Enhancing the Growth of Artemisia abrotanum by Magnesium and Tropaeolum majus Extract in a Field Experiment Along with the Antibacterial Activity of the Isolated Essential Oils

Abstract

Enhancing the growth and productivity of ornamental and horticultural plants is a major function of plant extracts and macronutrient elements. The growth properties of Artemisia abrotanum plants were evaluated in two successive seasons as affected by the magnesium (Mg) fertilizer added to the soil in the form of magnesium sulfate at four concentrations of 0 (as a control), 4, 6, and 8 g/L as well as Tropaeolum majus aqueous leaf extract (ALE) at concentrations of 0 (as a control), 4, 6, and 8 g/L as a foliar application. The chemical components of A. abrotanum essential oils (EOs) were analyzed using the gas chromatography–mass spectrometry (GC-MS) apparatus. The studied parameters, including plant height, total fresh weight, number of branches/plant, EO percentages, chlorophyll-a content, chlorophyll-b content, and carotenoid content, were enhanced by the application of Mg or T. majus ALE or their combinations. The highest plant heights, 48.83 cm, and 48.5 cm, were observed in the plants treated with Mg (8 g/L)+T. majus ALE (8 g/L) and Mg (6 g/L)+T. majus ALE (4 g/L), in both seasons, respectively. The highest values of total fresh weight, 54.80 and 60.59 g, were recorded in plants treated with Mg (8 g/L)+T. majus ALE (4 g/L) and Mg (8 g/L)+T. majus ALE (4 g/L), in both seasons, respectively. The highest number of branches/plant, 60.33 and 73.33, were measured in plants treated with Mg (8 g/L)+T. majus LAE (8 g/L), in both seasons, respectively. The highest EO percentages, 0.477% and 0.64%, were measured in plants treated with Mg (8 g/L)+T. majus ALE (8 g/L), in both seasons, respectively. The total fresh weight in both seasons (r = 0.96), the number of branches/plant in both seasons (r = 0.97), the number of branches/plant in the first season, and the number of branches/plant in both seasons (r = 0.96), the total fresh weight in the second season and the number of branches/plant in the first season (r = 0.95) and the second season (r = 0.94), and the number of branches/plant and the carotenoids in the first season (r = 0.90) were all found to be significantly and positively correlated. The major compounds in the EOs were 7-methoxy-4-methylcoumarin (4-methylherniarin), cedrol, endo-borneol, and 7-epi-silphiperfol-5-ene. The antibacterial activity of the EOs was evaluated against the growth of Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum, which causes soft rot of potato tubers. The EOs were found to be effective against P. carotovorum subsp. carotovorum with the inhibition zones ranging from 1 to 5 mm at the concentration of 100 μg/mL, and no inhibitions were found against P. atrosepticum at the studied concentrations. The minimum inhibitory concentration against P. carotovorum subsp. carotovorum was found at 75 μg/mL. In conclusion, using the combination treatments of Mg and T. majus ALE is highly suggested to enhance the growth of A. abrotanum plants.

1. Introduction

Southern wormwood, or Artemisia abrotanum L., (Family Compositae) is a plant species that has played a significant role in Asian and European medical history. This species is well known for its therapeutic properties in southeast and central Europe, as well as in Asia Minor and central Asia [1,2]. When utilized as an ingredient in nail gel for patients with irregularities of the nail plate surface, A. abrotanum leaf extract shows encouraging outcomes [3]. Furthermore, A. abrotanum extract has the potential to be an active ingredient in cosmetics for acne-prone skin due to its antibacterial activity against Propionibacterium acnes strains that are resistant to macrolides [4]. A. abrotanum leaves are used to flavor meats, salads, and cottage cheese because of their pleasant scent. They are occasionally used as flavorings in alcoholic beverages like liqueurs and vermouths, as well as in confections [5,6,7]. Teas contain the herb of A. abrotanum as an ingredient [7]. In European herbal therapy, A. abrotanum is frequently used to treat a variety of diseases, including fever. The crude extracts and some of the constituents of A. abrotanum were tested for antimalarial activity against Plasmodium falciparum in vitro using the technique of [3H]hypoxanthine incorporation [8].

The market for plant-derived biostimulants (PDBs) is expanding in importance, and environmental sustainability has a big impact on how they are used. PDBs are widely used to fortify plants, satisfy commercial needs, produce high-quality products, increase plant vitality, and make harvesting easier in a variety of horticultural applications, including ornamentals. Biostimulants can be regulated by proving their effectiveness and safety and identifying a general mechanism of action [9]. We may soon have a better grasp of the mechanisms and potentially possible modes of action of biostimulants due to the development of new molecular biotechnology tools [10].

When the development of quality features depends on Mg-driven photosynthesis and assimilated translocation within the plant, increasing the magnesium supply on sites where magnesium deficiency exists tends to improve the quality of agricultural harvests [11]. For horticultural crops to be healthy, productive, and high-quality, magnesium (Mg²⁺) is essential. Mg²⁺, an essential part of chlorophyll and a key player in plant physiology, drives photosynthesis, supporting biomass accumulation and growth [12]. Mg is recognized as an essential element of the plant, where it is essential for several fundamental physiological and metabolic processes. Key elements include the production, transport, and utilization of photoassimilates; protein synthesis; enzyme activation; and chlorophyll synthesis [13]. In fact, Mg is necessary for some critical physiological and biochemical functions that take place throughout plant growth and development [14]. Mg is the basic building block of the chlorophyll pigments in the light-capturing complex of the chloroplasts, which contributes to the uptake of CO₂ during photosynthetic processes [11,15].

Some research has demonstrated that PDBs have no detrimental effects on the environment or human health because their constituents have minimal biological toxicity, quick environmental degradation, poor food mobility, and low application rate [16,17,18,19]. Because they can encourage higher production with less environmental impact, their use can be crucial to enhancing agricultural sustainability [20]. Natural extracts can enhance and stimulate the growth and biochemical components present in treated horticultural, ornamental, and flowering plants [21,22]. PDBs can improve the efficiency of nutrient utilization, protect plants from abiotic stresses, and/or promote plant growth [23]. To sustainably increase productivity and quality, PDBs are receiving more and more attention and being included in high-value production systems (such as greenhouse production, fruit, vegetable, and floriculture) [22,24,25].

In the present work, Mg and Tropaeolum majus L. extract were used to enhance the growth of Artemisia abrotanum plants. T. majus L. (T. elatum Salisb., Tropaeolaceae), or nasturtium, comes from the mountainous areas of South and Central America. Nasturtium is native to Peru and Bolivia [26]. It has been cultivated in Europe since the seventeenth century because of its applications as a decorative plant and in herbal medicine. Sulfur compounds (glucotropaeolin), flavonoids (quercetin, isoquercetin and kaempferol glycosides), anthocyanins (delphinidin, cyanidin, and pelargonidin derivatives), carotenoids (lutein, zeaxanthin, and β-carotene), phenolic acids (chlorogenic acid), cucurbitacines, and vitamin C have all been mentioned in phytochemical studies of T. majus [26,27,28,29].

It has been proven that many edible flowers are a rich source of bioactive compounds, especially phenolic compounds such as flavonoids and anthocyanins, which have beneficial properties for human health [30]. The preliminary screening showed that the T. majus extracts have a variety of reducing phytochemicals including tannins, terpenoids, flavonoids, and cardiac glycosides, T. majus aqueous extract has a potential antioxidant effect [31]. The fresh leaves and flowers of T. majus are also used in feeding, especially in salads, and were pointed out by a study as an excellent source of the carotenoid lutein [27,28,32]. The extracts from edible flowers of T. majus are rich sources of polyphenols with marked antioxidant activity [33].

Pectobacterium spp. is the major cause of soft rot in potato tubers [34]. Potato (Solanum tuberosum L.) is considered one of Egypt’s vegetable crop values, after tomatoes, and is regarded as one of the country’s most significant crops [35]. Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum are pectinolytic bacteria that are facultatively anaerobic and Gram-negative and were previously classified as Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora [36]. These polyphagous bacteria induce bacterial soft rot in potatoes, and one of their host plants is the potato tuber. Tuber rot can be caused by bacteria in the soil right away, after harvest, or while being stored [37].

The quality, storage life, and yield of potato tubers are significantly reduced due to a strong odor that usually accompanies the subsequent decomposition of tubers [38]. The bacteria can live in contaminated tubers and contaminate surface water, weed roots, soil residues, and field crops. [39,40]. Therefore, protection is critical to prevent potato tuber infection. Attention is focused on using essential oils from different plant species or their purified components to control the causal against potato tubers’ soft rot [41].

In upcoming years, there could potentially be a greater need for biostimulants in low-input, conventional, and organic agriculture. Because of this, there is growing interest in creating a new generation of plant biostimulants for sustainable agriculture using various natural extracts that work in synergy. Furthermore, innovative products that can sustain yield under the conditions imposed by industrial global warming are becoming more and more necessary. Thus, this study aimed to determine how well magnesium and Tropaeolum majus aqueous leaf extract, a plant-derived biostimulant, might promote Artemisia abrotanum plant development and essential oil production. The second aim was to determine the primary effects of the essential oils as antibacterial agents against P. carotovorum subsp. carotovorum and P. atrosepticum.

2. Materials and Methods

The Artemisia abrotanum (Southernwood) and Tropaeolum majus ‘Empress of India’ plants were sourced from a botanical garden in Alexandria and were identified by the Plant Production Department, Faculty of Agriculture Saba Basha, Alexandria University. Voucher specimens were deposited at the Alexandria University Herbarium under accession numbers no. 4832 (Artemisia abrotanum) and no. 4891 (Tropaeolum majus), respectively.

2.1. Collection and Preparation of Tropaeolum majus Extract

Nasturtium (Tropaeolum majus L.) ‘Empress of India’ plants were collected in March 2022 and 2023 during the flowering stage from the nursery of the Department of Floriculture, Ornamental Horticulture, and Garden Design, Faculty of Agriculture, Alexandria University, Egypt, and the identification was confirmed at the Department, Faculty of Agriculture, Alexandria University, Egypt. T. majus leaves were washed with running tap water [21] to remove dust and any dirt over the leaves, then dried at room temperature, and ground to a fine powder using a grinder. The material powder was kept in dry conditions and ready for further use.

In a glass bottle, 100 g of T. majus leaf powders were soaked for 24 h at room temperature in 1000 mL of distilled water. After passing through Whatman filter paper No. 1, they were dried for 72 h at 50 °C in an oven [42]. The final dried extract was stored at 4 °C in a refrigerator [43]. The extraction yield of T. majus leaves was calculated by the following equation: extraction yield (%) = (W1/W2) × 100, where W1 is the mass of leaf crude extract and W2 is the mass of the dry leaf sample [44]. The extract percentage was 2.50%.

2.2. Field Experiment

Artemisia abrotanum L. (Southernwood) plants were arranged with two treatments performed in the randomized complete block design (RCBD) with three replications at two successive seasons (2022 and 2023) at the nursery of the Department of Floriculture, Ornamental Horticulture and Garden Design, Faculty of Agriculture, Alexandria University, Egypt. A. abrotanum cuttings were planted in March. Then, the rooted cuttings were transplanted after one month for both seasons (2022 and 2023), where three similar plants were selected for each replicate. Therefore, each treatment has nine plants. Figure S1 shows the arrangement of the experimental work.

2.3. Treatments

The first factor was magnesium (Mg) fertilizer in the form of magnesium sulfate at four concentrations of 0 (as a control), 4, 6, and 8 g/L added to the soil with water irrigation (500 mL/plant). The soil analysis is shown in

Table 1. Chemical analysis of soil.

Element	Value
pH	7.61
EC (ds/m)	3.80
Soluble cations (meq/L)
Na+	13.30
K+	3.80
Ca2+	11.00
Mg2+	9.00
Soluble anions (meq/L)
Cl−	10.50
HCO3−	12.00
CO32−	0.00
SO42−	15.50

[21]. The second factor, Tropaeolum majus aqueous leaf extract (ALE), was applied as a foliar application at the concentrations of 0 (as a control), 4, 6, and 8 g/L. A. abrotanum plants received these treatments four times (from May to August) at one-month intervals during the two seasons, starting one month after transplanting. In both seasons, measurements of the plant’s height (cm), number of branches/plant, total fresh weight (g) chlorophyll-a, chlorophyll-b, carotenoid content, and the percentage of essential oils were done.

2.4. Analysis of Phenolic and Flavonoid Compounds by HPLC

The phenolic compound from T. majus ALE was analyzed using HPLC (Agilent 1100, Agilent ChemStation, Santa Clara, CA 95051, USA) and a UV/Vis detector, two LC pumps, and a C18 column (125 mm × 4.6 mm, 5 μm film thickness) was used to gather and examine chromatograms. By using a gradient mobile phase of two solvents—solvent A (methanol) and solvent B [acetic acid in water (1:25)]. For the first 3 min, the gradient program was maintained at a concentration of 100% B. The concentration of eluent A was then raised to 80% for the following 2 min, then decreased to 50% once again for the following 5 min with detection wavelength at 250 nm. This was followed by 5 min of 50% eluent A. As a result, the order of phenolic compounds was established utilizing this mobile phase to verify standard compounds [45,46].

HPLC (Agilent 1100), which consists of two LC pumps, a UV/Vis detector, and a C18 column (250 mm × 4.6 mm, 5 μm), was used to identify the flavonoid components from T. majus ALE. With an isocratic elution (70:30) program, the mobile phase consisted of acetonitrile (A) and 0.2% (v/v) aqueous formic acid (B). The detection wavelength was 360 nm [45,46]. The phenolic and flavonoid compounds were identified by comparison with standard retention times.

2.5. Photosynthetic Pigments of Chlorophylls and Carotenoid Contents

The following extraction and analysis were carried out as previously mentioned [47]. In brief, the leaves were chopped into small pieces, and then, after adding 10 mL of absolute acetone, 0.5 g of the leaves were pulverized in a mortar and pestle. After centrifuging the homogenized material for 20 min at 4 °C at 10,000 rpm, 0.5 mL of the supernatant was combined with 4.5 mL of acetone absolute. The following suggested equations were then used to estimate the amounts of chlorophyll-a, chlorophyll-b, and carotenoid in a spectrophotometer using this solution [48,49]. All measurements were done in triplicate.

• Chlorophyll-a = 11.75 A662 − 2.350 A645 (μg/mL)
• Chlorophyll-b = 18.61 A645 − 3.960 A662 (μg/mL)
• Carotenoids = 1000 A470 − 2.270 Chl a − 81.4 Chl b/227 (μg/mL)

2.6. Essential Oil Extraction

The essential oils (EOs) from A. abrotanum leaves were extracted using the hydrodistillation process with the Clevenger apparatus. About 100 g of fresh leaves were placed in a 2 L flask with 1500 mL of distilled water for 3 h, then the EOs were collected and stored at 4 °C in a refrigerator until analysis [50].

2.7. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

The chemical composition of the EOs from the A. abrotanum leaves collected from the treated plants with the 16 treatments was performed using a Trace GC Ultra-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µm film thickness). The column oven temperature was initially held at 70 °C, then increased by 5 °C/min to 280 °C withheld for 5 min then increased to 300 at 5 °C/min. The injector and MS transfer line temperatures were kept at 250 °C. Helium was used as a carrier gas at a constant 1 mL/min flow rate. The solvent delay was 2 min and diluted samples of 1 µL were injected automatically using Autosampler AS1310 coupled with GC in the split mode. In full scan mode, EI mass spectra were collected at 70 eV ionization voltages over the m/z 40–600 range. The ion source was set at 200 °C.

The components of the EOs were identified by comparing their mass spectra with those of the WILEY 09 and NIST 14 mass spectral databases [51]. The Xcalibur 3.0 data system in the GC–MS with its threshold values was used to confirm that all the mass spectra of the identified compounds were attached to the library. The Xcalibur 3.0 data system of the GC–MS with threshold values was used to confirm that all the MS were attached to the library by measuring the Standard Index and Reverse Standard Index, where the value of the match factor (MF) with ≥650 was acceptable to confirm the compounds [52,53].

2.8. Antibacterial Activity of the Essential Oils

Two bacterial isolates, Pectobacterium carotovorum subsp. carotovorum (LN851554) and Pectobacterium atrosepticum (LT592258) [54], were obtained from the Department of Agricultural Botany, Faculty of Agriculture (Saba Basha), Alexandria University, Egypt. The bacterial isolates were renewed by growing on a nutrient agar (NA) medium at an optimal temperature of 26 ± 2 °C for 2 days. The EOs of A. abrotanum were prepared at the concentrations of 100, 75, 50, and 25 µL/mL by dissolving the respective amount of the EO in 10% dimethyl sulfoxide (DMSO), and then 0.5 mL of Tween 20 was added. The antibacterial assay of the EOs was determined using the disc diffusion susceptibility test [36]. Each of P. carotovorum subsp. carotovorum and P. atrosepticum inoculum at 100 µL was pipetted onto Petri dishes containing NA medium and uniformly spread with a sterile hockey stick rod. The sterile filter paper discs (10 mm diameter) were each filled with 75 µL of the prepared concentrations. The plates were incubated at the optimal temperature for bacterial growth, and, after 24 h, the diameters of the inhibition zones (IZs) were measured. The minimum inhibitory concentrations (MICs) were determined by serial dilution of the EOs ranging from 25 to 100 µg/mL [46]. Negative control (DMSO 10% and Tween 20) and positive control (Neomycin Sulphate 20 µg/disc) controls were used, and all the tests were done in triplicate.

2.9. Statistical Analysis

The experiment was conducted in a complete randomized block design (RCBD) containing 16 treatments with three replicates in two factors including Mg and the extract. The collected data from both seasons were subjected to analysis of variance (ANOVA) using the SAS program. For each of the seven factors examined in both seasons, simple linear regressions were used to determine a linear relationship that would characterize the correlation between an independent and potentially dependent variable.

3. Results

3.1. Phenolic Compounds from Tropaeolum majus Aqueous Leaf Extract

The phenolic compounds identified by the HPLC analysis in the Tropaeolum majus aqueous leaf extract (ALE) are shown in

Table 2. The phenolic compounds in Tropaeolum majus aqueous leaf extract.

RT * (min)	Compound	Height (mAU)	Area (mAU.s)	Concentration (μg/mL)
3.0	Chlorogenic acid	980.410	510.22	15.22
4.0	Catechol	640.471	450.02	10.62
5.0	Syringic acid	451.062	430.08	8.80
8.0	Caffeic acid	1105.036	588.79	17.46
9.0	Pyrogallol	630.400	439.77	11.78
10.7	Ferulic acid	98.012	75.04	3.30

*: RT: Retention time (min); mAU: milli-absorbance unit.

and Figure 1. The most detected abundant phenolic compounds were caffeic acid (17.46 μg/mL), chlorogenic acid (15.22 μg/mL), pyrogallol (11.78 μg/mL), and catechol (10.62 μg/mL).

3.2. Flavonoid Compounds from Tropaeolum majus Aqueous Leaf Extract

The flavonoid components found in T. majus ALE by HPLC analysis are displayed in

Table 3. The flavonoid compounds in Tropaeolum majus aqueous leaf extract.

RT * (min)	Compound	Height (mAU)	Area (mAU.s)	Concentration (μg/mL)
4.6	Naringin	202.102	215.68	18.45
5.2	Rutin	500.410	330.85	10.75
7.0	Quercetin	1201.011	448.52	8.51
8.1	Kaempferol	180.001	204.44	6.75
9.0	Luteolin	190.120	380.78	9.57
10.0	Apigenin	177.030	189.58	7.56
12.01	Catechin	195.255	320.09	8.70

*: RT: Retention time (min); mAU: milli-absorbance unit.

and Figure 2. Naringin (18.45 μg/mL), rutin (10.75 μg/mL), and luteolin (9.57 μg/mL) were the most prevalent flavonoid compounds.

3.3. Vegetative Parameters of A. abrotanum Plants

Table 4. The plant height, total fresh weight, and the number of branches/plant of A. abrotanum in both growing seasons.

Treatment		Plant Height (cm)		Total Fresh Weight (g)		No. Branches/Plant
Mg (g/L)	Extract (g/L)	First Season	Second Season	First Season	Second Season	First Season	Second Season
0	0	37.58 ± 5.00 cde	35.25 ± 3.54 f	32.57 ± 6.04 f	34.33 ± 1.31 f	30.83 ± 3.32 h	39.00 ± 1.80 g
0	4	37.33 ± 0.76 de	37.16 ± 2.08 ef	35.34 ± 6.83 ef	35.47 ± 8.38 f	33.50 ± 5.63 gh	42.16 ± 5.13 fg
0	6	43.33 ± 4.88 ab	41.16 ± 1.37 cde	36.74 ± 6.06 ef	38.21 ± 4.31 f	36.33 ± 4.64 gh	45.33 ± 3.05 fg
0	8	43.83 ± 1.94 ab	41.16 ± 3.75 cde	36.97 ± 1.85 ef	39.53 ± 1.78 ef	41.16 ± 6.42 defg	44.50 ± 5.41 fg
4	0	35.66 ± 5.39 e	38.66 ± 1.52 def	35.93 ± 5.33 ef	40.61 ± 3.32 ef	37.66 ± 4.64 fgh	44.50 ± 9.50 fg
4	4	40.91 ± 2.98 bcde	42.83 ± 1.77 bcd	36.37 ± 1.8 ef	40.63 ± 3.87 ef	39.83 ± 4.61 efg	45.00 ± 8.04 fg
4	6	43.25 ± 4.16 abc	45.25 ± 3.31 abc	45.65 ± 2.67 cd	47.52 ± 3.62 cde	44.66 ± 5.96 cdef	49.00 ± 4.35 ef
4	8	47.58 ± 4.30 a	45.75 ± 2.29 abc	45.45 ± 1.63 cd	46.74 ± 6.15 de	45.33 ± 2.75 cdef	57.00 ± 4.76 de
6	0	41.83 ± 1.90 bcd	45.08 ± 1.51 abc	41.84 ± 2.14 de	47.65 ± 1.71 cde	41.16 ± 3.78 defg	49.50 ± 7.05 ef
6	4	45.75 ± 5.81 ab	48.50 ± 3.25 a	46.69 ± 4.91 bcd	49.003 ± 6.26 cd	47.16 ± 5.34 cde	60.83 ± 3.40 bcd
6	6	47.58 ± 1.01 a	46.75 ± 2.94 ab	50.67 ± 7.29 abc	51.50 ± 5.36 bcd	57.66 ± 5.79 ab	66.66 ± 3.01 abc
6	8	45.25 ± 3.63 ab	47.58 ± 4.78 ab	49.94 ± 2.97 abc	50.57 ± 6.07 bcd	51.50 ± 1.32 bc	64.00 ± 1.50 bcd
8	0	41.41 ± 3.87 bcd	43.75 ± 2.88 abc	43.28 ± 8.14 cde	49.18 ± 4.73 cd	48.50 ± 3.46 cd	58.83 ± 3.21 cd
8	4	46.16 ± 2.75 ab	46.16 ± 1.37 ab	54.80 ± 5.84 a	60.59 ± 7.01 a	58.16 ± 4.36 ab	68.83 ± 5.75 ab
8	6	47.75 ± 2.70 a	46.25 ± 0.25 ab	51.08 ± 2.78 abc	55.18 ± 4.65 abc	57.33 ± 5.51 ab	66.00 ± 5.67 abc
8	8	48.83 ± 3.64 a	48.33 ± 6.52 a	53.75 ± 3.04 ab	57.78 ± 1.70 ab	60.33 ± 2.36 a	73.33 ± 1.04 a
LSD 0.05 *		5.702	4.975	8.096	8.241	7.806	8.284

*: LSD; Least significant difference. Values are means ± SD. Means with the same letter/s within the same column are not significantly different according to LSD at 0.05 level of probability.

shows the recorded data for the plant height, total fresh weight, and the number of branches/plant in both seasons.

In the first season, the highest plant height (cm) was observed in the plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (48.83 cm), 8+6 (47.75 cm), 6+6 (47.58 cm), 4+8 (47.58 cm), 8+4 (46.16 cm), 6+4 (45.75 cm) and 6+8 (45.25 cm). In the second season, the greatest heights of plants were observed in plants treated with Mg (g/L)+T. majus ALE (g/L) at 6+4 (48.5 cm), 8+8 (48.33 cm), 6+8 (47.58 cm), 6+6 (46.75 cm), 8+4 (46.16 cm) and 8+6 (46.25 cm).

For the weighted total fresh weight (g), the highest values observed in the first season were in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+4 (54.80 g), 8+8 (53.75 g), 8+6 (51.08 g), 6+6 (50.67 g), and 6+8 (49.94 g). In the second season, these were recorded in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+4 (60.59 g), 8+8 (57.78 g), 8+6 (55.18 g), 6+6 (51.50 g) and 6+8 (50.57 g).

In the first season, the highest numbers for branches/plant were recorded in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (60.33 branches/plant), 8+4 (58.16 branches/plant), 6+6 (57.66 branches/plant), 8+6 (57.33 branches/plant) and 6+8 (51.5 branches/plant). In the second season, the highest numbers for branches/plant were recorded in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (73.33 branches/plant), 8+4 (68.83 branches/plant), 6+6 (66.66 branches/plant), 8+6 (66 branches/plant), 6+8 (64 branches/plant) and 6+4 (60.83 branches/plant).

3.4. Essential Oil, Chlorophyll-a and -b, and Carotenoid Contents

The EO, chlorophyll-a and -b, and carotenoid contents of A. abrotanum in both growing seasons are shown in

Table 5. The essential oil, chlorophyll-a and -b, and carotenoid contents of A. abrotanum in both growing seasons.

Treatment		Essential Oil (%)		Chlorophyll-a Content (µg/mL)		Chlorophyll-b Content (µg/mL)		Carotenoid Content (µg/mL)
Mg (g/L)	Extract (g/L)	First Season	Second Season	First Season	Second Season	First Season	Second Season	First Season	Second Season
0	0	0.26 ± 0.02 d	0.32 ± 0.006 h	9.11 ± 1.19 g	14.77 ± 2.09 h	10.22 ± 2.46 a	6.85 ± 1.54 d	2.94 ± 0.04 g	5.51 ± 0.43 h
0	4	0.24 ± 0.05 d	0.36 ± 0.03 gh	16.46 ± 1.07 ef	15.51 ± 0.81 gh	6.97 ± 1.03 cd	6.18 ± 0.06 d	2.55 ± 0.24 g	6.69 ± 0.22 f
0	6	0.32 ± 0.09 cd	0.42 ± 0.07 fg	16.83 ± 0.84 def	15.50 ± 0.97 gh	6.73 ± 1.01 cd	6.71 ± 0.51 d	2.30 ± 0.34 g	7.19 ± 0.52 e
0	8	0.39 ± 0.04 abc	0.41 ± 0.07 fgh	14.96 ± 0.76 f	18.39 ± 0.58 cdef	6.03 ± 1.14 cd	6.87 ± 0.37 d	5.34 ± 0.21 de	6.61 ± 0.11 fg
4	0	0.31 ± 0.04 cd	0.43 ± 0.02 fg	17.11 ± 1.49 def	17.02 ± 0.45 efgh	7.78 ± 0.86 bcd	7.05 ± 0.77 d	2.99 ± 0.24 g	6.52 ± 0.47 fg
4	4	0.41 ± 0.07 abc	0.42 ± 0.04 fg	17.51 ± 1.39 cde	16.16 ± 0.82 fgh	6.68 ± 0.96 cd	7.55 ± 0.14 cd	5.75 ± 0.26 d	7.52 ± 0.25 de
4	6	0.45 ± 0.03 ab	0.54 ± 0.07 bcde	20.63 ± 0.37 ab	20.42 ± 0.35 bc	5.62 ± 1.04 d	10.76 ± 1.11 c	4.74 ± 0.74 ef	8.12 ± 0.14 ab
4	8	0.43 ± 0.01 ab	0.59 ± 0.07 abc	16.77 ± 1.05 ef	17.00 ± 0.77 efgh	8.09 ± 1.80 abc	7.64 ± 1.73 cd	5.16 ± 0.16 de	7.62 ± 0.19 bcde
6	0	0.39 ± 0.11 abc	0.49 ± 0.01 ef	15.95 ± 0.71 ef	16.26 ± 1.67 fgh	7.82 ± 0.69 bcd	6.19 ± 2.57 d	4.22 ± 0.74 f	6.15 ± 0.37 g
6	4	0.38 ± 0.08 abc	0.49 ± 0.02 def	18.96 ± 3.32 bcd	20.51 ± 1.21 bc	6.61 ± 0.55 cd	8.90 ± 0.94 cd	6.70 ± 0.38 c	8.04 ± 0.18 abc
6	6	0.47 ± 0.05 a	0.62 ± 0.05 bc	19.48 ± 1.79 abc	19.22 ± 2.82 cde	6.82 ± 1.98 cd	8.54 ± 1.29 cd	7.44 ± 0.19 abc	7.33 ± 0.47 e
6	8	0.48 ± 0.07 a	0.58 ± 0.09 abcd	20.58 ± 1.05 ab	20.00 ± 1.26 cd	6.24 ± 0.70 cd	8.92 ± 1.13 cd	7.51 ± 0.35 ab	8.12 ± 0.21 ab
8	0	0.37 ± 0.11 abc	0.54 ± 0.03 bcde	16.57 ± 1.25 ef	17.68 ± 1.41 defg	8.14 ± 2.08 abc	7.16 ± 1.24 cd	6.79 ± 0.49 bc	6.63 ± 0.29 fg
8	4	0.34 ± 0.03 bcd	0.52 ± 0.04 cde	19.95 ± 0.89 ab	20.25 ± 1.67 c	9.78 ± 0.85 ab	8.52 ± 1.20 cd	6.82 ± 0.34 bc	7.99 ± 0.25 abcd
8	6	0.44 ± 0.02 ab	0.57 ± 0.08 abcde	20.89 ± 0.52 ab	22.67 ± 2.76 ab	7.71 ± 1.10 bcd	14.79 ± 5.84 b	7.88 ± 0.39 a	7.61 ± 0.32 cde
8	8	0.47 ± 0.07 a	0.64 ± 0.005 a	21.27 ± 0.34 a	25.04 ± 1.57 a	7.45 ± 1.26 cd	23.73 ± 4.27 a	7.61 ± 1.01 a	8.36 ± 0.07 a
LSD 0.05 *		0.113	0.092	2.182	2.393	2.205	3.687	0.764	0.503

*: LSD; Least significant difference. Values are means ± SD. Means with the same letter/s within the same column are not significantly different according to LSD at 0.05 level of probability.

. In the first season, the highest percentages of the EOs were measured in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (0.47%), 6+6 0.47%), 8+6 (0.44%), 4+6 (0.44%), 4+8 0.43%), and 6±8 (0.42%). In the second season, the highest percentages of the EOs were measured in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (0.64%), 6+6 (0.62%), 4+8 (0.59%), 6+8 (0.58%), and 8+6 (0.57%).

The plants that were treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (21.27 µg/mL), 8+6 (20.89 µg/mL), 4+6 (20.63 µg/mL), and 6+8 (20.58 µg/mL) had the highest chlorophyll-a content in the first season. In the second season, the highest values of chlorophyll-a content were measured in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (25.04 µg/mL), 8+6 (22.67 µg/mL), 6+4 (20.51 µg/mL), 4+6 (20.41 µg/mL), 8+4 (20.25 µg/mL), and 6+8 (20.00 µg/mL).

The highest values of chlorophyll-b content in the first season were shown in untreated plants (10.22 µg/mL) followed by plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+4 (9.78 µg/mL), 8+0 (8.14 µg/mL) and 4+8 (8.09 µg/mL). In the second season, the highest values were recorded in plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (23.73 µg/mL), 8+6 (14.79 µg/mL), 4+6 (10.76 µg/mL), and 6+4 (8.90 µg/mL).

The highest contents of carotenoid in the first season were measured in the plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+6 (7.88 µg/mL), 8+8 (17.61 µg/mL), 6+8 (7.51 µg/mL), and 6+6 (7.44 µg/mL). In the second season, the highest carotenoid contents were found in the plants treated with Mg (g/L)+T. majus ALE (g/L) at 8+8 (8.36 µg/mL), 6+8 (8.12 µg/mL), 4+6 (8.12 µg/mL), and 6+4 (8.04 µg/mL).

3.5. Correlations Among the Studied Variables in Both Seasons

Observing the possible relationships among the studied parameters measured for the growing A. abrotanum plants as affected by the Mg and T. majus ALE treatments at 0, 4, 6, and 8 g/L, see Figure 3 and

Table 6. The regression equations with the coefficient of determination (R²) and the correlation (r) between the predictor variable, x, and the response variable, y.

Factors		Regression Equation	R2	r	p-Value
X	Y
Height S1	Height S2	y = 6.3951 + 0.8606x	0.74	0.86	0.00002
	FW S1	y = −21.8903 + 1.509x	0.704	0.83	0.00005
	FW S2	y = −17.9014 + 1.4854x	0.58	0.76	0.0005
	N.B S1	y = −37.1651 + 1.9102x	0.68	0.82	0.00007
	N.B S2	y = −43.0503 + 2.2525x	0.67	0.82	0.00009
	EO% S1	y = −0.2267 + 0.0141x	0.58	0.76	0.0005
	EO% S2	y = −0.3337 + 0.0191x	0.66	0.81	0.0001
	Chl.a S1	y = −3.2193 + 0.482x	0.40	0.63	0.0078
	Chl.a S2	y = −3.7352 + 0.5132x	0.52	0.72	0.0014
	Chl.b S1	y = 9.6826 − 0.0522x	0.028	−0.16	0.53
	Chl.b S2	y = −17.819 + 0.6217x	0.31	0.56	0.0227
	Carotenoid S1	y = −10.6151 + 0.3697x	0.58	0.76	0.0006
	Carotenoid S2	y = 0.8259 + 0.1481x	0.52	0.72	0.0015
Height S2	FW S1	y = −25.7819 + 1.586x	0.77	0.88	0.00001
	FW S2	y = −26.1982 + 1.6633x	0.73	0.85	0.00002
	N.B S1	y = −39.1855 + 1.9412x	0.71	0.84	0.00004
	N.B S2	y = −46.9577 + 2.3238x	0.72	0.85	0.00003
	EO% S1	y = −0.2882 + 0.0154x	0.69	0.83	0.00005
	EO% S2	y = −0.4086 + 0.0207x	0.77	0.87	0.00001
	Chl.a S1	y = −8.5727 + 0.6006x	0.63	0.79	0.0002
	Chl.a S2	y = −4.5155 + 0.5269x	0.55	0.74	0.0009
	Chl.b S1	y = 11.203 − 0.0866x	0.07	−0.27	0.2970
	Chl.b S2	y = −15.029 + 0.5529x	0.25	0.50	0.0476
	Carotenoid S1	y = −12.0081 + 0.3986x	0.67	0.82	0.00009
	Carotenoid S2	y = 0.1582 + 0.1622x	0.63	0.79	0.0002
FW S1	FW S2	y = 1.0336 + 1.0443x	0.93	0.96	0.0000
	N.B S1	y = −7.9196 + 1.2306x	0.92	0.96	0.00000
	N.B S2	y = −9.107 + 1.4635x	0.92	0.96	0.00000
	EO% S1	y = 0.0769 + 0.0071x	0.47	0.69	0.0031
	EO% S2	y = −0.0102 + 0.0116x	0.78	0.88	0.00000
	Chl.a S1	y = 3.1416 + 0.3339x	0.63	0.79	0.0002
	Chl.a S2	y = 4.1985 + 0.3288x	0.70	0.83	0.00005
	Chl.b S1	y = 7.3813 + 0.0008x	0.0000	0.004	0.9857
	Chl.b S2	y = −7.0378 + 0.3715x	0.36	0.60	0.0127
	Carotenoid S1	y = −4.369 + 0.2247x	0.69	0.83	0.00006
	Carotenoid S2	y = 3.5142 + 0.0858x	0.57	0.75	0.0007
FW S2	N.B S1	y = −7.0251 + 1.133x	0.91	0.95	0.00000
	N.B S2	y = −7.1665 + 1.3286x	0.88	0.94	0.00000
	EO% S1	y = 0.1014 + 0.0061x	0.41	0.64	0.0074
	EO% S2	y = 0.0163 + 0.0103x	0.72	0.84	0.00003
	Chl.a S1	y = 3.9408 + 0.2955x	0.57	0.75	0.0007
	Chl.a S2	y = 4.6567 + 0.298x	0.67	0.81	0.0001
	Chl.b S1	y = 6.629 + 0.017x	0.01	0.10	0.6969
	Chl.b S2	y = −6.6775 + 0.3401x	0.35	0.59	0.0143
	Carotenoid S1	y = −4.2315 + 0.2074x	0.69	0.83	0.00006
	Carotenoid S2	y = 3.9409 + 0.0711x	0.45	0.67	0.0041
N.B S1	N.B S2	y = 2.0483 + 1.1513x	0.94	0.97	0.0000
	EO% S1	y = 0.1222 + 0.0057x	0.51	0.71	0.0017
	EO% S2	y = 0.0836 + 0.009x	0.78	0.88	0.00001
	Chl.a S1	y = 6.0548 + 0.2546x	0.60	0.77	0.0004
	Chl.a S2	y = 6.527 + 0.2626x	0.73	0.85	0.00002
	Chl.b S1	y = 7.4919 − 0.0016x	0.0001	−0.011	0.9650
	Chl.b S2	y = −4.9707 + 0.309x	0.41	0.64	0.0068
	Carotenoid S1	y = −3.2686 + 0.1902x	0.82	0.90	0.00000
	Carotenoid S2	y = 4.4365 + 0.0616x	0.48	0.69	0.0028
N.B S2	EO% S1	y = 0.1454 + 0.0044x	0.42	0.65	0.0064
	EO% S2	y = 0.0832 + 0.0076x	0.77	0.87	0.00001
	Chl.a S1	y = 6.5574 + 0.2037x	0.54	0.73	0.0011
	Chl.a S2	y = 6.9288 + 0.2122x	0.67	0.82	0.00009
	Chl.b S1	y = 7.0394 + 0.0069x	0.003	0.06	0.8228
	Chl.b S2	y = −4.8816 + 0.2567x	0.40	0.63	0.0080
	Carotenoid S1	y = −3.0475 + 0.155x	0.76	0.87	0.00001
	Carotenoid S2	y = 4.4112 + 0.052x	0.48	0.69	0.0028
EO% S1	EO% S2	y = 0.0779 + 1.0884x	0.72	0.85	0.00003
	Chl.a S1	y = 6.9857 + 27.8469x	0.45	0.67	0.0039
	Chl.a S2	y = 8.6785 + 25.6169x	0.44	0.66	0.0048
	Chl.b S1	y = 10.3866 − 7.7228x	0.21	−0.45	0.0757
	Chl.b S2	y = −2.7306 + 30.9023x	0.26	0.51	0.0409
	Carotenoid S1	y = −2.3366 + 20.183x	0.58	0.76	0.0005
	Carotenoid S2	y = 4.4249 + 7.3516x	0.43	0.66	0.0052
EO% S2	Chl.a S1	y = 5.549 + 24.4642x	0.58	0.76	0.0006
	Chl.a S2	y = 7.5068 + 22.2029x	0.54	0.74	0.0010
	Chl.b S1	y = 8.5239 − 2.2283x	0.02	−0.16	0.5327
	Chl.b S2	y = −4.5419 + 27.5856x	0.34	0.58	0.0162
	Carotenoid S1	y = −2.5636 + 16.0903x	0.61	0.78	0.0003
	Carotenoid S2	y = 4.2883 + 5.9695x	0.47	0.68	0.0032
Chl.a S1	Chl.a S2	y = 5.7074 + 0.7246x	0.60	0.77	0.0004
	Chl.b S1	y = 10.6952 − 0.1853x	0.20	−0.44	0.0805
	Chl.b S2	y = −5.3134 + 0.8175x	0.31	0.56	0.0239
	Carotenoid S1	y = −1.9722 + 0.418x	0.42	0.65	0.0061
	Carotenoid S2	y = 3.0351 + 0.2383x	0.77	0.88	0.00001
Chl.a S2	Chl.b S1	y = 8.9343 − 0.0818x	0.03	−0.18	0.4913
	Chl.b S2	y = −15.6155 + 1.3367x	0.73	0.85	0.00002
	Carotenoid S1	y = −4.495 + 0.5353x	0.61	0.78	0.0004
	Carotenoid S2	y = 3.2825 + 0.2142x	0.54	0.74	0.0010
Chl.b S1	Chl.b S2	y = 10.6977 − 0.209x	0.003	−0.05	0.8280
	Carotenoid S1	y = 6.5495 − 0.1521x	0.009	−0.09	0.7184
	Carotenoid S2	y = 9.1987 − 0.2626x	0.16	−0.40	0.1246
Chl.b S2	Carotenoid S1	y = 3.2634 + 0.2359x	0.28	0.53	0.0319
	Carotenoid S2	y = 6.2895 + 0.1051x	0.32	0.56	0.0221
Carotenoid S1	Carotenoid S2	y = 5.8067 + 0.2663x	0.39	0.63	0.0088

S1: First season; S2: Second season; Chl.: Chlorophyll; EO: Essential oil; N.B: Number of branches/plant; FW: Total fresh weight.

, the most positive and good correlations were found between the number of branches/plant in both seasons (r = 0.97), the total fresh weight in both seasons (r = 0.96), the total fresh weight in the first season and the number of branches/plant in both seasons (r = 0.96), the total fresh weight in the second season and the number of branches/plant in the first season (r = 0.95) and the second season (r = 0.94), and the number of branches/plant and the carotenoids in the first season (r = 0.90).

Additionally, good correlation results were found between the plant height in the second season with the total fresh weight in the first season (r = 0.88), the total fresh weight in the first season, and the EO% in the second season (r = 0.88), chlorophyll-a in the first season and the carotenoids in the second season (r = 0.88), and the number of branches/plant in the first season and the EO% in the second season (r = 0.88).

Furthermore, the results also reported good correlation results between the plant height with the EO% in the second season (r = 0.87), the number of branches/plant in the second season and the carotenoids in the first season (r = 0.87), the number of branches/plant and the EO% in the second season (r = 0.87), the plant height in both seasons (r = 0.86), the EO% in both seasons (r = 0.85), the plant height and the number of branches/plant in the second season (r = 0.85), and chlorophyll-a and chlorophyll-b in the second season (r = 0.85).

3.6. Chemical Compounds of the Essential Oils from Artemisia abrotanum Leaves

Table 7. Variation in the chemical compounds of the essential oils from Artemisia abrotanum plants as affected by the treatments of magnesium element and T. majus aqueous leaf extract.

Compound	Percentage of Chemical Compounds of the Essential Oils from Plants Treated with Mg (g/L) + T. majus ALE (g/L) at
	0+0	0+4	0+6	0+8	4+0	4+4	4+6	4+8	6+0	6+4	6+6	6+8	8+0	8+4	8+6	8+8
2-Heptanol	nd	nd	nd	nd	0.77	nd	nd	nd	0.55	0.50	nd	nd	nd	nd	nd	nd
Camphene	nd	nd	0.64	0.80	0.71	0.65	0.41	1.51	nd	0.54	1.03	0.37	0.90	nd	0.32	0.45
α-Terpinene	nd	nd	nd	nd	nd	nd	nd	0.46	nd	nd	nd	nd	0.47	nd	nd	nd
m-Cymene	nd	nd	1.96	1.32	nd	1.16	2.70	2.90	nd	1.16	1.19	0.78	2.86	nd	0.62	0.78
Eucalyptol	3.10	nd	4.94	6.00	7.04	4.89	5.41	7.18	1.62	4.19	6.01	3.98	7.20	2.47	4.08	4.28
cis-Sabinene hydrate	nd	nd	nd	0.43	nd	nd	0.60	0.46	nd	nd	nd	nd	0.56	nd	nd	nd
trans-para-2-Menthen-1-ol	nd	nd	nd	nd	nd	nd	0.47	0.33	nd	nd	nd	nd	0.39	nd	nd	nd
Camphor	0.93	nd	1.05	1.90	2.45	1.34	1.64	1.36	1.25	1.36	1.49	1.36	1.98	1.45	1.45	1.10
endo-Borneol	6.33	4.05	8.40	10.77	9.57	8.96	9.79	9.02	9.23	8.38	10.06	8.95	9.49	10.91	10.21	7.16
trans-Piperitol	nd	nd	0.56	0.62	nd	nd	0.89	0.83	0.81	nd	0.62	0.55	0.90	nd	0.70	0.62
Ascaridole	nd	nd	0.95	1.16	nd	0.52	4.99	2.51	1.05	nd	0.85	0.82	3.30	0.71	1.37	0.54
Bornyl acetate	0.58	nd	0.72	0.54	0.61	0.52	0.59	0.73	0.59	0.56	0.68	0.59	0.64	0.60	0.63	0.65
Isoascaridol	nd	nd	nd	nd	nd	nd	0.25	0.50	nd	nd	0.47	nd	0.62	nd	0.56	nd
Silphiperfol-5-ene	0.52	nd	0.67	0.47	nd	0.56	0.45	0.48	nd	0.57	0.46	0.53	0.48	0.51	0.34	0.50
2,2,8-Trimethyltricyclo[6.2.2.01,6]dodec-5-ene	0.50	nd	0.63	0.46	nd	0.53	0.41	0.45	0.37	0.54	0.44	0.49	0.44	0.49	nd	0.47
Thujopsene-I3	0.69	nd	0.77	0.46	nd	0.54	0.45	0.61	nd	0.64	0.50	0.62	0.57	0.50	0.35	0.62
Silphiperfola-5,7(14)-diene	nd	nd	0.35	nd	nd	nd	0.23	0.30	nd	nd	nd	nd	0.29	nd	nd	nd
á-Neoclovene	0.58	nd	0.67	0.52	nd	0.61	0.50	0.53	0.37	0.61	0.50	0.55	0.50	0.56	0.38	0.54
alpha-patchoulene	nd	nd	0.48	0.31	nd	0.38	0.34	0.34	nd	0.43	nd	nd	0.32	nd	nd	0.36
Caryophyllene	0.65	nd	0.47	0.37	nd	0.43	0.38	0.40	0.43	0.48	nd	nd	0.45	nd	nd	0.47
Germacrene D	2.52	1.58	2.80	2.34	1.07	2.80	3.33	1.72	2.13	3.30	1.61	1.62	1.23	1.72	1.10	1.67
Isoaromadendrene epoxide	1.35	0.44	1.38	1.26	1.07	1.33	1.35	1.22	1.32	1.29	1.30	1.35	1.24	1.44	1.36	1.37
Drimenol	0.72	nd	0.70	0.71	0.55	0.71	0.70	0.66	0.69	0.85	0.66	0.69	0.67	0.73	0.73	0.71
Cedrol	11.06	13.25	10.85	10.98	9.57	11.64	9.90	9.77	10.56	10.67	10.87	11.03	9.73	12.62	11.52	11.13
Ledene alcohol	2.86	3.34	2.79	2.74	2.35	2.85	2.79	2.49	2.72	2.73	2.69	2.78	2.51	3.07	2.83	2.86
7-epi-Silphiperfol-5-ene	6.39	7.01	6.27	6.12	5.42	6.28	5.96	5.83	6.32	6.03	6.07	6.13	5.96	6.74	6.35	6.37
1,5,9,9-Tetramethyl-2-methylene-spiro[3.5]non-5-ene	2.52	2.71	2.14	2.24	1.98	2.07	2.17	2.28	2.19	2.08	2.29	2.40	2.17	2.39	2.11	2.49
α-Costol	5.04	5.20	4.70	3.90	3.62	3.85	4.37	4.72	4.57	4.27	4.35	4.83	4.30	4.13	4.61	5.08
7-methoxy-4-methylcoumarin(4-Methylherniarin)	41.94	55.45	30.45	31.03	45.37	36.38	23.29	29.44	41.00	34.67	35.04	38.70	28.32	38.71	37.92	39.13
Longiverbenone	3.86	5.90	3.85	3.77	4.77	4.00	3.74	3.47	3.73	3.61	3.73	3.60	3.58	4.25	4.08	3.85
β-Caryophyllene oxide	1.15	nd	1.15	1.24	0.92	1.21	1.10	1.12	1.12	1.11	1.16	1.14	1.11	1.30	1.12	1.15
Widdrol	0.95	nd	0.86	0.79	nd	0.82	0.84	0.90	0.96	0.86	0.86	0.96	0.86	0.87	0.91	0.99
cis-Lanceol	0.65	nd	0.97	0.77	0.53	0.69	0.93	0.49	0.69	0.96	0.63	0.54	0.50	0.68	0.54	0.52
Caryophylla-4(12),8(13)-dien-5α-ol	2.86	3.09	3.01	2.71	1.64	2.52	2.40	2.70	2.72	2.93	2.66	2.85	2.58	2.63	2.64	2.86
Santalol, cis, alpha	0.63	nd	1.29	0.68	nd	0.56	0.80	0.51	0.69	1.30	0.66	0.46	0.51	0.53	nd	nd
Costunolide	nd	nd	0.38	0.37	nd	nd	0.45	0.33	nd	0.44	nd	nd	0.32	nd	nd	nd
Bisabolone oxide	0.58	nd	0.50	0.45	nd	0.48	0.48	0.54	0.64	0.44	0.46	nd	0.56	nd	0.51	0.52
α-Bisabolol oxide A	1.02	nd	0.88	0.67	nd	0.71	0.75	0.91	0.94	0.81	0.65	0.77	0.80	nd	0.66	0.76
2,3-Diphospho-D-glyceric acid	nd	nd	0.64	0.39	nd	nd	0.63	nd	nd	0.84	nd	nd	nd	nd	nd	nd

nd: Not detected.

shows the chemical analysis of the essential oils (EOs) extracted from Artemisia abrotanum as affected by Mg and T. majus ALE treatments.

Table 8. The match factors (MFs) for the identified compounds from the essential oils of Artemisia abrotanum plants by Xcalibur 3.0 data system in the GC–MS as affected by the treatments of magnesium and T. majus aqueous leaf extract.

Compound	The Match Factors of the Chemical Compounds of the Essential Oils from Plants Treated with Mg (g/L)+T. majus ALE (g/L) at
	0+0	0+4	0+6	0+8	4+0	4+4	4+6	4+8	6+0	6+4	6+6	6+8	8+0	8+4	8+6	8+8
2-Heptanol	nd	nd	nd	nd	756*	nd	nd	nd	836	837	nd	nd	nd	nd	nd	nd
Camphene	nd	nd	966	960	928	964	967	964	nd	958	968	953	962	nd	962	959
α-Terpinene	nd	nd	nd	nd	nd	nd	nd	944	nd	nd	nd	nd	944	nd	nd	nd
m-Cymene	nd	nd	927	893	nd	936	949	940	nd	933	929	898	941	nd	926	926
Eucalyptol	932	nd	933	937	934	933	944	938	940	931	944	930	932	934	943	940
cis-Sabinene hydrate	nd	nd	nd	929	nd	nd	950	933	nd	nd	nd	nd	927	nd	nd	nd
trans-para-2-Menthen-1-ol	nd	nd	nd	nd	nd	nd	916	901	nd	nd	nd	nd	912	nd	nd	nd
Camphor	925	nd	929	954	918	948	955	954	948	941	938	940	937	928	936	928
endo-Borneol	954	945	953	954	935	953	950	953	957	957	959	958	950	956	956	952
trans-Piperitol	nd	nd	938	935	nd	nd	926	934	937	nd	936	934	937	nd	933	938
Ascaridole	nd	nd	873	881	nd	851	917	909	863	nd	860	859	913	865	878	854
Bornyl acetate	921	nd	937	904	835	901	929	931	852	914	911	891	908	900	903	911
Isoascaridol	nd	nd	nd	nd	nd	nd	884	887	nd	nd	858	nd	853	nd	872	nd
Silphiperfol-5-ene	897	nd	917	911	nd	900	921	907	nd	914	897	879	916	896	887	904
2,2,8-Trimethyltricyclo[6.2.2.01,6]dodec-5-ene	848	nd	854	892	nd	841	853	855	916	849	844	843	851	840	nd	855
Thujopsene-I3	855	nd	858	869	nd	870	880	875	nd	870	865	872	877	850	866	866
Silphiperfola-5,7(14)-diene	nd	nd	916	nd	nd	nd	914	915	nd	nd	nd	nd	925	nd	nd	nd
á-Neoclovene	816	nd	895	819	nd	874	846	845	822	842	826	841	839	812	864	836
alpha-patchoulene	nd	nd	811	818	nd	836	826	812	nd	835	nd	nd	822	nd	nd	812
Caryophyllene	942	nd	939	933	nd	925	940	934	934	938	nd	nd	941	nd	nd	943
Germacrene D	951	816	960	961	878	951	962	931	941	954	941	942	942	939	933	942
Isoaromadendrene epoxide	779	934	786	784	761	778	760	796	762	773	791	781	763	780	764	768
Drimenol	722	nd	768	737	713	734	739	723	764	734	742	754	760	731	758	754
Cedrol	783	787	776	780	789	777	769	784	779	777	779	784	780	771	777	780
Ledene alcohol	793	792	797	787	783	791	789	790	789	788	807	795	771	792	809	809
7-epi-Silphiperfol-5-ene	790	795	788	792	788	789	784	787	758	792	794	760	752	791	788	787
1,5,9,9-Tetramethyl-2-methylene-spiro[3.5]non-5-ene	815	823	822	825	811	823	819	824	822	814	818	822	821	819	822	824
α-Costol	835	836	843	831	824	841	836	842	841	848	848	839	839	818	836	816
7-methoxy-4-methylcoumarin(4-Methylherniarin)	878	871	860	878	816	873	876	879	811	857	856	882	849	871	876	859
Longiverbenone	821	821	823	820	843	846	823	843	828	846	826	823	825	843	824	845
β-Caryophyllene oxide	912	nd	909	923	884	903	927	916	906	897	918	905	920	905	914	917
Widdrol	769	nd	775	787	nd	789	772	777	783	783	792	773	773	779	766	771
cis-Lanceol	776	nd	760	764	779	776	775	775	758	761	768	766	780	763	775	773
Caryophylla-4(12),8(13)-dien-5α-ol	904	902	944	904	848	912	945	909	898	942	940	941	910	946	945	951
Santalol, cis, alpha	791	nd	771	797	nd	809	797	800	812	777	784	785	790	786	nd	nd
Costunolide	nd	nd	839	837	nd	nd	842	852	nd	879	nd	nd	881	nd	nd	nd
Bisabolone oxide	794	nd	728	881	nd	878	887	896	882	861	870	nd	895	nd	875	888
α-Bisabolol oxide A	903	nd	907	909	nd	888	922	913	897	899	885	900	913	nd	890	894
2,3-Diphospho-D-glyceric acid	nd	nd	935	933	nd	nd	943	nd	nd	933	nd	nd	nd	nd	nd	nd

nd: Not detected.

presents the match factors for the identified compounds from the EOs of A. abrotanum plants by the Xcalibur 3.0 data system in the GC–MS. The abundant compounds in the EOs were 7-methoxy-4-methylcoumarin (4-methylherniarin), cedrol, α-costol, eucalyptol, camphor, endo-borneol, camphene, m-cymene, ascaridole, germacrene D, isoaromadendrene epoxide, ledene alcohol, 7-epi-silphiperfol-5-ene, 1,5,9,9-tetramethyl-2-methylene-spiro[3.5]non-5-ene, longiverbenone, β-caryophyllene oxide, and caryophylla-4(12),8(13)-dien-5α-ol. The GC–MS chromatograms of the isolated EOs extracted from A. abrotanum leaves treated with the 16 treatments are shown in Figure S2.

These main compounds had different concentrations in the EOs according to the treatment used. The ranges of these compounds were 0–1.51, 0–2.90, 0–7.20, 0–2.45, 4.05–10.91, 0–4.99, 1.10–3.33, 0.44–1.44, 9.57–13.2, 2.35–3.34, 5.42–7.01, 1.98–2.71, 3.62–5.20, 28.32–55.45, 3.58–5.90, and 1.64–3.09% for camphene, m-cymene, eucalyptol, camphor, endo-borneol, ascaridole, germacrene D, isoaromadendrene epoxide, cedrol, ledene alcohol, 7-epi-silphiperfol-5-ene, 1,5,9,9-tetramethyl-2-methylene-spiro[3.5]non-5-ene, α-costol, 7-methoxy-4-methylcoumarin (4-methylherniarin), longiverbenone, and caryophylla-4(12),8(13)-dien-5α-ol, respectively.

The highest percentage of camphene (1.51%) was obtained in the EO of treated plants with Mg (4 g/L)+T. majus ALE (8 g/L) followed by treated plants with Mg (6 g/L)+T. majus ALE (6 g/L) (1.03%). For m-cymene, the highest percentages were found in the plants treated with Mg (g/L)+T. majus ALE (g/L) at 4+8, 8+0, and 4+6, with values of 2.90, 2.86, and 2.70%, respectively. The highest values of eucalyptol in the EOs were observed in the plants treated with Mg (8 g/L)+T. majus ALE (0 g/L) (7.20%), followed by Mg (4 g/L)+T. majus ALE (8 g/L) (7.18%) and Mg (4 g/L)+T. majus ALE (0 g/L) (7.04%). The highest percentage values of camphor in the EOs were found in plants treated by Mg (4 g/L)+T. majus ALE (0 g/L) (2.45%) and Mg (8 g/L)+T. majus ALE (0 g/L) (1.98%). The highest percentages of endo-borneol in the EOs were obtained in the plants treated by Mg (8 g/L)+T. majus ALE (4 g/L) (10.91%) followed by Mg (0 g/L)+T. majus ALE (8 g/L) (10.77%) and Mg (8 g/L)+T. majus ALE (6 g/L) (10.21%). Ascaridole was observed at a high percentage in the EO of plants treated with Mg (4 g/L)+T. majus ALE (6 g/L) (4.99%), followed by Mg (8 g/L)+T. majus ALE (0 g/L) (3.30%), and Mg (4 g/L)+T. majus ALE (8 g/L) (2.51%). The high percentages of germacrene D were found in the EOs of plants treated with Mg (4 g/L)+T. majus ALE (6 g/L) (3.33%), followed by Mg (0 g/L)+T. majus ALE (6 g/L) (2.80%), Mg (4 g/L)+T. majus ALE (4 g/L) (2.80%), and untreated plants (2.52%).

The highest values of isoaromadendrene epoxide were found in the EOs from the plants treated with the concentrations of Mg (8 g/L)+T. majus ALE (4 g/L) (1.44%), Mg (0 g/L)+T. majus ALE (6 g/L) (1.38%), Mg (8 g/L)+T. majus ALE (8 g/L) (1.37%), and Mg (8 g/L)+T. majus ALE (6 g/L) (1.36%). The highest percentages of cedrol were obtained in the EOs of plants treated by Mg (0 g/L)+T. majus ALE (4 g/L) (13.25%), Mg (8 g/L)+T. majus ALE (4 g/L) (12.62%), Mg (4 g/L)+T. majus ALE (4 g/L) (11.64%), and Mg (8 g/L)+T. majus ALE (6 g/L) (11.52%). The highest concentrations of ledene alcohol in the EOs were observed in plants treated with Mg (0 g/L)+T. majus ALE (4 g/L) (3.34%), and Mg (8 g/L)+T. majus ALE (4 g/L) (3.07%). The highest percentages of 7-epi-silphiperfol-5-ene were observed in the EOs from plants treated with Mg (0 g/L)+T. majus ALE (4 g/L) (7.01%), Mg (8 g/L)+T. majus ALE (4 g/L) (6.74%), untreated plants (6.39%), and Mg (8 g/L)+T. majus ALE (8 g/L) (6.37%). The highest percentages of 1,5,9,9-tetramethyl-2-methylene-spiro[3.5]non-5-ene were observed in the EOs from treated plants with Mg (0 g/L)+T. majus ALE (4 g/L) (7.01%), Mg (8 g/L)+T. majus ALE (4 g/L) (6.74%), untreated plants (6.39%), and Mg (8 g/L)+T. majus ALE (8 g/L) (6.37%). The high percentages of α-costol were found in the EOs from plants treated with the combination treatments of Mg (0 g/L)+T. majus ALE (4 g/L) (5.20%), Mg (8 g/L)+T. majus ALE (8 g/L) (5.08%), and from untreated plants (5.04%).

The abundant concentrations of the main compound, 7-methoxy-4-methylcoumarin (4-methylherniarin), were observed in the EOs extracted from plants treated with Mg (0 g/L)+T. majus ALE (4 g/L) (55.45%), Mg (0 g/L)+T. majus ALE (8 g/L) (45.37%), untreated plants (41.94%), and Mg (6 g/L)+T. majus ALE (0 g/L) (41.00%). The highest percentages of longiverbenone were observed in the EOs from plants treated with Mg (0 g/L)+T. majus ALE (4 g/L) (5.90%), Mg (4 g/L)+T. majus ALE (0 g/L) (4.77%), Mg (8 g/L)+T. majus ALE (4 g/L) (4.25%) and Mg (8 g/L)+T. majus ALE (6 g/L) (4.08%). The highest percentages, 3.09 and 3.01%, of caryophylla-4(12),8(13)-dien-5α-ol were observed in the EOs from treated plants with Mg (0 g/L)+T. majus ALE (4 g/L) and Mg (0 g/L)+T. majus ALE (6 g/L), respectively.

3.7. Antibacterial Activity

A. abrotanum EOs were found to be effective against only P. carotovorum subsp. carotovorum, unlike Pectobacterium atrosepticum. Figure 4 shows the overall effect of A. abrotanum EOs against P. carotovorum subsp. carotovorum isolate. The minimum inhibitory concentration (MIC) of A. abrotanum EOs was 75 μg/mL against P. carotovorum subsp. carotovorum bacterial isolate and the inhibition zones (IZs) ranged from 1 to 5 mm for a concentration of 100 μg/mL compared to 6 mm from the positive control Neomycin Sulphate (20 µg/disc). With increasing A. abrotanum EO concentration, the activity was increased at 100 and 75 μg/mL, as measured by the IZ. No IZs were found against the growth of P. atrosepticum as the MIC was MIC > 1000 μg/mL.

4. Discussion

Using Mg in conjunction with T. majus ALE as biostimulants encouraged the growth of A. abrotanum.

4.1. Role of Mg in Enhancing A. abrotanum Plants

Most of the investigated parameters (plant height, total fresh weight, number of branches/plants, essential oil (EO%), chlorophyll-a content, chlorophyll-b content, and carotenoid content) rose in the treated A. abrotanum plants with Mg in the form of magnesium sulfate at 4, 6, and 8 g/L as compared to the untreated A. abrotanum plants. The application of Mg significantly improved the plant’s growth and yield.

Mg is a necessary cofactor for several enzymes involved in photosynthetic CO₂ fixation and chlorophyll production [55,56]. Additionally, Mg is necessary for several biological functions, including pollen production, energy metabolism, N intake, plant-microbe interactions, stress tolerance, and sucrose transport [56,57,58,59,60]. Magnesium sulfate (4 g/L) produced the highest aerial biomass production and EO of Dracocephalum moldavica [61]. Mg supplements are necessary for increasing the antioxidant capacity of tea plants, decreasing growth inhibition brought on by acid stress in the root environment, and increasing the amount of chlorophyll and the vitality of the roots [62]. Mg as a foliar application for Mentha crispa decreased lipid peroxidation and osmotic stress while increasing root development, plant biomass, EO production, leaf area, chlorophyll content, soluble sugar synthesis, and antioxidant enzyme activity [63].

Furthermore, it is well recognized that Mg is crucial to the plant’s ability to produce the EO. This element is a crucial cofactor of terpene synthase enzymes (TPS) in addition to its well-known functions. For instance, sesquiterpene synthases prefer Mg, but monoterpene synthases have less stringent requirements for divalent cations [64,65]. Instead of an overabundance of Mn, a shortage of Mg increased the fraction of monoterpenes [66].

The foliar application of Fe (3 g/L), Mg (8 g/L), or Mn (300 mg/L) significantly improved the plant’s growth, yield, EO constituents, and chemical composition of A. abrotanum, when compared to the control [67]. Mg (370 and 740 mg/L MgSO₄) has a beneficial effect on the structural cultural growth of Chamomilla recutita as well as the quantity and quality of the EO produced [68]. Mg at 0.8 mM dramatically raised water-soluble extracts, amino acids, and polyphenols in tea leaves under acid stress.

4.2. Role of T. majus ALE in Enhancing A. abrotanum Plants

In the present work, T. majus ALE was characterized by the presence of several compounds, including chlorogenic acid, catechol, syringic acid, caffeic acid, pyrogallol, ferulic acid, naringin, rutin, quercetin, kaempferol, luteolin, apigenin, and catechin. Through its bioactive substances to reduce abiotic stress, or by providing the treated plants with important components like vitamins and menials that can dissolve in water extraction, T. majus ALE, as a biostimulant, could increase the development and production of A. abrotanum plants. Its growth-stimulating phytohormones enhance apical meristem activity leading to cell division and elongation. Therefore, chemical fertilizers and manufactured growth regulators can be safely replaced with plant extracts. When sprayed on treated plants, natural extracts also contain minerals like Fe, N, K, Zn, Mg, P, and Ca, as well as carbohydrates that are absorbed by the leaves and increase the plants’ growth activity.

In a previous study, it was reported that T. majus ALEs were characterized by the presence of esters of quinic acid with cinnamic acids (chlorogenic acids, p-coumaroylquinic acids) and flavonoids [26]. The major phenolic acids from T. majus ALE, identified by HPLC-RP with UV detection were gallic, caffeic, and p-coumaric, and the predominant flavonoids were quercetin, epicatechin, and luteolin [33]. Additionally, this plant contains various flavonoids, including kaempferol glucoside and quercetin-3-O-glucoside [69].

Sustainable horticultural production techniques require the use of natural plant biostimulants. Extracts from leaves are among the many plant-derived biostimulants that are attracting interest worldwide. Biostimulants generated from natural plants have the potential to enhance horticultural products’ growth, productivity, and quality after harvest. Under abiotic stress, biostimulants, a new class of crop management chemicals, can increase the yield of crops [70]. According to several studies, using extracts from plants could boost their defenses and make them more resilient to abiotic stressors [71,72]. In addition to the previously mentioned phytohormones, mineral components, and vitamins, this is most likely caused by plant-derived extracts that contain antioxidants, osmoprotectants, and secondary metabolites [73]. Exogenous administration of plant extracts like moringa leaf extract, for instance, reduces the negative effects of gamma rays, high temperatures, salt and dehydration, and heavy metals [74,75,76].

4.3. The Effect of the Combination Treatments of Mg with T. majus ALE

In the present work, the combination treatments of Mg with T. majus ALE enhanced the growth parameters of A. abrotanum plants. Their interactions have the potential to greatly improve A. abrotanum’s growth characteristics and biochemical synthesis. Similarly, moringa leaf extract alone, and in combination with K and Zn, can improve fruit quality parameters, such as soluble solid contents, vitamin C, sugars, total antioxidant and phenolic contents, and activities of superoxide dismutase and catalase enzymes in ‘Kinnow’ mandarin [77].

Additive, synergistic, or antagonistic interactions may result from the combination of two or more biostimulants. Furthermore, the combined effect may differ from the individual effects of each biostimulant [78]. When Diplotaxis tenuifolia plants were treated with a mixture of three biostimulants, their dry biomass and total yield increased by an average of 48.1% and 37.2%, respectively. Additionally, the plants’ chlorophyll, K, Mg, and Ca contents increased in comparison to the untreated plants when using combination treatments [79,80,81]. Foliar magnesium combined with maize grain extract increased the levels of macronutrients, such as magnesium in plants, which is depleted in sandy environments. It also increased the levels of antioxidants, plant metabolites, and hormones in sunflower plants grown in sandy environments [82].

4.4. The Chemical Compounds of the Essential Oils

By the analysis of the GC–MS apparatus, the abundant compounds in the EOs extracted by the hydrodistillation method from A. abrotanum leaves as affected by Mg and T. majus ALE treatments were 7-methoxy-4-methylcoumarin (4-methylherniarin), cedrol, α-costol, eucalyptol, camphor, endo-borneol, camphene, m-cymene, ascaridole, germacrene D, isoaromadendrene epoxide, ledene alcohol, 7-epi-silphiperfol-5-ene, 1,5,9,9-tetramethyl-2-methylene-spiro[3.5]non-5-ene, longiverbenone, β-caryophyllene oxide, and caryophylla-4(12),8(13)-dien-5α-ol. Figure 5 presents the chemical structure of the most abundant compounds.

7-Methoxy-4-methylcoumarin (4-methylherniarin), a coumarin compound, was identified as the main compound in the EOs from A. abrotanum leaves by the hydrodistillation method. This method is used primarily to extract EOs, including those containing coumarins. In this method, steam is passed through the source material, vaporizing compounds. The steam and extracted compounds are condensed and collected separately, with the EO enriched in coumarins obtained as the distillate [83]. It was also reported that the primary constituents extracted from the EO of aerial parts of A. abrotanum were camphor, β-eudesmol, and 2-hydroxy-1,8-cineole [67]. The main chemicals found in A. abrotanum included borneol, cymene, camphor, terpineol, 1,8-cineole, and aromadendrene [84].

4.5. The Antibacterial Activity of the Essential Oils

The antimicrobial activities of the EOs from A. abrotanum showed weak activity against the plant pathogen bacterium Pectobacterium carotovorum subsp. carotovorum and no activity was found against Pectobacterium atrosepticum. However, most of the antimicrobial activities of the EOs from A. abrotanum as shown in the literature were assayed against human or animal pathogens.

Among some EOs, the higher sensitivity of soft rot of potato tuber bacteria was manifested in clove (P. carotovorum subsp. carotovorum and P. atrosepticum), mint (P. carotovorum subsp. carotovorum), oregano (P. atrosepticum), and thyme (P. atrosepticum) EOs., while rosemary EO was the least effective [36]. It was found that P. carotovorum subsp. brasiliensis was suppressed by EOs from rosemary, tea tree, and clove at 1%, 0.75%, and 0.25%, respectively, after 48 h of incubation [85]. These findings are consistent with our conclusions about the effect of wormwood on P. carotovorum. The EOs of cinnamon (MIC 128 mg/L) and oregano (MIC 256 mg/L) were the most efficient against P. carotovorum subsp. carotovorum after harvest [86]. Rosemary and tea tree (MIC > 1024 mg/L) were ineffective at the concentrations used. These results are consistent with our previous findings that wormwood had little impact on P. atrosepticum.

The EO isolated from the herb of A. abrotanum showed a potential activity against the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, and Aspergillus flavus [87]. The EOs’ chemicals found in A. abrotanum plants were responsible for inhibiting the growth of Staphylococcus aureus, Bacillus subtillis, Salmonella typhi, Escherichia coli, and Candida albicans [84].

Finally, natural biostimulant feedstocks include leaf, root, or seed extracts, either individually or in combination with others. Their positive effect on horticultural production is mostly due to plant growth-enhancing bioactive compounds such as phytohormones, amino acids, and nutrients.

The usage of surfactants for the crude extract is one of the study’s limitations; more investigation and testing with various surfactants are required. In the future, this can be accomplished using several formulation studies. It is crucial to remember that some factors can impact the bioactivity of the applied extract, which requires more research. This work therefore paves the way for additional investigation into the long-term effects, or shelf life, of plant extracts when used in the field.

5. Conclusions

The application of magnesium in the form of magnesium sulfate to the soil and Tropaeolum majus aqueous leaf extract as a foliar application significantly influenced the plant yield and essential oil content of Artemisia abrotanum plants. This study recommends the combination treatments of Mg (g/L)+Tropaeolum majus aqueous leaf extract (g/L) at 8+4, 8+6, or 8+8 g/L, with no significant difference among them to enhance the vegetative growth (plant height, total fresh weight, and number of branches/plant) compared to the untreated plants. For the essential oils and photosynthetic pigments, this study recommends the combination treatments of Mg (g/L)+Tropaeolum majus aqueous leaf extract (g/L) at 8+6 or 8+8 g/L, compared to the untreated plants. As a natural source of a complex mixture of micronutrients, polysaccharides, and plant growth hormones, natural plant extracts can improve horticultural product quality, boost plant resistance to abiotic stresses, increase plant growth, flowering, and productivity, decrease fertilizer application rates, and replace synthetic plant regulators. One essential sustainable method for increasing cropping systems’ efficiency and productivity—and, more critically, their environmental impact—is using plant extracts, and elemental nutrients in particular, as biostimulants in horticulture crops.