Alternations in Physiological and Phytochemical Parameters of German Chamomile (Matricaria chamomilla L.) Varieties in Response to Amino Acid Fertilizer and Plasma Activated-Water Treatments

Abstract

Plasma-activated water (PAW) is an emerging and promising green technology in agriculture in recent years. This study aimed to examine the influence of the spraying of PAW and amino acid fertilizer concentrations on German chamomile varieties’ physiological, biochemical, and phytochemical characteristics under field conditions. Method: The experiment was performed during 2020–2021 as a factorial using a randomized complete block design with three replications in an arid and semi-arid region east of the Persian Gulf. The factors contained five fertilizer levels (0 (control), 1, 2, 3 mL L⁻¹ amino acid and PAW) and three German chamomile cultivars Bona, Bodegold, and Lianka). Physiological, biochemical, and phytochemical traits such as plant height, fresh and dried flower weight, chlorophyll a, b, carotenoids, CHN elements: N ratio, total protein, amino acid profile, essential oil yield, apigenin content, and major secondary metabolites were examined. Results: The ANOVA indicated that the impact of the cultivar and fertilizer was significant on all physiological, biochemical, and phytochemical studied parameters. The amino acid fertilizer and PAW enhanced physiological features, hydrogen, C: N ratio, essential oil yield, apigenin content, and main phytochemical compositions like chamazulene, and α-bisabolol, but it had no incremental effect on the carbon, nitrogen, and total protein percentage. Conclusion: Findings revealed that applying foliar amino acid fertilizer and PAW treatments improves physiological, biochemical, and phytochemical parameters in German chamomile cultivars under field conditions.

1. Introduction

Chamomile is one of the most essential and well-known medicinal plants worldwide, which has many uses in the pharmaceutical, hygienic, cosmetic, and food industries [1,2]. Botanically, chamomile is an annual plant belonging to the Asteraceae family, native to Iran, and it vegetates as a wild plant in Europe. In Iran, chamomile is expanded in the south, southwest, west, and northwest of the country, and its application has an ancient history in the traditional medicine of Iran [3]. Chamomile essential oil is widely used in the pharmaceutical, cosmetics, and food industries [4]. Chamomile is a good source of bioactive compounds, and its phytochemical compositions of essential oils and plant parts have valuable pharmaceutical properties [5]. Chamomile’s characteristic important oil content is even connected to a definitive level of differentiation within the development of flower heads [6]. It is a popular treatment for numerous ailments, including sleep disorders, anxiety, digestion/intestinal conditions, skin infections/inflammation (including eczema), wound healing, infantile colic, teething pains, and diaper rash [3]. The essential oil content and compositions are various in the chamomile flower, and they depend on plant genetics and environmental factors [6,7].

The largest group of medically important compounds forming the essential oil are α-bisabolol, chamazulene, and bisabolol oxide. Many medical properties of chamomile are associated with its essential oil. In the analyzed essential oils, over 120 constituents have been identified, which the most components present in essential oil include α-bisabolol, chamazulene, (Z)-spiroether, ß-farnesene, bisabolol oxides A and B and α-bisabolone oxide A [8,9]. Active constituents of German chamomile consist of flavonoids: luteolin, apigenin, quercetin; spiroethers: en-yn-dicycloether; terpenoids: α-bisabolol, α-bisabolol oxide A and B, chamazulene, sesquiterpenes; coumarins: umbelliferone; and other components [10].

The oil constituents inbred and wild chamomile populations contain α-bisabolol (24.0–41.5%), bisabolone oxide (2.0–7.0%), bisabolol oxide a (1.0–36.2%), bisabolol oxide B (3.6–20.42%) and chamazulene (5.0–24.0%). The content of sesquiterpenoid compounds was more than 70% of the total essential oil [11,12]. In one research, 77 components were identified that include 99% of essential oil of chamomile consisting of farnesene (71.1%), chamazulene (8.4%), bisabolol oxide A (11.2%), and Spathulenol (11.3%) [2].

Amino acids synthesize other organic compounds, such as proteins, amines, alkaloids, vitamins, enzymes, terpenoids, and plant hormones that control various plant processes [13]. Amino acids are crucial to stimulating cell growth, acting as buffers, providing a source of carbon and energy, and protecting the cells from ammonia toxicity amid formation [14].

Nowadays, amino acids are also helpful stimulants for plant growth and development. Amino acids incite the plant metabolic processes and metabolism [15]. The amino acids have a crucial role in the secondary metabolites biosynthesis. Accordingly, the application of amino acids can be utilized as a significant agent in enhancing the quantity and quality of medicinal plants [13]. Omer et al. studied the application of amino acid’s impact on yield, essential oil, and phytochemical compounds of chamomile planted in salty soil at Sinai, Egypt. Their results showed that four significant compositions such as bisabolone oxide, α-bisabolol oxide A, α-bisabolol oxide B, and cis-β-farnesene in the essential oil were increased in combined treatment (250 ppm amino acids + 11.28 ds/m salinity. The highest amount of proline was attained from higher dosages of both amino acids and salinity treatment [13].

Velĭcka et al. investigated the effect of foliar application of amino acids on volatile oil, flavonoid compounds, and secondary metabolite profile of some mint varieties under field conditions in Kaunas, Lithuania. The findings indicated that the efficacy of amino acid treatments differed on the phytochemical compounds in the various mint varieties; thus, amino acid type and dosage should be chosen based on the targeted variety [16].

In recent years, researchers have widely noticed plasma-based agriculture [17,18]. “Plasmas in agriculture and food processing” is a new, emerging interdisciplinary field of plasma applications [17,19,20,21,22]. Plasma is the last known form of matter formed by inserting energy such as electricity, intense radiation, radio frequencies, or heat [23]. Plasma is a complete or partly ionized gas composed of charged ions, electrons, and neutral and reactive species that drive its chemistry [20,24]. The plasmas can be created under atmospheric conditions and are widely used in liquids on material surfaces to kill or inactivate microorganisms, bacteria, and spores. Many studies have demonstrated that Non-thermal plasmas can generate PAW by treating water under specific conditions [25]. Through the plasma activating process, nitrogen from the air can be captured and reacted with water, forming nitrogen resources capable of sustaining plants. Also, reactive oxygen species decrease the stress of pathogens in the soil.

Škarpa et al. investigated the effect of PAW foliar application on some growth parameters of maize [26]. Mandici et al. reported that the PAW treatment raises the quality of Triticum aestivum L. cv. Glosa sprouts., and it also increases the antioxidant enzyme activity [27].

It has been reported that wetting properties of the surfaces of some seeds, including wheat, lentils, and beans, can be modified using cold radiofrequency air plasma treatment. A significant reduction in apparent contact angle and improvement in germination rate and germination rate was achieved during air plasma treatment of seeds [28].

This experiment aimed to assess the influence of the spraying of PAW and amino acid fertilizer treatments on the physiological, biochemical, and phytochemical parameters of German chamomile (Matricaria chamomilla L.) cultivars under field conditions.

2. Material and Methods

2.1. Field Experiment Description

The experiment was performed at the Darya Zist Kavosh Co. Ltd. research farm, Abad City, Tangestan County, Bushehr province, Iran, during the 2020–2021 season. The meteorological data during the experimental year (2020–2021) for the planting of chamomile are presented in

Table 1. Meteorological data during the experimental year (2020–2021) for the planting of chamomile in Abad City, Tangestan County, Bushehr province, Iran.

Evaporation (mm)	Average of Sunny Hours (h)	Precipitation (mm)	Average of Relative Humidity (%)		Average of Temperature (°C)		Month and Year
			Max	Min	Max	Min
5.4	7.5	39.3	62	26	30.8	17.9	November 2020
2.8	7.9	62.4	83	46	21.7	10.6	December 2020
2.9	6.8	32.2	77	44	22.4	10.8	January 2021
3.5	5.6	33.9	76	38	21.6	10.5	February 2021
5.3	6.9	22.2	68	28	24.1	11.3	March 2021
8.9	6.7	10.5	65	22	32.2	19.1	April 2021

. The experimental area was located at Latitude 29°03′24″ N and Longitude 51°13′39″ E, at an elevation of about 65 m above mean sea level (Figure 1). The soil chemical properties of the experimental location are presented in

Table 2. Chemical features for the soil of research field.

Mn (ppm)	Cu (ppm)	Zn (ppm)	Fe (ppm)	K (ppm)	P (ppm)	N (%)	O.C (%)	T.N.V (%)	pH	SP (%)	EC (ds·m−1)	Texture	Soil Depth (cm)
2.3	0.74	2	3.14	124	0	0.02	0.23	52.5	7.5	51	10.68	Sandy loam	0–30

EC: Electrical Conductivity, SP: Saturation Percentage, T.N.V: Total Neutralizing Value, O.C: Organic Carbon.

. The chamomile seeds were provided by the Department of Ecology, Faculty of Humanities and Natural Sciences, University of Prešov, Prešov, Slovakia.

2.2. Experimental Design

A field experiment consisted of two factors: (i) chamomile cultivars (Bona, Bodegold, Lianka) and (ii) fertilizer levels (0 (control), 1, 2, 3 mL L⁻¹ foliar amino acid and PAW were designed and performed. The factors mentioned above were combined, and the experiments were set up as a factorial scheme with 15 treatments (5 fertilizer levels × 3 cultivars) replicated thrice in a randomized complete block design (RCBD). The experimental site had 45 plots (including 15 in each block). Each experimental plot size was 1 m × 1 m, and in each plot, the plants were grown in three equidistant rows with adjacent rows 30 cm apart. It is necessary to mention that the recommended dosage of the foliar amino acid fertilizer was 2 mL L⁻¹ by the manufacturer’s factory. Therefore, we have chosen one dosage lower and one higher than the recommended dosage, along with distilled water as a control. The PAW had a fixed dosage, explained in Section 2.4 and Section 2.5.

2.3. Field Management

The seeds were sown superficially by hand and then were covered through a very thin layer of sandy soil. Meanwhile, we had not added organic matter or chemical fertilizers to the experimental field. The irrigation was performed immediately after seed sowing using an installed pipeline and dropping-tube system. The experimental field was irrigated every four days during cool months from 10:30 a.m. until 11:30 a.m. completely and uniformly. The chemical composition of irrigation water is presented in

Table 3. Chemical characteristics of the irrigation water used in this study.

SAR	TDS	TH	Alkalinity	Na+	Ca2+ + Mg2+	SO4−	Cl−	CO3	HCO3−	pH	EC (ds m−1)
meq L−1
2.22	4467.2	2850	240	11.9	57	40.6	24	0	4	6.78	6.98

EC: Electrical Conductivity, TH: Total Hardness, TDS: Total Dissolved Solids, SAR: Sodium Adsorption Ratio.

. Weeding was performed two times during the vegetative and reproductive phases by hand. Plant diseases and pests were not observed in the field, so herbicides and pesticides were not used during this experiment.

2.4. Preparation of Plasma Activated Water (PAW)

The main PAW production method involves creating plasma on the surface or inside the water to produce UV, shock waves, ions, and reactive species [29]. The main components of our setup include a high-voltage power supply, an air pump, and electrodes. A schematic diagram of the experimental setup has been presented in Figure 2. The device is designed to activate the water precisely by creating plasma under the water’s surface using an electrode and blowing air through an air pump. Based on the optimization of the device, the parameters were set as follows; water volume: 100 mL, voltage: 15 kV, frequency: 50 Hz, and airflow: 5 L/min. The time of plasma activation was 30 min. When plasma is applied to water, the characteristics of the water change and the resulting liquids are named PAW. After activation, the PAW was transferred to the chamomile farm.

The results of nitrate, nitrite, and pH levels of PAW have been presented in

Table 4. Chemical properties of PAW used in this experiment.

Water Sample	pH	Nitrate mg/kg	Nitrite μg/kg
Untreated water	7.58	6.16 ± 0.31	10.12 ± 0.21
30 min treated water	3.16	45.92 ± 1.33	52.32 ± 1.3

. After the treatment, the pH of the solution was measured using a pH meter. Nitrite concentration in PAW was determined using the standard USEPA diazotization method, and absorbance was read by spectrophotometer at 507 nm [30]. Nitrate concentrations were measured photometrically using the Spectroquant^® nitrate assay kit (Merck Chemicals, Darmstadt, Germany) [31]. The pH value of PAW dropped significantly from 7.58 to 3.16 during 30 min of treatment, as shown in Table 4, implying that the water has undergone acidification. The results show that the concentration of nitrate and nitrite increases with treatment time.

2.5. Application of Amino Acid Fertilizer and Plasma Activated Water Treatments

Amino acid concentrations including four levels (0 (control), 1, 2, 3 mL L⁻¹ and PAW treatment were prepared and used on three chamomile cultivars (Lianka (diploid), Bona (diploid) and Bodegold (tetraploid). The Fuego Base™ (Amino acid foliar fertilizer) was purchased from Grow More^® Co. (Gardena, CA, USA). The compositions of Fuego Base (Amino acid) foliar fertilizer according to the guaranteed analysis presented on the fertilizer bottle are presented in

Table 5. Compositions of Fuego Base (Amino acid) foliar fertilizer according to the guaranteed analysis presented on the fertilizer bottle, Manufactured by Grow More^®, Gardena, California, USA.

Compositions	Content (%)
Total Nitrogen (water-soluble organic nitrogen)	4.50
A phosphate (P2O5)	0.50
Soluble Potash (K2O)	2.90
Chelated Iron (Fe)	0.10
Total Amino Acid	22.70
Total Carbon	11.70

. The Fuego Base™ is composed of all-natural vegetable extracts, amino acids, and polypeptides complex that supplies organic nitrogen, natural chelating and complexing agents, naturally occurring minor nutrients, and growth-stimulating co-enzymes. Foliar spray using Fuego Base™ (Foliar Amino acid Fertilizer) and PAW was done during chamomile plants’ growth and flowering stages. The amino acid fertilizer and PAW treatments were used three times in the vegetative and reproductive phases every 30 days. The first amino acid and PAW foliar spray stage was 30 days after planting. The spray was done from 15:30 p.m. until 17:30 p.m. for each treatment and plot. Spraying was done to the shoots evenly using a hand pump sprayer. The total volume of the amino acid solution for different concentrations was 4 L sprayed for each block (including 15 plots). Sprayed PAW also was 4 L for each block. Precise dates of foliar spraying using amino acid and PAW solutions are presented in

Table 6. Spraying dates of chamomile plants using amino acid fertilizer and PAW treatments under field conditions.

Times of Spraying	Growth Stage	Date of Spraying
1	30 days after planting	15 December 2020
2	Vegetative phase	15 January 2020
3	Flowering phase	15 February 2020

. Harvesting dried flowers was carried by hand, and just the flowers were picked up, and then they were dried at room temperature (around 20–25 °C) after harvest.

2.6. Physiological Characteristics

The evaluated physiological and biochemical characters included plant height (cm), fresh and dried flower weight (g m⁻²), Chlorophyll a, b, carotenoids, CHN elements percentage, C:N ratio, total protein, and amino acids profile (mg g⁻¹). The plant height was measured at the full bloom period as a mean height of 10 plants per plot using the meter. After harvest, samples were weighed (±0.001 g) using a balance set, and after drying at room temperature (20–25 °C) dried flower weight of each plot was calculated [3]. The chamomile flowering was initiated in late February 2021 and continued for two months. Harvesting was performed once time.

2.7. Leaf Chlorophyll a, b, and Carotenoids Contents

To estimate the chlorophyll a, b, and carotenoids, leaf samples were collected and dried for 48 h at 75 °C. Chlorophyll measurement was conducted according to the method presented by Lichtenthaler et al. [32].

2.8. Elemental Analysis and Carbon-to-Nitrogen Ratio

Elemental analysis was performed to determine/quantify carbon, hydrogen, and nitrogen content/level by combustion analysis with a CHNOS Elemental Analyzer Model-ECS 4010 (Costech, Italy). Samples were prepared by loading 1–3 mg of dried leaf tissue into a tin wrapper and were then placed into the analyzer for combustion. Triplicates were prepared from each sample, and data generated by the analyzer were collected from the software package EA Data Manager (Costech, Italy). Data collected from EA Data Manager were then transferred to Excel to calculate carbon-to-nitrogen (C: N) ratios. Total protein contents (%w/w) were estimated using formula = %N × 4.78 [33].

2.9. Amino Acids Content Measurement

The fresh leaves of chamomile (M. Chamomilla L.) were collected from the field (Abad city, Tangestan, Bushehr, Iran). The HPLC system was an Agilent HPLC Infinity Isocratic LC 1220 (Agilent; Palo Alto, CA, USA). The assay of amino acid compositions was carried out according to [34].

2.10. Flower Harvesting and Oil Extraction

The harvesting of flowers was performed by hand, and then the flowers were dried at a temperature of 25 °C. The essential oil of 30 g dried flower was extracted by hydrodistillation for 4 h, using a Clevenger system in a 500 mL round-bottom flask with 300 mL distilled water according to the method described by British Pharmacopeia [10]. The essential oil was dried with anhydrous sodium sulfate, kept in dark glass bottles, and stored in the refrigerator (4 °C) until the next analysis [1,3].

2.11. Chamomile Oil Analysis

The chamomile essential oil analysis was performed using a gas chromatograph connected with a mass spectrometry (GC/MS) model (QP2010 SE, Shimadzu, Kyoto, Japan) equipped with fused silica capillary HP-5 column (50 m length × 0.20 mm i.d., 0.25 μm film thickness). Sample volume 1.0 μL was utilized manually type injection. The carrier gas was Helium at a 1 mL/min flow rate, and the oven temperature was 60 °C. Then the column was sequentially heated from 60 °C to 150 °C at a rate of 10 °C min⁻¹, held for 5 min. Then, the column was heated from 150 °C to 180 °C at a rate of 5 °C min⁻¹ for 3 min. Finally, it was heated from 180 °C to 280 °C a rate of 7 °C min⁻¹, for 25 min. Detector, injection, and temperature were programmed at 250 °C and 280 [35]. The chamomile oil constituent’s identification and quantification in detail in Ghasemi et al. [3].

2.12. Determination of Total Apigenin Content

Total apigenin quantification was conducted using the HPLC method according to the procedure described in United States Pharmacopoeia [24]. This procedure has been presented in detail by Baghalian et al. [36]. The HPLC system was an Agilent HPLC Infinity Isocratic LC 1220, (Agilent; Palo Alto, CA, USA).

2.13. Statistical Analysis

After physiological, biochemical and phytochemical evaluation, statistical analyses were performed using DSAASTAT software version 1.022, Perugia, Italy. The variables included were fertilizer and cultivar with three replications. Data were analyzed using ANOVA to determine any significant variation between the means, and the mean results were compared using Duncan’s multiple range test (p ≤ 0.05).

3. Results

The ANOVA for physiological, biochemical, and phytochemical parameters showed that all the studied traits were significantly influenced by chamomile cultivars, fertilizer and the interaction of cultivar × fertilizer treatments (p ≤ 0.05) (Tables S1 and S3).

3.1. Physiological Characteristics

The analysis of variance indicated that the physiological parameters (plant height, fresh and dried flower weight) traits were significantly impressed by cultivars, fertilizer and interaction between cultivars and fertilizer treatments (p ≤ 0.01) (Table S1). The mean comparison for the interaction between cultivars and fertilizer treatments demonstrated that the Bona cultivar had the maximum plant height with an average of 54.67 and 54.50 cm at the concentration of 2 mL L⁻¹ amino acid fertilizer and PAW treatments, respectively. On the other hand, the Bodegold cultivar had the minimum plant height with an average of 31.00 cm under 3 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (

Table 7. Mean comparison of the interaction between cultivar and fertilizer on physiological and biochemical parameters of German chamomile under amino fertilizer and PAW treatments.

Car (μg/g dw)	Chl b (mg/g dw)	Chl a (mg/g dw)	Dried Flower Weight (g m−2)	Fresh Flower Weight (g m−2)	Plant Height (cm)	Fertilizer	Cultivar
701.08 g,h	2.22 b	5.06 a,b	31.1 e,f	124.00 g	40.70 b,c,d	0	Bona
598.89 g,h	2.15 b	4.96 b	36.51 c	146.00 c	42.50 b,c	1
479.52 h	2.19 b	4.99 a,b	33.15 d	132.00 e,f	54.67 a	2
810.64 f,g	1.03 d	5.18 a	38.68 b	154.70 b	43.50 b	3
1010.18 e,f	0.99 d	4.99 a,b	33.56 d	134.00 e	54.50 a	PAW
3832.02 b	1.01 d	3.82 d	23.57 i	99.00 i	35.33 e,f	0	Bodegold
3699.03 b	2.55 a	3.99 c,d	31.74 e	133.30 e	37.80 d,e	1
3388.64 c	2.57 a	4.00 c,d	21.3 j	90.00 j	31.27 g	2
3352.85 c	2.54 a	4.12 c	27.1 h	114.60 h	31.00 g	3
4656.06 a	1.14 c	2.61 f	30.7 f	130.00 f	32.60 f,g	PAW
1187.35 e	1.15 c	3.57 e	36.24 c	154.00 b	43.00 b	0	Lianka
1770.47 d	1.14 c	3.59 e	41.91 a	177.67 a	41.80 b,c	1
1893.44 d	1.03 d	3.43 e	33.42 d	142.00 d	32.70 f,g	2
1940.15 d	1.02 d	3.41 e	29.65 g	126.00 g	38.67 c,d,e	3
1121.58 e	1.03 d	3.83 d	41.92 a	178.00 a	40.07 b,c,d	PAW
EO Yield (%w/w)	Total Protein (%)	C:N Ratio	N (%)	H (%)	C (%)	Fertilizer	Cultivar
0.93 c,d	46.08 f	7.62 h	9.65 d	5.75 c,d	73.44 a	0	Bona
0.69 g	39.87 g	8.22 g	8.34 e,f	6.04 b,c	68.58 c,d	1
0.80 e	32.03 j	9.01 e,f	6.63 h	4.61 f,g	60.35 i	2
0.73 f	38.34 h	8.65 f,g	8.02 e,f,g	6.12 b,c	69.4 c	3
0.74 f	39.91 g	7.15 h	8.35 e	5.95 b,c	59.72 i	PAW
0.96 c	63.38 a	5.14 j,k	13.26 a	5.24 d,e	68.14 d	0	Bodegold
1.27 a	53.01 c	5.9 i	11.09 b	4.88 e,f,g	65.44 e	1
1.11 b	61.28 b	5.01 k	12.82 a	5.04 e,f	64.28 f	2
0.90 d	48.66 e	6.29 i	10.18 c,d	4.41 g	64.03 f	3
1.09 b	50.62 d	5.72 i,j	10.59 b,c	4.80 e,f,g	60.56 i	PAW
0.34 h	27.1 l	11.11 c	5.70 i	6.36 a,b	62.98 g	0	Lianka
0.95 c,d	28.68 k	10.27 d	6.00 h,i	6.86 a	61.63 h	1
0.81 e	16.6 m	16.87 b	3.36 j	6.29 a,b,c	56.68 k	2
0.96 c	15.77 m	17.56 a	3.30 j	6.43 a,b	57.94 j	3
0.73 f,g	36.57 i	9.29 e	7.65 e,g	6.38 a,b	71.09 b	PAW

Fertilizer levels: 0 = control, 1 = 1 mL L⁻¹, 2 = 2 mL L⁻¹, 3 = 3 mL L⁻¹ foliar amino acid, and PAW, different letters indicate significant differences between treatments according to Duncan’s multiple range test (p ≤ 0.05).

). The PAW and amino acid fertilizer increased the plant height in the Bona cultivar, while these treatments had no significant effect on the Lianka and Bodegold cultivars.

Mean comparison for the interaction between cultivar × fertilizer showed that the Lianka cultivar gained the highest fresh flower weight with an average of 178.00 and 177.67 g m⁻² under PAW and 1 mL L⁻¹ amino acid fertilizer treatment, respectively. In contrast, the lowest fresh flower weight was obtained for the Bodegold cultivar with an average of 90.00 g m⁻² at the concentration of 2 mL L⁻¹ amino acid fertilizer (p ≤ 0.01) (Table 7). Duncan’s analysis for the interaction between the cultivar and fertilizer showed that the Lianka cultivar had the greatest dried flower yield with averages of 41.92 and 41.91 g m⁻² under PAW and 1 mL L⁻¹ amino acid fertilizer treatments, respectively. Also, the Bodegold cultivar had the lowest dried flower yield with an average of 21.30 g m⁻² at the concentration of 2 mL L⁻¹ amino acid fertilizer (p ≤ 0.01) (Table 7). Also, the lowest dried flower yield was yield for the Bodegold cultivar, with an average of 21.30 g m⁻² at the concentration of 2 mL L⁻¹ amino acid fertilizer (p ≤ 0.01) (Table 7).

3.2. Chlorophyll a, b, and Carotenoids Content

The analysis of variance showed that the chlorophyll a, b, and carotenoids content was significantly impacted by the cultivars, fertilizer, and the interaction of cultivar and fertilizer (p ≤ 0.01) (Table S1). The mean comparison for the chlorophyll content showed that the maximum was 5.18 mg/g dw at the 3 mL L⁻¹ amino acid fertilizer treatment concentration. While the minimum chlorophyll content was related to Bodegold cultivar with an average of 2.61 mg/g dw under PAW treatment. The highest chlorophyll b content belonged to the Bodegold cultivar with an average of 2.57 mg/g dw under 2 mL L⁻¹ amino acid fertilizer treatment. In contrast, the lowest chlorophyll b content attained was an average of 0.99 mg/g dw under PAW treatment. The Duncan analysis for the interaction of cultivar and fertilizer showed that the Bodegold cultivar had the maximum carotenoid content with an average of 4656.06 μg/g dw under PAW treatment. In contrast, the minimum amount of carotenoid content belonged to the Bona cultivar with an average of 479.52 μg/g dw at the concentration of 2 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 7).

3.3. Carbon, Hydrogen, and Nitrogen Percentage

The analysis of variance indicated that the carbon, hydrogen, and nitrogen percentage was significantly impressed by the cultivar, fertilizer, and interaction between cultivar and fertilizer (p ≤ 0.01) (Table S1). The mean comparison of the interaction between cultivar and fertilizer illustrated that the Bona cultivar had the highest carbon percentage with an average of 73.44% (w/w) under control treatment and the lowest carbon content with an average of 56.88% (w/w) belonged to the Lianka cultivar at the concentration of 2 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 7). Duncan’s analysis for the interaction between cultivar and fertilizer demonstrated that the Lianka cultivar had the greatest hydrogen percentage with an average of 6.88% (w/w) at the concentration of 1 mL L⁻¹ amino acid fertilizer treatment. While the lowest hydrogen percentage, with an average of 4.41% (w/w), belonged to the Bodegold cultivar at the concentration of 3 mL L⁻¹ amino acid fertilizer treatment (Table 7). The mean comparison of the interaction between cultivar and fertilizer showed that the Bodegold cultivar had the highest nitrogen percentage, averaging 13.26 and 12.82% (w/w) under control and 2 mL L⁻¹ amino acid fertilizer treatment, respectively. In contrast, the lowest nitrogen percentage, with an average of 3.30% (w/w), belonged to the Lianka cultivar at the concentration of 3 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 7).

3.4. C: N Ratio and Total Protein Content

The analysis of variance demonstrated that the C: N ratio and total protein content were significantly affected by the cultivar, fertilizer, and interaction between cultivar and fertilizer (p ≤ 0.01) (Table S1). Duncan’s analysis of the interaction between the cultivar and fertilizer displayed that the Lianka cultivar had the greatest C: N ratio with an average of 17.56 at the concentration of 3 mL L⁻¹ amino acid fertilizer treatment. While the lowest C: N ratio with an average of 5.01 belonged to the Bodegold cultivar at the concentration of 2 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 7). The mean comparison of the interaction between cultivar and fertilizer indicated that the Bodegold cultivar had the maximum total protein percentage, averaging 63.38% (w/w) under control treatment. In contrast, the minimum total protein percentage with an average of 15.77% (w/w) belonged to the Lianka cultivar at the concentration of 3 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 7).

3.5. Essential Oil Yield

The analysis of variance revealed that the essential oil yield was significantly impacted by the cultivar, fertilizer, and interaction between cultivar and fertilizer (p ≤ 0.01) (Table S1). Duncan’s analysis for the interaction between cultivar and fertilizer showed that the Bodegold cultivar had the greatest essential oil yield with an average of 1.27% (w/w) at the 1 mL L⁻¹ amino acid fertilizer treatment concentration. While the lowest essential oil yield, with an average of 0.34% (w/w), belonged to the Lianka cultivar under control treatment (p ≤ 0.01) (Table 7).

3.6. Amino Acids Profile Analysis

In this study, we measured 16 types of amino acids, including Aspartic acid, Serine, Glutamic acid, Glycine, Histidine, Arginine, Threonine, Proline, Alanine, Tyrosine, Valine, Methionine, Lysine, Isoleucine, Leucine and Phenylalanine in dry powder of leaf chamomile cultivars. Significant changes were observed in the chamomile amino acids profile under applying foliar amino acid fertilizer and PAW treatments, shown in Figure 3, Figure 4 and Figure 5. The concentration of some amino acids composition increased after foliar application of amino acid and PAW treatment.

The Proline and Alanine amino acids were dominated among 16 in every three chamomile cultivars. Among 16 amino acids assayed in Bona, Bodegold and Lianka cultivars, most increased under amino acid fertilizer and PAW treatments compared with the control. The Proline, arginine, glycine, alanine, and valine showed the most changes under amino acid fertilizer and PAW treatments.

The amino acid fertilizer and PAW treatments increased the total amino acid content in the Bona cultivar compared to the control. In the Bona cultivar, seven types of amino acids increased under the influence of PAW treatment. The 13 amino acid types increased at 1 mL L⁻¹ amino acid fertilizer treatment concentration. The nine types of amino acids were raised at the concentration of 2 mL L⁻¹ amino acid fertilizer treatment. The concentration of 1- and 2-mL L⁻¹ amino acid fertilizer treatments showed the highest increase in the number of amino acids in the Bona cultivar (Figure 3).

The amino acid fertilizer and PAW treatments increased the total amino acid content in the Bodegold cultivar compared to the control. The 11 amino acid types were increased in the Bodegold cultivar due to the effect of amino acid fertilizer and PAW treatments. The concentration of 1 mL L⁻¹ and PAW treatments also demonstrated the greatest increase in amino acid amounts (Figure 4).

The amino acid fertilizer and PAW treatments decreased the total amino acid content in the Lianka cultivar compared to the control. But the amounts of proline, alanine, valine, lysine, phenylalanine and tyrosine increased under amino acid fertilizer and PAW treatments. The 4 types of amino acids increased at the concentration of 1 mL L⁻¹ amino acid fertilizer treatment. The PAW treatment increased six kinds of amino acids in the Lianka cultivar (Figure 5).

3.7. Secondary Metabolite Profile Analysis

In this study, the dried chamomile flowers generated dark blue essential oil in the range of 0.34% to 1.27% (w/w). Thirty-six compounds were identified in chamomile cultivars under foliar amino acid fertilizer and PAW treatments. Identified volatile constituents in the essential oils of the chamomile flower samples, which represented 92.91% of the oils, have been shown in Table S2.

The ANOVA for phytochemical compositions indicated that the identified constituents (including chamazulene, α-bisabolol, α-bisabolol oxide B, α-bisabolone oxide A, (E)-β-farnesene and (E)-α-farnesene) were significantly influenced by cultivar, fertilizer and the interaction of cultivar × fertilizer at 99% level of probability (Table S3). While α-bisabolol oxide A was significantly impacted by cultivar, fertilizer, and the interaction of cultivar × fertilizer at a 95% level of probability (Table S3).

The maximum percentage of major volatile constituents of the German chamomile oil were obtained as following: chamazulene (1.23%), α-bisabolol (2.99%), α-bisabolol oxide A (4.84%), α-bisabolol oxide B (6.80%), α-bisabolone oxide A (5.79%), (E)-β-farnesene (38.96%), (E)-α-farnesene (11.15%), (Z)-spiroether (5.88%), germacrene D (6.57%), α-pinene (4.50%) and (E)-β-ocimene (4.20%) (

Table 8. Mean comparison for major phytochemical compositions of German chamomile cultivars under PAW and amino acid fertilizer treatments.

α-Fa (%)	β-Fa (%)	α-BnA (%)	α-BoB (%)	α-BoA (%)	α-Bo (%)	Ch (%)	Fertilizer (mL/L)	Cultivar
10.60 b	34.52 b,c,d,e	4.80 f	5.89 e,f	4.28 c,d,e,f	2.81 a,b	0.73 g	0	Bona
9.67 e	33.55 d,e	5.00 e	6.03 c,d,e	4.12 d,e,f	2.43 e,f	1.19 a,b,c	1
10.37 c	35.04 b,c,d	4.94 e,f	5.92 d,e,f	4.48 a,b,c,d,e	2.26 f	0.69 g	2
8.23 h	33.95 c,d,e	5.37 b,c,d	6.06 c,d,e	4.53 a,b,c,d	2.48 e,f	0.61 g	3
10.04 d	35.40 b,c	5.31 c,d	5.67 g	4.05 e,f	2.82 a,b	0.85 f	PAW
10.59 b	29.64 f	5.43 b,c	5.81 f,g	4.84 a	2.62 b,c,d,e	1.11 b,c	0	Bodegold
11.15 a	30.88 f	5.79 a	5.90 e,f	4.77 a,b	2.78 a,b,c,d	1.14 a,b,c	1
10.38 c	33.94 c,d,e	5.40 b.c	6.40 b	4.36 b,c,d,e,f	2.79 a,b,c	1.11 b,c	2
8.60 f	36.01 b	4.95 e,f	6.23 b,c	4.10 d,e,f	2.31 f	0.90 e,f	3
9.51 e	33.01 e	5.46 b	6.80 a	4.18 d,e,f	2.99 a	1.21 a,b	PAW
10.09 d	35.67 b,c	5.04 e	5.44 h	3.98 f	2.56 d,e	0.99 d,e	0	Lianka
8.39 g,h	37.64 a	5.26 d	4.64 j	4.12 d,e,f	2.34 f	1.23 a	1
8.53 f,g	35.18 b,c,d	5.42 b,c	6.12 c,d	4.69 a,b,c	2.62 b,c,d,e	0.72 g	2
7.88 i	37.81 a	5.04 e	6.42 b	3.97 f	2.57 c,d,e	0.65 g	3
8.47 f,g	38.96 a	4.99 e	4.95 i	3.93 f	2.70 b,c,d	1.09 c,d	PAW

Ch (chamazulene), α-Bo(α-bisabolol), BoA (α-bisabolol oxide A), BoB (α-bisabololoxide B), BnA (α-bisabolonoxide A), βFa (β-farnesene), αFa (α-farnesene), Fertilizer levels: 0 = control, 1 = 1 mL L⁻¹, 2 = 2 mL L⁻¹, 3 = 3 mL L⁻¹ foliar amino acid and PAW, different letters indicate significant differences between treatments according to Duncan’s multiple range test (p < 0.05).

).

Based on GC/MS analysis, the percentage of oil constituents, including chamazulene, α-bisabolone oxide A and (E)-α-farnesene, were increased using foliar amino acid fertilizer treatments while PAW treatment improved α-bisabolol, α-bisabolol oxide B and (E)-β-farnesene (Table 8).

3.7.1. Chamazulene

Mean comparison for the interaction of cultivar × fertilizer demonstrated that the highest percentage of chamazulene was attained with an average of 1.23% at the concentration of 1 mL L⁻¹ amino acid treatment in the Lianka cultivar. In contrast, the lowest percentage was obtained with an average of 0.61% at the concentration of 3 mL L⁻¹ amino acid treatment in the Bona cultivar (p ≤ 0.01) (Table 8). The GC/MS analysis of chamazulene of German chamomile essential oil is presented for the Lianka cultivar treated with 1 mL L⁻¹ amino acid treatment (Figure 6).

3.7.2. α- Bisabolol

The maximum α-bisabolol percentage (2.99%) belonged to Bodegold cultivar under PAW treatment. In contrast, the lowest α-bisabolol percentage (2.26%) was obtained for the Bona cultivar under 2 mL L⁻¹ amino acid treatment (p ≤ 0.01) (Table 8). The GC/MS analysis of α-bisabolol of German chamomile essential oil is presented for the Bodegold cultivar under PAW treatment (Figure 7).

3.7.3. Bisabolol Oxide A and B

Duncan’s test results of the interaction of cultivar × fertilizer indicated the Bodegold cultivar had the maximum bisabolol oxide A percentage (4.84%) under control treatment. While the minimum bisabolol oxide A amount (3.93%) belonged to the Lianka variety under PAW treatment (p ≤ 0.05) (Table 8).

The maximum bisabolol oxide B percentage (6.80%) belonged to the Bodegold variety under PAW treatment. While the minimum bisabolol oxide B percentage (6.64%) was achieved for the Lianka variety under 1 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 8). A typical GC-MS chromatogram of bisabolol oxide A is presented for the Bodegold cultivar under control treatment (Figure 8). Also, bisabolol oxide B is presented for the Lianka cultivar treated with 1 mL L⁻¹ amino acid treatment (Figure 9).

3.7.4. Bisabolone Oxide A

The maximum bisabolone oxide A average (5.79%) was obtained in the Bodegold variety under 1 mg L⁻¹ amino acid fertilizer treatment. Whereas the minimum bisabolone oxide A percentage (4.80%) was attained under control treatment in the Bona cultivar (p ≤ 0.01) (Table 8). A typical GC-MS chromatogram of bisabolone oxide A of German chamomile oil is displayed for the Bodegold variety under 1 mL L⁻¹ amino acid treatment (Figure 10).

3.7.5. α- and β- Farnesene

Duncan’s analysis of the interaction of the cultivar × fertilizer demonstrated the Lianka variety had the maximum β-farnesene percentage (38.96%) under PAW treatment. While the Bodegold variety showed the minimum β-farnesene percentage (29.64%) under control treatment (p ≤ 0.01) (Table 8). The GC/MS analysis of β-farnesene of German chamomile essential oil is presented for the Lianka cultivar under PAW treatment (Figure 11).

The highest percent of α-farnesene, with an average of 11.15%, belonged to Bodegold cultivar under 1 mL L⁻¹ amino acid fertilizer treatment. In contrast, the Lianka variety displayed the minimum α-farnesene percentage (7.88%) under 3 mL L⁻¹ amino acid fertilizer treatment (p ≤ 0.01) (Table 8).

3.8. Apigenin Content Evaluation

The ANOVA indicated that the apigenin content was significantly impacted by German chamomile varieties and fertilizer treatments, and the interaction of cultivar × fertilizer was significant on its at a 99% level of probability (Table S3). The highest average of apigenin was obtained, with an average of 16.68 mg g⁻¹ under PAW treatment in the Bodegold cultivar. Whereas the minimum apigenin percentage (5.95 mg g⁻¹) was attained under 2 mL L⁻¹ amino acid fertilizer treatment in the Bona cultivar (p ≤ 0.01) (Figure 12). A typical HPLC chromatogram of apigenin of German chamomile oil is presented for the Bodegold cultivar under PAW treatment (Figure 13).

4. Discussion

Producing plasma inside the water volume along with airflow increased the amount of nitrate and nitrite and decreased pH. Depending on what type of the discharge gas is used, reactive oxygen species (ROS–ozone, O₃, H₂O₂, ·OH) and reactive nitrogen species (RNS–ONOO⁻, NO₃⁻, NO₂⁻, and the corresponding acids, nitrogen oxides NO_x) are produced in the solution [37].—the reactions taking place between the chemical species formed in the plasma and water result in acidification. In addition, it was known that acidic solutions are highly effective in bacterial inactivation [29]. Several researchers have studied the impact of acidification on reducing bacterial colony formation [9,25,29]. The results show that the concentration of nitrate increases with treatment time which is due to capturing nitrogen from air that acts as a fertilizer [17]. The antimicrobial property of a solution containing nitric ions with a pH of around 4–5 is high. Both ROS and RNS play an essential role in bacteria inactivation. The major evidence for the production of RNS is the presence of nitrates and nitrites compounds in the PAW. Therefore, this solution would be a good fertilizer candidate which can act similarly to conventional nitrogen-based fertilizers. Reactions between gas-phase species and water molecules advance the generation of aqueous species like hydrogen peroxide nitrite and nitrate.

This study demonstrated that the foliar spraying of PAW and amino acid fertilizer significantly impacted physiological traits and volatile oil yield in chamomile cultivars under field conditions. These findings are in accordance with Omer et al. [13] and Kučerová et al. [38]. Alexander (2005) reported that the plant height had a higher value in Bona as a diploid variety than in the Goral and Lutea as tetraploid varieties [35]. Omer et al. reported that the plant height, fresh and dried flower weight of chamomile (Matricaria recutita L.) were increased under the foliar application of amino acid fertilizer in Egypt [13].

In the current work, the Lianka variety (diploid) indicated the highest dried and fresh flower weight under PAW and 1 ml L⁻¹ amino acid fertilizer treatments. It has been expressed the German chamomile diploid varieties showed the greatest dried and fresh flower weight and yield [39,40]. In all three German chamomile cultivars, PAW has increased the dried flower weight. Amino acid treatment has also increased the dried flower weight in most concentrations. Therefore, it is concluded that the plasma-activated water and amino acid fertilizer treatments can be increased the dried flower weight in appropriate concentration.

The content of Chlorophyll a did not change significantly under the influence of amino acid and PAW treatments. The amount of Chlorophyll b also increased only in Bodegold cultivar under the application of amino acid treatments. Also, the amount of carotenoid has raised significantly under the effect of PAW in Bona and Bodegold cultivars. The amino acid fertilizer treatments also increased carotenoid content only in Lianka cultivar in all used concentrations. Regarding the results of the other investigations, the chamomile varieties response is varied to amino acid fertilizer doses. Kučerová et al. [38] showed that PAW improves the wheat leaves’ chlorophyll a, b, and carotenoid content. Chlorophylls and carotenoids as photosynthetic pigments are involved in physiological processes such as conservation versus oxidative stress, photosynthesis, and metabolic reactions [41].

In this experiment, PAW and amino acid fertilizer treatment enhanced the chlorophyll b and carotenoid content in chamomile cultivars. H₂O₂ and NO₃⁻ are key species in boosting and expanding photosynthetic pigments. H₂O₂ raises the opening of stomata, thus promoting CO₂ uptake, photosynthetic rate, and aggregation of photosynthetic pigments [19]. The effect of NO₃⁻ is ascribed to the up-regulation of the genes encoding δ-amino levulinic acid dehydratase, an effective enzyme in chlorophyll biosynthesis [27]. Stoleru et al. demonstrated that the utilization of PAW had no remarkable effect on the chlorophyll content in the lettuce leaves. Sajib et al. reported that the PAW treatment significantly affected the enhancement of leaf chlorophyll levels in black gram plants [21].

The carbon, hydrogen, nitrogen, oxygen, and sulfur (CHNOS) are major precursors for protein, carbohydrates, and lipids in plant and algal cells, which are measured using combustion analysis [35]. The carbon content was increased in Lianka cultivar under the PAW treatment. The amino acid fertilizer sprayed has elevated the hydrogen percentage in Bona and Lianka cultivars. While the content of nitrogen has increased in Lianka cultivar under PAW treatment.

The C:N ratio has increased in Bona and Lianka cultivars under amino acid fertilizer treatments. While the C:N ratio increased under PAW and amino acid fertilizer treatments in the Bodegold cultivar. The strong interactions between C and N assimilation have been reported in metabolic processes and energy levels [42,43]. Both C and N contents were increased in the Lianka cultivar under PAW treatment. This is probably due to the linkage between carbon and nitrogen metabolism because they share organic carbon and energy provided by respiration, CO₂ fixation, or photosynthetic electron transport [43].

Proteins are essential elements of plant enzymes and fundamental players in plant growth [29]. In the present work, total protein content via % nitrogen has almost decreased in all three chamomile cultivars under the influence of amino acid fertilizer treatments. While the amount of total protein has increased in Lianka cultivar under PAW treatment. The increase in protein content is due to the presence of NO3⁻ and NO2⁻ ions in PAW; both species are crucial nitrogen sources, necessitating protein synthesis [30]. Kucerová et al. also reported an enhancement in the soluble protein content in the roots and shoots of wheat plants under PAW produced from tap water [38]. Sajib et al. observed an increase in the content of total soluble protein in the roots and above-ground parts of black gram plants germinated from the seeds under PAW treatment [21]. Furthermore, nitrates are essential in plant nutrition, given their pivotal role in amino acids, proteins, and chlorophyll synthesis [44].

Amino acids are incorporated in synthesising some organic compositions involving plant hormones, vitamins, alkaloids, amines, enzymes, terpenoids, and protein. These compounds control various plant processes [13]. Nitrogen is used directly to synthesize amino acids through a primary process involving the synthesis of glutamine and glutamate via the 2-oxoglutarate-glutamate synthase pathway [45,46], and the increment of free amino acids could thus be attributed to the NO₂⁻ and NO₃⁻ that produced in PAW.

Proline and alanine can ameliorate various abiotic stress tolerance in different plants via osmotic adjustment, chlorophyll metabolism, and free radical scavenging [47,48]. According to this, the enhancement of proline and alanine contents of chamomile under PAW and amino acid fertilizer in the present investigation was supposed to be related to the oxidative stress and osmotic stress caused by NO₂⁻, NO₃⁻ and low pH of PAW [45]. Proline is one of the indices for plant defence response against stress, which has increased in this experiment for all three chamomile cultivars. The proline increasing under PAW treatment for the tetraploid Bodegold cultivar shows that PAW probably has been able to activate the genes involved in the defence mechanism of the chamomile plant and the signaling pathway for tolerance to abiotic stresses. The proline increasing under PAW treatment indicates the ability to induce or stimulate the increase of plant defence response by PAW. There has been a difference between chamomile diploid and tetraploid cultivars and cultivar indices at the tetraploid and diploid levels. Based on this, it can be seen that the important characteristics related to the quality of chamomile essential oil have increased significantly in the tetraploid variety.

The reaction of German chamomile varieties was varied to amino acid fertilizer and PAW treatments under field circumstances. The various responses of chamomile varieties can be ascribed to plant genetic and environmental situations [7]. So, the percentage of α-bisabolol and chamazulene were enhanced in Bona, Bodegold, and Lianka cultivars under foliar amino acid fertilizer and PAW treatments at the specific concentration.

The chamomile essential oil yield has significantly increased under PAW treatment in Bodegold and Lianka cultivars. Also, amino acid treatments have made a considerable enhancement in the oil yield in Bodegold and Lianka chamomile cultivars, so it has increased about three times in the Lianka cultivar. The biosynthesis of secondary metabolites is not solely controlled genetically but is also severely influenced by environmental factors [8]. Based on the previous studies, the essential oil content varied between 0.24% to 2.0% in chamomile-dried flowers [6]. Alternation in oil content and compounds in Iran is attributed to the impact of genetic, environmental factors, and agricultural practices [11]. The chamomile essential oil yield depends on genotype, climate, and agro-technical practices [11].

Furthermore, it may be due to various responses of German chamomile varieties to certain doses and/or specific ranges of foliar amino acid. Nitrogen affected essential oil production via acetyl-CoA formation and carbon metabolism through the mevalonate pathway [16]. Generally, PAW and amino acid fertilizers may impress the quantity and quality of chamomile oil. Probably, the ROS species (H₂O₂, NO₂⁻, NO₃⁻ and dissolved O₃⁻) generated by PAW causes to be active mechanisms of production of the antioxidant enzymes.

Plant secondary metabolites and essential oils have increased, resulting from stress metabolisms. Velicka et al. [16] have investigated the changes in oil and flavonoid contents of various mint species after foliar application of amino acids in field conditions. They reported that the foliar spray with aromatic amino acids could enhance the essential oil content and total flavonoids and alter the mint essential oil odor profile. The effect of amino acids on the oil content was solely discovered in M. piperita ‘Granada’ plants [16].

The extracted oil color was blue to dark blue in our work which was in accordance with the previous works [49,50]. The essential oil’s blue color is imputed to the presence of chamazulene [3]. In total, thirty-six compositions were recognized in chamomile varieties under foliar amino acid fertilizer and PAW treatments, where the most important ones include chamazulene, α-bisabolol, bisabolol oxides A and B, farnesene and α-bisabolonoxide A. Factors that influence the components, quantity, and quality of extracted essential oil include isolation method, environmental conditions, nutrient condition, and some stresses [51]. Flowering and the type of essential oil profile are also genetically controlled, but their amount depends on external factors.

Medicinal plant production is mostly dependent on ecological conditions. So, monitoring and management of environmental parameters are very important [7]. Some researchers showed that the response of the bis-aboloids to these situations was very intense, while environmental conditions had no or only little effect on the yield of essential oil accumulation as well as on chamazulene content. However, they did not report any qualitative changes in essential oil composition due to experimental conditions [6,7].

The bisabolol amount depends on environmental conditions of growth, but the amount of chamazulene is more affected genetically in the chamomile essential oil [7,52,53]. Besides the main effect of genetic factors, the environment has a significant influence on the quantity and quality of essential oil. The increase of chamazulene may be attributed to the interaction between genotype and environment and/or the impact of amino acid fertilizer on the expression of chamazulene controller genes, functional proteins, and metabolic pathways. In addition, the amino acids signaling pathway is involved in the biosynthesis of terpenoids, including sesquiterpenoids (such as chamazulene), triterpenoids, and diterpenoids [54]. It has been reported that the amount of chamazulene varies in various chamomile cultivars in varied years and climate conditions [55].

The α-bisabolol content rises until the full blooming phase due to the decrease in the amount of dicycloether and is not related to the metabolism of the other substances [56]. The temperature conditions seem to impact α-bisabolol content in chamomile flowers significantly, so the highest amount occurs during sunlight and sunset [53].

The environmental situation, which is altered by chamomile plant ontogeny, and many other parameters are also known to impact chamomile oil yield and its compounds [57]. Findings illustrated the relative impact of amino acid fertilizer and PAW treatments on the essential oil, chamazulene, and α-bisabolol contents. In total, the amino acid fertilizer and PAW treatments were altered amount and compositions of the chamomile essential oil under field conditions.

The response of German chamomile cultivars for α-bisabolol and bisabolol oxide A had the same trend, while there was no such trend for chamazulene. Metabolites of α-bisabolol and bisabolol oxide A illustrated the maximum percentage under PAW treatment in the Bodegold tetraploid cultivar uniformly, which indicates the genetic capacity of German chamomile tetraploid cultivars to produce sesquiterpene secondary metabolites such as α-bisabolol and chamazulene. In general, PAW treatment increased the number of secondary metabolites, such as α-bisabolol, in tetraploid cultivars of German chamomile than in its diploid cultivars.

If the goal is to increase chamazulene, it can be recommended to the German chamomile growers on a wide scale as a practical recommendation to use the foliar application of amino acid fertilizer at the concentration of 1 mL L⁻¹ after verification and confirmation during at least three years research.

As a flavonoid, changes in apigenin are considered a phytochemical adaptation to the abiotic and biotic environment [58]. Previous research shows that the synthesis of apigenin is affected by various factors such as UV radiation, drought, ozone, plant pathogens, and insecticides [36,59]. According to HPLC analysis, the apigenin content was improved under amino acid fertilizer and PAW treatments. Considering the importance of apigenin use in the pharmaceutical and cosmetic industries, it is recommended to utilize tetraploid cultivars (such as Bodegold) under PAW treatment with a certain dosage or specific characteristics if the objective is to increase apigenin of German chamomile.

5. Conclusions

The findings of this experiment demonstrated the foliar application of amino acid fertilizer and PAW treatments made a remarkable effect on the physiological traits, including plant height, fresh and dried flower weight, photosynthetic pigments (chlorophyll a, b, carotenoids), and biochemical parameters including total protein content and essential oil yield and phytochemical compounds of German chamomile under field conditions. But it had no considerable impact on the carbon and hydrogen. Nevertheless, the amino acid contents were enhanced after applying foliar amino acid fertilizer and PAW. It can be due to various responses of German chamomile varieties to special dosages or specific ranges of foliar amino acids fertilizer and PAW treatments. However, the response of German chamomile varieties was varied to foliar amino acid fertilizer and PAW treatments under field conditions.

Regarding these results, PAW accompanying foliar amino acid fertilizer can be proposed as a good candidate to produce an admissible yield in medicinal plants, especially German chamomile. Results of the current study may support the positive impact of foliar amino acid fertilizer and PAW on the improvement of quality and quantity of German chamomile oil yield and phytochemical compositions under field conditions. According to the results, applying PAW and amino acid fertilizer treatments caused great changes in amino acid values. Now, depending on the desired research goals, to increase the amount of each measured amino acid in German chamomile cultivars, PAW with special features and amino acid fertilizer with specific and optimal concentrations can be selected and then applied. The results also showed that PAW has the potential to be a promising candidate to use as fertilizer to reduce the use of chemical products. Recently, PAW has focused on agriculture as an alternative to chemical fertilizer, controlling microorganisms and plant diseases to increase agricultural production. Production of PAW is a green technology with many advantages such as high efficiency, being portable, user-friendly, without the need for chemicals, and no residue. Moreover, more studies and experiments are needed to clarify the biochemical functions of amino acid fertilizer concentrations and PAW in German chamomile cultivars. By comparing the results obtained from amino acid fertilizer and PAW, it can be concluded that PAW represents a promising method as a green technology in the medicinal plants and crop production process.