Investigate the impact of microorganism species richness, carrier materials, and nitrogen fertilizer on (Eruca vesicaria subsp. Sativa) growth and its active compounds content: Running Title; Fertilizer and Arugula

Using microorganisms is one of the most important tools to increase plant production without harming our environment and health. However, little is known about microbial diversity and related that to soil nutrients in arugula plants. Therefore, we hypothesize that mixing more microorganisms with suitable carrier materials will improve arugula growth and its active compounds. Using two factors biofertilizer and chemical fertilizer, biofertilizer including: mycorrhizae (30g) Trichoderma asperellum (30 g), Bacillus subtilis (50 ml/L), and a combination of microorganisms (Bacteria and fungi Trichoderma and Mycorrhizae) in that order (B1, B2, B3, B4, B5, and B6). Chemical fertilizer including nitrogen, comparison, and at a rate of 15 kg per acre (or one-fourth of the recommended rate), and 30 kg per acre (or half the recommended rate), respectively (A1, A2, A3). The results showed that:Adding bacteria and their carrier materials was significantly superior to the leaf content of active compounds (phenols, flavonoids, and tannins), whose values reached 1.083 (mg.g-1dry weight), 35.98(mg.g-1dry weight), and 1.086(mg.g-1dry weight) and sequentiall, and addition of Trichoderma and and their carrier materials was significantly superior to the leaves’ content of elements (N, P, and K), whose values reached 3.98% and 0.88% and 4.92%, respectively, and quantity of yield in the first harvest increased upon the addition of combination of microorganisms and nitrogen fertilizer (urea). The use of environmentally friendly fertilizers is critical to promoting both plant development and the increasing of beneficial active compounds, and this research provides useful insights into how to best cultivate arugula organically. Using such environmentally friendly methods is crucial to promote sustainable agriculture.


Introduction
The (Eruca vesicaria subsp.Sativa) plant is among the most medicinal and nutritionally valuable of plants.It belongs to the Brassicaceae family and is cultivated in moderate regions throughout the year, excluding extremely hot months [1].Arugula is a highly nutritious leafy vegetable that also serves as an oil plant, with seeds that contain 27.8% oil.Their edible leaves are rich in potassium, sulfur, iron, and vitamins A and C, making them a favored food [2].Furthermore, it has a diploid genetic makeup (n = 11), which makes it an intriguing and fascinating plant.The antioxidant properties of arugula have demonstrated that it has medicinal properties in recent times [3].The medicinal properties of its leaves are highly valued due to the abundance of beneficial compounds they contain.These compounds, particularly glucosinolates, serve as potent antioxidants [2].Consequently, the use of appropriate growing techniques is crucial for optimizing vegetable quality.Increasing the yield and nutrition of plants is now possible with a variety of organic and biofertilizers spread throughout the world [4].By incorporating these fertilizers into the soil, the heavy soil granules are reduced, resulting in significant enhancements to its physical, chemical, and biological properties.This process effectively dissolves mud particles, increasing water retention capacity within the soil [5].The environmental advantages, plant nutrition efficiency, and cost-effectiveness of these fertilizers have recently made them popular in soilless growing systems.Further, they increase plant growth and yield as well as resistance to biotic and abiotic stresses [4].Bacteria, fungi, and algae are essential sources of organic fertilizers, and their interaction with plants contributes to nutrient enrichment and resistance against environmental stresses [6,7].In addition, the bacteria present in the root zone play a significant role in enhancing nutrient absorption and synthesizing vital substances like antibiotics and indole acetic acid, which are essential for the growth and development of plants [8].Moreover, the use of chemical fertilizers is a major source in agriculture and has been widely used by farmers for its important role in increasing crop productivity, as it contains some soil nutrients such as nitrogen, phosphorus, and potassium in a ready-toabsorb form, thus achieving high crop productivity [9].On the other hand, the excessive use of chemical fertilizers negatively impacts soil ecosystems and reduces microbial diversity [10].The accumulation of toxic chemicals in the soil caused by chemical fertilizers contributes to environmental pollution [11].A sustainable agriculture method and biofertilization are essential to reducing environmental pollution [12].Therefore, this study aimed to investigate the effect of adding Bacillus subtilis and Trichoderma asperellum bacteria, nitrogen fertilizer, and their interaction on the growth and yield arugula.

Site of experiment and plant material
The experiment was carried out in a private field in Al-Azaouia, Al-Musayyib district during the fall of 2022-2023 to investigate the effects of bacterial, Trichoderma fungus, and nitrogen fertilizer additions as well as their interactions on the growth indicators and yield of arugula plant.To represent the root zone, soil samples were collected from the field at various locations at a depth of 0 -30 cm.Following air drying, grinding, and sieving through a 2 mm mesh sieve, the samples were examined to determine some of the soil's chemical and physical characteristics.The experimental field was leveled, plowed, and smoothed before use.The specified experimental soil was then split into three replicates with 18 samples each.Seeds were sown in the form of lines inside the panels, at a rate of 5 lines per experimental unit, where the distance between one line and another is (50 cm), and between one plant and another is (15 cm).The experiment included two factors, comparison, and 40% degraded palm residues, 30% decomposed sheep residues 5% charcoal, 5% wood shavings, and 20% decomposed chicken leftovers were used as the only carrier materials in the control treatment, adding bio fertilizer including mycorrhizae (30g) Trichoderma asperellum (30 g), and Bacillus subtilis (50 ml/L) and a combination of microorganisms (Bacteria and fungi Trichoderma and Mycorrhizae).Adding nitrogen fertilizer (urea) includes; comparison, and at a rate of 15 kg per acre (or one-fourth of the recommended rate), and 30 kg per acre (or half the recommended rate).

The yield of the experimental unit of leaves (kg)
The plants were stuffed by cutting the plant with the level of the soil surface from each experimental unit, where they were weighed directly in the field after harvesting using a two-ranked field scale type (SF -400 C).

Total Phenolic Content (mg/g -1 Dry Weight)
The total phenolic content in the leaves was estimated using the method described by [13] and modified by Peredo [14].One milliliter of the extract was mixed with one milliliter of distilled water, and then 5 milliliters of Folin-Ciocâlteu reagent(FCR) (10% v/v) were added.The samples were left for 8 minutes, followed by the addition of 4 milliliters of 7.5% sodium carbonate.After 90 minutes at 25°C, the absorbance was measured using a Spectrophotometer at a wavelength of 765 nanometers.Blank was prepared similarly, but without adding the plant extract.A standard curve was prepared using Gallic acid (C7H6O5) at concentrations ranging from 10 to 100 micrograms per milliliter.The readings were then normalized to mg/g -1 dry weight.

Total Flavonoids Content (mg/g -1 Dry Weight)
The total flavonoid content in the leaves was determined following the method by [15].0.5 milliliters of the extract were mixed with 1.5 milliliters of methanol and then 0.5 milliliters of 10% aluminum chloride solution were added.Afterward, 0.1 milliliters of 1M potassium acetate were added, followed by 2.8 milliliters of distilled water.The samples were left at room temperature for 30 minutes, and then the absorbance was measured at a wavelength of 510 nanometers using a Spectrophotometer.A standard curve was prepared using Catechin at concentrations ranging from 5 to 45 micrograms per milliliter.The readings were normalized to mg/g -1 dry weight.

Total Tannins Content (mg/g -1 Dry Weight)
The method by [16] was adopted for the estimation of total tannins content.Fifty microliters of the extract were mixed with 1.5 milliliters of 4% Vanillin solution (diluted with methanol) and 750 microliters of concentrated HCl.The samples were incubated in the dark at room temperature for 20 minutes.The absorbance was measured at a wavelength of 500 nanometers using a Spectrophotometer.

Percentage of Nitrogen (%)
The percentage of total nitrogen was determined using the Micro Kjeldahl method following [17] and the following formula: Nitrogen % = (Dilution factor × 14 × Acid normality × Volume of consumed acid) / (1000 × Sample weight after digestion × Volume taken for distillation) × 100.

Percentage of Phosphorus (%)
The percentage of phosphorus in the plant was determined using the ammonium molybdate-vanadate method.Ammonium molybdate (22.5 g) was dissolved in 400 mL of distilled water (solution A), and ammonium vanadate (1.25 g) was dissolved in 300 mL of distilled water (solution B).Solution B was added to solution A, and the mixture was left to cool to room temperature.Then, 250 mL of concentrated nitric acid (HNO3) was added, and the volume was completed to 1 liter with distilled water.Five milliliters of the digested sample were mixed with 5 milliliters of ammonium molybdate-vanadate solution, and the volume was completed to 50 milliliters with distilled water.The sample was left for 30 minutes, and the absorbance was measured at a wavelength of 410 nanometers [18] using a Spectrophotometer.Blank was prepared similarly but without adding the sample.A standard curve was prepared using potassium dihydrogen phosphate (KH 2 PO 4 ) to calibrate the readings, and the concentrations were converted to percentages.

Percentage of Potassium (%)
The percentage of potassium in the plant was determined according to [19] using a Flame Photometer.The instrument was self-calibrated using a standard curve for potassium provided by the manufacturer.

Statistical Analysis
The data obtained from the experiment were statistically analyzed to assess the effects of all factors, including the addition of organic and nitrogenous fertilizers and their interactions, as well as all studied characteristics.The analysis of variance (ANOVA) with a randomized complete block design (RCBD) was performed, and significant differences between means were compared using the least significant difference test at a probability level of 5%.The statistical software [20] was used for the analysis.

The yield of the experimental unit of leaves for the first cutting (kg)
The results of Table 1showed the significant effect of the addition of biofertilizer and chemical fertilizer (nitrogen) and the interaction between them on the yield of the experimental unit of leaves, as the treatment of biofertilizer B6 achieved the highest increase of 6.52 (kg) compared to the rest of the treatments.As for the nitrogen fertilizer, the results showed the superiority of the nitrogen fertilizer treatment A3, which amounted to 4.14 (kg) compared to treatments A1 and A2, which amounted to 3.67 and 3.90 (kg).As for the Interaction, the Interaction treatment A3B6 showed the highest value of 6.76 (kg) compared to the comparison treatment A1B1, which had a value of 1.24 (kg).

phenolic Total (mg/g -1 dry weight)
The results in Table 2 indicate that all study factors, including the addition of bio and nitrogen fertilizers and their interaction, had a significant effect on the total phenolic content (mg/g -1 dry weight) of the leaves.The addition of biofertilizer showed a significant effect, with the highest value observed in treatment B3, reaching 1.046 (mg/g - 1 dry weight), compared to other treatments.Similarly, the addition of nitrogen fertilizer had also a significant effect on the total phenolic content, with the highest mean value observed in treatment A3, reaching 0.896 (mg/g -1 dry weight) compared to treatments A1 and A2 with values of 0.805 and 0.862 (mg/g -1 dry weight) respectively.Furthermore, the interaction between bio and nitrogen fertilizers also showed a significant effect, with the highest value observed in treatment A3B3, reaching 1.083 (mg/g -1 dry weight), compared to the reference treatment A1B1 with a value of 0.513 (mg/g -1 dry weight)."

Flavonoids Total (mg/g -1 dry weight)
Table 3 results showed that all study indicators, represented by the addition of biofertilizer, nitrogen fertilizer, and their interaction, significantly influenced the total flavonoid content in the leaves (mg/g -1 dry weight).It is observed that the effect of adding biofertilizer had a significant impact, with the highest mean at treatment B3, reaching 35.92 (mg/g -1 dry weight) compared to the other treatments.Similarly, the addition of nitrogen fertilizer had a significant effect on the total flavonoid content in the leaves (mg/g -1 dry weight), with the highest mean at treatment A3, which reached 35.56 (mg/g -1 dry weight) compared to treatments A1 and A2, which reached values of 35.29 and 35.49(mg/g -1 dry weight), respectively.Moreover, it can be noticed from the same table that the interaction between adding biofertilizer and nitrogenous fertilizer had a significant effect.The highest value was observed for treatment A3B3, reaching 35.98 (mg/g -1 dry weight) compared to the control treatment A1B1, which reached a value of 34.47 (mg/g -1 dry weight).4 results showed that all study indicators, represented by the addition of biofertilizer, nitrogen fertilizer, and their interaction, significantly influenced the total tannin content in the leaves (mg/g -1 dry weight).It is observed that the effect of adding biofertilizer had a significant impact, with the highest mean at treatment B3, reaching 1.012 (mg/g -1 dry weight) compared to the other treatments.Similarly, the addition of nitrogen fertilizer had a significant effect on the total tannin content in the leaves (mg/g -1 dry weight), with the highest mean at treatment A3, which reached 0.707 (mg/g -1 dry weight) compared to treatments A1 and A2, which reached values of 0.578 and 0.646 (mg/g -1 dry weight), respectively.Moreover, it can be noticed from the same table that the interaction between adding biofertilizer and nitrogenous fertilizer had a significant effect.The highest value was observed for treatment A3B3, reaching 1.086 (mg/g -1 dry weight) compared to the control treatment A1B1, which reached a value of 0.326 (mg/g -1 dry weight).

Nitrogen Percentage (%)
Table 5 results showed that all study indicators; represented by the addition of biofertilizer, nitrogen fertilizer, and their interaction, significantly influenced the increase in nitrogen concentration in arugula leaves as a percentage.It is observed that the effect of adding biofertilizer had a significant impact on increasing nitrogen concentration, with the highest value at treatment B4, which reached 3.89% compared to the other treatments.Similarly, the addition of nitrogen fertilizer had a significant effect on increasing nitrogen concentration in arugula leaves as a percentage, with the highest value at treatment A3, reaching 2.85%, compared to treatments A1 and A2, which reached values of 2.62% and 2.73%, respectively.Moreover, it can be noticed from the same table that the interaction between adding biofertilizer and nitrogen fertilizer had a significant effect on increasing nitrogen concentration in arugula leaves as a percentage.The highest value was observed for treatment A3B4, reaching 3.98%, compared to the control treatment A1B1, which reached a value of 1.20%.6 results showed that all study indicators, represented by the addition of biofertilizer and nitrogenous fertilizer, and their interaction, significantly influenced the increase in phosphorus concentration in arugula leaves as a percentage.It is observed that the effect of adding biofertilizer had a significant impact on increasing phosphorus concentration, with the highest value at treatment B4, which reached 0.81% compared to the other treatments.Similarly, the addition of nitrogenous fertilizer had a significant effect on increasing phosphorus concentration in arugula leaves as a percentage, with the highest value at treatment A3, reaching 0.51%, compared to treatments A1 and A2, which reached values of 0.43% and 0.48%, respectively.Moreover, it can be noticed from the same table that the interaction between adding biofertilizer and nitrogenous fertilizer had a significant effect on increasing phosphorus concentration in arugula leaves as a percentage.The highest value was observed for treatment A3B4, reaching 0.88%, compared to the control treatment A1B1, which reached a value of 0.20 %.

Potassium content (%)
Table 7 results showed that all study indicators, represented by the addition of biofertilizer and nitrogenous fertilizer, and their interaction, significantly influenced the increase in the percentage of potassium concentration in arugula leaves.It is observed that the effect of adding biofertilizer had a significant impact on increasing potassium concentration, with the highest value at treatment B4, which reached 4.81% compared to the other treatments.Similarly, the addition of nitrogenous fertilizer had a significant effect on increasing potassium concentration in arugula leaves as a percentage, with the highest value at treatment A3, reaching 4.03%, compared to treatments A1 and A2, which reached values of 3.83% and 3.95%, respectively.Moreover, it can be noticed from the same table that the interaction between adding biofertilizer and nitrogenous fertilizer had a significant effect on increasing potassium concentration in arugula leaves as a percentage.The highest value was observed for treatment A3B4, reaching 4.92%, compared to the control treatment A1B1, which reached a value of 2.14%.

Discussion
Through tables (1, 2, 3, 4, 5, 6, and7), it was observed that the addition of bacteria, Trichoderma fungus, and nitrogen fertilizer, as well as their interactions, had a significant impact on the content of active compounds in the leaves.The reason for this effect can be attributed to the increased accumulation of dry matter resulting from enhanced photosynthetic processes, which play a fundamental role in primary metabolism.This, in turn, led to an increased accumulation of intermediate compounds involved in secondary metabolisms, such as phenolic and flavonoid compounds [21].Furthermore, there is a significant positive association between indicators of vegetative growth and the rate of photosynthetic processes, resulting in an increased need for antioxidant compounds to maintain enzymatic reactions involved in the synthesis of dry matter from free radicals [22].This relationship becomes evident when biological inoculation is accompanied by increased concentrations of ascorbic acid, which plays a crucial role in the Ascorbate-  [23,24].
The increase in content can also be attributed to the accumulation of carbohydrates in the plant, which directly contributes to the production of phenols via Shikimic acid and others [25].It is worth noting that the increase in plant content of flavonoids depends on the availability of elements from their source in the soil [26].Recent studies have shown that biological fertilizers play a role in increasing the availability of phosphorus in the soil by the ability of bacterial inoculation to dissolve precipitated phosphate compounds and release them into the soil solution as H 2 PO4 1-and HPO4 2-, and Bacillus bacteria can increase phosphorus availability through soil pH reduction [27].Several studies have also demonstrated the ability of Bacillus spp.bacteria, when applied as a safe environmental biological agent, to enhance growth standards and increase organic acids in plants, and they have been shown to inhibit both natural and artificial plant diseases [28].
The increase in leaf content of major nutrients (N, P, and K), can be attributed to the effect of Trichoderma fungus inoculation, which improves nutrient availability and leads to an increased root-soil contact area.It also enhances the activity of external enzymes, resulting in an increased production of organic acids in the rhizosphere, improving the efficiency of enzyme activity in the soil [29].Moreover, the experiment's treatments had significant effects on leaf phosphorus content, possibly due to the role of phosphatesolubilizing fungi, including Trichoderma, which possesses various properties and characteristics that promote plant growth [30].These properties include acidification, production of chelating metabolites, redox activity, as well as the production of growth regulators like ethylene, gibberellins [31], and IAA [32], which enhance plant growth and develop a dense and strong root system capable of extracting nutrients such as phosphorus.In this context, inoculation with Trichoderma spp.enhances the efficiency of nutrient absorption [33].Additionally, certain isolates of Trichoderma spp.possess the ability to penetrate plant roots and form fungal structures in the roots [34].Among these characteristics is their ability to dissolve insoluble phosphate compounds by producing organic acids such as gluconic, lysinic, humic, as well as inorganic acids like nitric, sulfuric, and phosphoric acids, transforming them from non-readily available forms to readily available forms for plant uptake [35,36].
As for the addition of nitrogen fertilizer, it had a positive effect on the nutritional content of the green mass due to its high solubility.Moreover, adding nitrogen at the appropriate level led to an increase in the plant's mineral content, which aligns with the findings of [37] and [38].The addition of nitrogen stimulates vital biological processes within the plant and its role in forming substances required by the plant, such as the formation of amino acids and protein compounds in the green mass [39,40].The nitrogen fertilizer contains readily available elements that are directly absorbed by the plant, facilitating the formation of a good green mass, which positively affects the plant's nutrient content, including nitrogen.This allows the plant to perform its vital functions effectively, thus improving the quality and yield of the harvest [41].

Conclusion
This study investigated the effect of adding Bacillus subtilis and Trichoderma asperellum bacteria, along with nitrogen fertilizer, and their interactions on the growth and yield of arugula.The results demonstrated that these interventions had a significant positive impact on arugula's growth and the content of active compounds in its leaves.The addition of Bacillus subtilis and its carrier materials significantly increased the amount of phenolic compounds, flavonoids, and tannins in arugula leaves.On the other hand, adding Trichoderma asperellum and its carrier substances dramatically raised the levels of nitrogen, phosphorus, and potassium in the leaves.The application of both organic and nitrogen fertilizers greatly improved various growth indicators for arugula.The use of eco-friendly strategies, such as organic and bio fertilizers, proved to be effective in enhancing plant growth and the content of active compounds.The findings highlight the importance of sustainable agricultural practices in maximizing the nutritional and medicinal value of arugula.

Table 1 .
Effect of adding biofertilizer and nitrogen fertilizer and the interaction between them on the yield of the experimental unit from the first cutting (Kg)

Table 2 .
Effect of adding biofertilizer and chemical fertilizers and their interaction on the total phenolic content in the leaves (mg/g -1 dry weight)

Table 3 .
Effect of adding biofertilizer and chemical fertilizers and their interaction on the total flavonoid content in the leaves (mg/g -1 dry weight)

Table 4 .
Effect of adding biofertilizer and chemical fertilizers and their interaction on the total tannin content in the leaves (mg/g -1 dry weight)

Table 5 .
Effect of adding biofertilizer and chemical fertilizers and their interaction on the nitrogen content in arugula leaves %

Table 6 .
Effect of adding biofertilizer and chemical fertilizers and their interaction on the percentage of phosphorus content in arugula leaves %

Table 7 .
Effect of adding bio and chemical fertilizers and their interaction on the potassium content in arugula leaves % Cycle Glutathione pathway to counteract H 2 O 2 produced by increased biological activities.The linear correlation coefficient describes the strong positive association statistically