of vegetative growth and the yield and thus the biological yield.Table 6.Effect of salicylic acid on biological yield (ton ha -1 ) of broad bean cultivars.

Harvest Index (%)
It is clear from Table (7) that plants sprayed with a concentration of 200 mg/Lof salicylic acid achieved the highest average harvest index value of 49.23% and significantly outperformed the comparison plants that gave the lowest value for this trait 44.88%.The reason for the concentration exceeding 200 mg/Lis due to the fact that it was distinct in the seed yield (Table 5), which reflected positively on the increase in the value of the harvest.As for the varieties, the Spanish Cultivar achieved the highest value of the harvest index amounting to 56.20% compared to the Turkish Cultivar, which gave the lowest value of 40.17%.The reason for the difference in varieties in the quality of the harvest guide may be attributed to differences among themselves and in their ability to change within the prevailing environmental conditions as well as in the extent of their response to the effect of spray concentrations on some characteristics of the yield and this is consistent with [26].
Table 7.Effect of salicylic acid on harvest index (%) of broad bean cultivars.a high yield of beans due to its adaptation to the iraqi environment and the extent of its interaction with foliar feeding with salicylic acid, and the spraying of salicylic acid led to an increase in the amount of total chlorophyll(a+b) and increased the effectiveness of an enzyme CAT and POD, which led to improving plant growth and regulating physiological and enzyme processes and thus reducing the harmful effect of salinity, so we recommended conducting more studies in this field.

Table 1 .
Effect of salicylic acid 1-Total chlorophyll content of the leaves (mg 100 g -1 soft weight)of faba bean Cultivars.

Table 2 .
Effect of salicylic acid on Concentration of the amino acid free proline in leaves (μg g-1 dry matter): of faba bean Cultivars.

Table 4 .
Effect of salicylic acid on Estimation of Peroxidase Enzyme (Peroxidase -POX g -1 Soft Weight Absorption Unit): of faba bean Cultivars.