Biomass Production and Volatile Oil Accumulation of Ocimum Species Subjected to Drought Stress

Abstract

Ocimum, commonly known as basil, is a group of aromatic plants extensively cultivated for their aromatic leaves, flavorful seeds, and essential oils, finding applications in food and herbal medicine. Drought stress is a crucial environmental factor that has a considerable impact on basil growth and the accumulation of bioactive compounds. This study aims to evaluate how drought stress affects biomass production and volatile oil accumulation in selected Ocimum species over two consecutive years in an open-field cultivation setting. Five distinct basil genotypes, O. basilicum L. ‘Ohře,’ O. basilicum L. ‘Genovese,’ O. × africanum Lour., O. americanum L., and O. sanctum L., were evaluated under two levels of water supply, with one group receiving irrigation as a control and the other exposed to non-irrigated conditions to induce drought stress. Consistent negative impacts of drought stress on biomass production were observed in both years. The reduction in fresh herb yield varied from 16.5 g plant⁻¹ (10.3%) for O. sanctum to 118 g plant⁻¹ (41.7%) for O. basilicum ‘Ohre.’ Across the study years, drought stress slightly increased the essential oil content of O. × africanum and O. basilicum ‘Genovese’ by 9.8% and 26%, respectively. The essential oil composition varied considerably among the different Ocimum species and cultivars. Cultivars Ohře and Genovese had linalool as a major component, exceeding 40%. O. americanum was rich in citral compounds—neral and geranial—accounting for 26–37%, which contribute to its strong lemon-like fragrance. The hybrid O. × africanum contained high proportions of 1,8-cineole (32–38%) and limonene (14–16%), while O. sanctum was characterized by its elevated levels of eugenol (36.4–50.3%) and β-caryophyllene (26.4–38.5%). The influence of water availability on essential oil content and composition was inconsistent across species. Similarly, variations were observed in total phenolic content (TPC) and antioxidant capacity (AOC) depending on both species and growing year. Notably, the highest TPC (341.4 mg GAE g⁻¹ DM) and AOC (122.9 mg AAE g⁻¹ DM) were recorded for O. sanctum grown under drought stress during the first experimental year. In conclusion, it is recommended to irrigate the studied basil species at least twice a week under open-field conditions to minimize the negative effects of drought stress.

1. Introduction

The Ocimum L. genus, commonly known as basil and belonging to the Lamiaceae family, derives its name from the Greek word “basilikos”, signifying “royal” [1]. This genus encompasses a diverse range of annual and perennial herbs and shrubs distributed across various geographical regions. The primary areas of Ocimum diversity are tropical and subtropical regions of the world. Despite its regional distribution, basil is extensively cultivated on a global scale [2]. The genus exhibits significant genetic diversity, with around 64 species [3,4], including notable species such as Ocimum basilicum (sweet basil), Ocimum gratissimum (African basil), Ocimum americanum (American basil), and Ocimum sanctum (holy basil). Numerous varieties, related species, and hybrids exist [5,6]. The Ocimum genus is renowned for its essential oils, primarily composed of monoterpenes such as camphor, limonene, citral, geraniol, and linalool [5,7,8]. However, certain species feature essential oils rich in phenolic compounds like eugenol, methyl chavicol (also known as estragole), and methyl cinnamate, often with varying amounts of linalool [9]. Basil plants also contain polyphenolic compounds including phenolic acids (e.g., rosmarinic, caffeic, caftaric, and chicoric acids) and flavonoids [10,11,12]. These essential oils and polyphenols find applications across industries such as flavoring, fragrances, cosmetics, aromatherapy, and pharmaceuticals due to their various functional properties, including antioxidant, anti-inflammatory, antimicrobial, and immunomodulatory effects [7,13,14].

The biosynthesis, accumulation, and distribution of secondary metabolites are strongly affected by genetic, ontogenic, morphogenetic, and environmental factors and processing methods [15,16]. Drought stress leads to a reduction in plant growth, which is a well-documented phenomenon across various Ocimum species. Furthermore, drought stress has been shown to influence the accumulation of bioactive compounds, although in a complex manner that varies across different studies and species [17,18]. Some studies report an increase in the concentration of secondary metabolites under mild to moderate drought stress. For example, drought stress has been associated with enhanced antioxidant activities and increased essential oil content in certain Ocimum species [18,19,20,21]. Conversely, severe drought stress can negatively affect the accumulation of secondary metabolites or have no effect [18,22,23]. The balance between enhanced accumulation under mild stress and reduced synthesis under severe stress highlights the complex interaction between drought stress and secondary metabolite production in Ocimum species.

Given the economic and therapeutic significance of Ocimum species, understanding their diversity, essential oil composition, and the effects of environmental stresses such as drought is crucial. Most existing research findings regarding morpho-chemical alterations in basil induced by drought primarily concentrate on O. basilicum in a controlled pot experiment. Thus, there is a scarcity of research outcomes about drought responses specific to different taxa in outdoor cultivation settings. Hence, the objective of this research article is to investigate the impact of water supply on the biomass production and biochemical characteristics of four Ocimum species under open-field cultivation. This knowledge can guide the development of improved cultivation practices, the selection of drought-tolerant cultivars, and enhanced utilization of Ocimum species in various industries.

2. Material and Methods

2.1. Experimental Site and Design

A field experiment was conducted over two consecutive years, 2021 and 2022, at the Experimental and Research Farm of the Hungarian University of Agriculture and Life Sciences (MATE) in Soroksár, Budapest. These investigations involved five different basil genotypes, O. basilicum ‘Ohře’, O. basilicum ‘Genovese’, O. × africanum Lour, O. americanum, and O. sanctum (purple variety), along with two levels of water supply, one being irrigated as a control and the other subjected to non-irrigated conditions to induce drought stress. The experiments were structured using a randomized complete block design (RCBD) with three replications. The seeds of the Ocimum species were obtained from the gene bank of the Department of Medicinal and Aromatic Plants of MATE. In mid-March of 2021 and 2022, the seeds were sown in seed trays measuring 27 × 57 × 7 cm within a greenhouse. When the seedlings developed two leaves, they were transferred into 0.1 L pots. Then, during the first and second weeks of June, vigorous, healthy, and uniformly sized seedlings were transplanted into open-field plots with a 40 × 40 cm plant spacing. The plots consisted of four rows and six plants per row. There was a half-meter distance between plots and a meter between blocks. Drought treatment began two weeks after transplanting and lasted 30 days in both years. A spraying hose connected to a water meter device was utilized to apply 20 mm of water twice weekly for the irrigated treatments. Natural precipitation, which reached 220 mm in 2021 and 234 mm in 2022 throughout the experiment, was the only source of moisture for the drought treatment. The average air temperatures recorded during the experiment were 21 °C in 2021 and 20 °C in 2022. The average relative humidity was 69% and 64%, respectively. The daily weather conditions during the experiment are indicated in Figure 1 below. In addition, the soil chemical attributes are illustrated in

Table 1. Soil characteristics of experimental field.

pH H2O	Humus %	KA	NO3-N mg kg−1	P2O5 mg kg−1	K2O mg kg−1	Na mg kg−1	Mg mg kg−1	Mn mg kg−1	Zn mg kg−1	Cu mg kg−1	SO4 mg kg−1
2021
7.99	1.75	26	11.97	473.86	377.31	45.76	146.91	22.14	2.13	1.88	24.77
2022
7.58	1.7	<25	11.36	544.43	177.36	30.11	377.93	58.48	4.90	2.46	71.58

Note: K_A stands for Arany Soil Plasticity Index [24].

.

2.2. Measurement of Growth Parameters

Nine plants, chosen at random, from the middle rows were used to measure the selected growth parameters. The height of each plant was measured from its base to the tip of the shoot, and the canopy diameter was measured before the plants were harvested at the widest point. After harvesting at full flowering stage, the fresh herb yield (g plant⁻¹) was determined. Additionally, each sample plant was dried at room temperature in a well-ventilated room until reaching a constant weight, and the dry herb yield (g plant⁻¹) was measured.

2.3. Determination of Secondary Compounds

Essential oil content (EOC): The dried samples were used to extract essential oils through hydro-distillation. The content of essential oil (mL 100 g⁻¹ of dry mass) was measured across six replications using a bulk sample of dried leaves and inflorescences, excluding the stem. Each sample, weighing 20 g, was hydro-distilled in 500 mL of distilled water using a Clevenger-type apparatus, as recommended by the Hungarian Pharmacopeia [25]. Once the essential oils were collected, any residual water was removed, and the oils were stored in a sealed vial in the refrigerator at 4 °C for one week before analysis.

Essential oil composition: The composition of the essential oils was analyzed using Gas Chromatography–Mass Spectrometry (GC-MS). The GC analysis was performed using an Agilent Technologies 6890 N instrument with an HP-5MS capillary column (30 m length × 0.25 mm diameter, with a film thickness of 0.25 μm). The analysis started at an initial temperature of 60 °C and gradually increased to 240 °C at a rate of 3 °C per minute and the final temperature was maintained for 5 min. The injector and detector were both subjected to a temperature of 250 °C. Helium gas was used as the carrier, maintaining a consistent flow rate of 1 mL per minute, a split ratio of 30:1, and an injection volume of 0.2 μL (1%, n-hexane). To determine the relative proportions of individual compounds, the total area percentages were calculated. An Agilent Technologies MS 5975 detector (Agilent Technologies, Inc., Waltham, MA, USA) was used for component identification. The energy of ionization was 70 eV. The chromatograms obtained from full-scan mass spectra represented the total ion current (TIC). The Van Den Dool and Kratz [26] equation was used to calculate linear retention indices (LRIs). The LRIs and mass spectra were compared between homemade library mass spectra, Adams [27], and commercial databases such as NIST and Wiley. The SPME and GC samples were repeated three times for reliability.

Total phenolic content (TPC): The total phenolic content of the extracts was determined using the Folin–Ciocalteu method with slight modifications [28]. Firstly, half a gram of powdered and sieved plant material, with a diameter of 500 µm, was prepared. The extracts were obtained by adding 50 mL of boiling distilled water and allowing it to steep for 24 h. Subsequently, the extracts were filtered using filter paper with specific characteristics: a pore size of 10–12 µm, a weight of 85 g m⁻², and a thickness of 200 µm. The filtered extracts were then stored in a freezer until further measurements were taken. During the measurement process, a test tube was filled with 40 µL of the test sample, 460 µL of distilled water, and 2.5 mL of Folin–Ciocalteau’s reagent (10% v:v). After an incubation period of 1 min, sodium carbonate (0.7 M) was added to the mixture. The resulting mixture was placed in hot water at a temperature of 50 °C for 5 min. Following this, the absorbance was measured at 760 nm using a Thermo Evolution 201 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). For calibration purposes, gallic acid (0.3 M) was used as a chemical standard. A blank was prepared from distilled water. The total phenol content of the samples was expressed in terms of gallic acid equivalent (mg GAE g-1 DW). The measurements were repeated six times.

Antioxidant capacity (AOC): The FRAP assay, which measures ferric-reducing antioxidant power, was conducted following the procedure established by Benzie and Strain [29] with slight modifications. In this study, the FRAP reagent was prepared freshly by combining sodium acetate buffer (pH 3.6), TPTZ (2, 4, 6-tripyridyl-s-triazine) in HCl and a solution of FeCl₃ · 6H₂O (20 mmol L⁻¹) in a ratio of 10:1:1, respectively. To perform the assay, 10 μL of the previously extracted sample and 40 μL of distilled water were added to 1.5 mL of the active FRAP reagent and thoroughly mixed. After 5 min, the absorbance was measured at 593 nm using the same spectrophotometer. In the blank, distilled water was used instead of the sample extract. As a chemical standard for calibration, ascorbic acid was employed. The antioxidant capacity of the samples was expressed in terms of ascorbic acid equivalent (mg AAE g⁻¹ DW). The measurements were repeated six times.

2.4. Statistical Analysis

The analysis was conducted using both a one-way analysis of variance (ANOVA) and a t-test. The normality of the distribution was assessed using Shapiro–Wilk’s test, while variance homogeneity was evaluated by using Levene’s test. Tukey’s honest significant difference (HSD) test was employed to identify any significant differences in means, with a significance level set at p < 0.05. IBM SPSS 29 software was utilized for all statistical analyses.

3. Results

3.1. Effect of Water Supply on Biomass Production

Water supply had a significant effect on plant height, canopy diameter, and the fresh and dry herb yield of various basil species over multiple years of cultivation, as indicated in

Table 2. Effect of irrigation on plant height and canopy diameter of Ocimum species in 2021/22.

Species	TRT	Plant Height (cm)		Canopy Diameter (cm)
		2021	2022	2021	2022
O. basilicum ‘Ohře’	I	43.8 ± 2.5 Aab	50.3 ± 3.8 Aa	39.3 ± 2.5 Ab	50.2 ± 4.4 a
	NI	40.3 ± 4.2 Ba	41.7 ± 4.0 Ba	34.4 ± 4.4 Ba	42.0 ± 3.8 a
O. basilicum ‘Genovese’	I	47.3 ± 4.9 Aa	53.3 ± 4.0 a	38.7 ± 4.7 Ab	47.7 ± 3.7 ab
	NI	40.8 ± 3.6 Ba	43.7 ± 2.5 a	34.6 ± 4.5 Ba	39.3 ± 5.4 a
O. × africanum	I	44.5 ± 2.2 Aab	50.2 ± 1.9 a	45.7 ± 4.2 Aa	43.3 ± 4.0 b
	NI	39.7 ± 5.6 Ba	41.2 ± 1.8 a	39.9 ± 5.6 Ba	34.2 ± 3.7 b
O. americanum	I	35.7 ± 1.4 Ac	38.8 ± 2.1 c	47.1 ± 4.2 Aa	50.1 ± 2.7 a
	NI	30.2 ± 4.2 Bb	35.4 ± 2.9 b	36.7 ± 7.2 Ba	41.4 ± 5.1 a
O. sanctum	I	42.0 ± 1.9 Ab	44.5 ± 2.6 b	30.9 ± 4.8 Ac	33.3 ± 2.6 c
	NI	39.3 ± 4.9 Aa	35.8 ± 2.9 b	25.7 ± 5.1 A	24.2 ± 3.2 c

Values are expressed as mean ± SD, where TRT represents treatment, I denotes the irrigated condition, and NI denotes the non-irrigated condition. Different letters indicate significantly different groups. Capital letters differentiate between drought stress under fixed species and small letters differentiate between species under fixed drought stress.

and

Table 3. Effect of irrigation on herb production among Ocimum species in 2021/22.

Species	TRT	Fresh Herb Yield (g plant−1)		Dry Herb Yield (g plant−1)
		2021	2022	2021	2022
O. basilicum ‘Ohře’	I	180.1 ± 28.4 A	379.7 ± 97.7 Aa	38.4 ± 6.5 A	74.4 ± 16.7 Aa
	NI	142.4 ± 43.9 B	221.2 ± 6.0 Ba	32.1 ± 7.2 B	39.1 ± 10.5 Ba
O. basilicum ‘Genovese’	I	157.8 ± 31.9 Aa	273.8 ± 53.0 Aab	34.7 ± 6.4 A	51.0 ± 10.2 Ab
	NI	118.0 ± 26.1 B	192.2 ± 64.5 Bb	26.9 ± 5.2 B	32.9 ± 9.8 Bab
O. × africanum	I	142.7 ± 57.5 Aa	269.0 ± 37.4 Ab	42.2 ± 19.0 A	67.7 ± 7.5 Aa
	NI	108.1 ± 79.2 B	164.0 ± 47.8 Babc	32.2 ± 17.4 B	38.0 ± 10.8 Ba
O. americanum	I	146.9 ± 30.1 Aa	214.8 ± 51.7 Ab	45.9 ± 8.5 A	43.3 ± 9.9 Abc
	NI	97.7 ± 65.8 B	152.7 ± 33.9 Bbc	30.7 ± 16.5 B	35.4 ± 5.8 Bab
O. sanctum	I	85.0 ± 30.3 Ab	134.8 ± 11.6 Ac	32.4 ± 11.3 A	32.0 ± 2.9 Ac
	NI	82.9 ± 28.8 A	111.2 ± 14.7 Bc	30.6 ± 9.9 A	24.3 ± 5.7 Bb

Values are expressed as mean ± SD, where TRT represents treatment, I denotes the irrigated condition, and NI denotes the non-irrigated condition. Different letters indicate significantly different groups. Capital letters differentiate between drought stress under fixed species and small letters differentiate between species under fixed drought stress.

. Consequently, basil plants exposed to drought stress showed shorter plant heights, narrower canopies, and lower herb yields. Based on the yearly average, height reduction ranged from 4.5 cm (19%) to 8 cm (16%) in O. americanum and O. basilicum ‘Genovese’, respectively. Similarly, the reduction in canopy diameter ranged from 6.2 cm (14.3%) in ‘Genovese’ to 9.6 cm (19.6%) in O. americanum. Hence, taxa-specific responses were observed.

Furthermore, basil plants subjected to drought treatment showed a notable decline in biomass, as depicted in Table 3. The combined average over two years indicated that the reduction in fresh herb yield ranged from 16.5 g plant⁻¹ (10.3%) for O. sanctum to 118.0 g plant⁻¹ (41.7%) for O. basilicum ‘Ohře’. Similarly, the decrease in dry herb yields due to drought stress varied from 4.8 g plant⁻¹ (15%) for O. sanctum to 20.8 g plant⁻¹ (32.0%) for O. basilicum ‘Ohře’ plants. Additionally, significant variations were observed between species and cultivation year in herb production of the Ocimum species. Ocimum basilicum ‘Ohře’ displayed the highest fresh and dry herb yield, while O. sanctum exhibited the lowest. Interestingly, all basil species reached their maximum plant height, canopy diameter, and peak biomass (both fresh and dry) production in the second year compared to the initial cultivation year.

3.2. Effect of Water Supply on Essential Oil Production

The effect of water supply on EOC and essential oil yield (EOY) in basil varied by species and year (see

Table 4. Effect of water supply on essential oil production of Ocimum species.

Species	TRT	Essential Oil Content (mL 100 g−1)		Essential Oil Yield (mL plant−1)
		2021	2022	2021	2022
O. basilicum ‘Ohře’	I	1.3 ± 0.2 Ab	1.2 ± 0.1 Ab	0.5 ± 0.1 Ab	0.9 ± 0.0 Ab
	NI	1.3 ± 0.3 Ab	1.2 ± 0.3 Ab	0.4 ± 0.1 Ab	0.5 ± 0.1 Bb
O. basilicum ‘Genovese’	I	0.8 ± 0.1 Ac	0.5 ± 0.2 Bc	0.3 ± 0.0 Ab	0.3 ± 0.1 Ac
	NI	0.9 ± 0.2 Ab	0.7 ± 0.0 Abc	0.3 ± 0.1 Ab	0.2 ± 0.1 Abc
O.× africanum	I	3.2 ± 0.5 Ba	2.8 ± 0.0 Ba	1.9 ± 0.8 Aa	2.0 ± 0.1 Aa
	NI	3.7 ± 0.1 Aa	2.9 ± 0.2 Aa	1.6 ± 0.3 Aa	1.1 ± 0.1 Ba
O. americanum	I	0.7 ± 0.1 Ac	0.7 ± 0.1 Ac	0.3 ± 0.0 Ab	0.3 ± 0.1 Ac
	NI	0.9 ± 0.3 Ab	0.6 ± 0.1 Ac	0.3 ± 0.1 Ab	0.2 ± 0.0 Abc
O. sanctum	I	1.0 ± 0.1 Abc	0.6 ± 0.0 Ac	0.4 ± 0.5 Ab	0.2 ± 0.0 Ac
	NI	1.0 ± 0.0 Ab	0.7 ± 0.0 Abc	0.3 ± 0.0 Bb	0.2 ± 0.0 Ac

Values are expressed as mean ± SD, where TRT represents treatment, I denotes the irrigated condition, and NI denotes the non-irrigated condition. Different letters indicate significantly different groups. Capital letters differentiate between drought stress under fixed species and small letters differentiate between species under fixed drought stress.

below). Accordingly, drought stress increased EOC in O. × africanum in both years and O. basilicum ‘Genovese’ in the second year, but no significant differences were seen in other cases. In contrast, O. sanctum had over a 40% EOC reduction in the first year, and O. basilicum ‘Ohre’ and O.× africanum showed a 45% EOY decrease in the second year. Other species showed no significant EOY changes. Notably, O.× africanum had the highest EO content (3.0 mL 100 g⁻¹ DM) and yield (2.0 mL plant⁻¹).

3.3. Influence of Water Supply on Essential Oil Composition

The examined basil species exhibited varied compositions of essential oils (see

Table 5. Effect of water supply on essential oil composition of sweet basil cultivars.

Components	RT	LRI	O. basilicum ‘Ohře’				O. basilicum ‘Genovese’
			2021		2022		2021		2022
			I	NI	I	NI	I	NI	I	NI
limonene	8.19	1028	0.2	0.3	0.2	0.2	0.3	0.5	0.5	0.4
1,8-cineole	8.38	1034	2.8	3.5	5.2	4.7	6.4	8.9	8.9	10.9
linalool	10.76	1097	43.2	40.7	57.5	40.9	40.8	41.0	47.2	50.2
camphor	12.68	1144	0.5	1.2	0.3	0.3	0.9	0.9	0.8	0.6
α-terpineol	14.55	1189	0.5	0.6	0.6	0.6	1.2	1.6	1.1	1.3
nerol	16.15	1227	0.1	0.1
geraniol	17.29	1252	16.6	10.7	10.0	12.2	0.4	0.2	0.1
iso-bornyl acetate	18.52	1284	0.1	0.5	0.2	0.2	1.7	3.1	1.5	1.7
eugenol	21.44	1361	0.7	1.4	0.8	0.5	2.3	2.5	1.4	1.8
ß-elemene	22.55	1391	4.8	5.4	2.9	3.2	3.3	2.0	1.6	1.4
methyl eugenol	23.31	1411				13.7			0.2	0.2
(E)-ß-caryophyllene	23.68	1419	1.4	1.7	0.6	0.7	0.8	0.4	0.3	0.2
trans-α-bergamotene	24.36	1437	0.1	0.2	2.0	4.2	4.8	6.4	5.4	6.8
α-humulene	25.07	1454	0.9	1.1	0.5	0.5	1.1	0.8	0.7	0.5
germacrene D	26.18	1482	2.7	3.1	2.0	1.9	4.2	2.7	2.7	2.1
α-bulnesene	27.23	1508	2.5	2.9	1.4	1.7	3.0	1.7	1.5	1.4
cis-γ-cadinene	27.80	1515	2.4	2.9	2.1	2.1	3.4	2.5	2.4	2.6
caryophyllene oxide	30.20	1590	0.2	0.2		0.2	0.1	0.1	0.1	0.1
1,10-di-epi-cubenole	31.67	1621	1.4	1.6	0.9	0.9	1.5	1.3	1.0	1.0
tau-cadinol	32.26	1644	9.7	11.2	7.5	7.0	10.0	8.8	7.9	7.9
Others (< 1%)			6.2	7.9	4.1	5.6	9.7	10.8	19.1	8.3
Total identified (%)			97.0	97.2	98.8	97.6	95.9	96.2	97.6	99.4
Monoterpenes			0.9	1.3	1.1	1.1	1.4	2.4	1.9	2.1
Oxygenated monoterpenes			65.3	59.6	75.0	60.4	53.2	58.2	61.3	66.4
Sesquiterpenes			17.5	20.5	12.9	16.6	25.5	20.8	18.0	18.1
Oxygenated sesquiterpenes			12.3	13.9	9.0	8.9	13.3	11.9	10.0	10.4
Phenylpropanes			1.0	1.9	0.8	14.3	2.5	2.9	13.2	2.4

RT is retention time. LRI is the linear retention index relative to C9–C23 n-alkanes on an HP-5MS capillary column. I is for irrigated, and NI is for non-irrigated. Different letters indicate significantly different groups.

and

Table 6. Effect of water supply on essential oil composition of O. americanum, O. × africanum Lour., and O. sanctum.

Components	RT	LRI	O. americanum				O. × africanum Lour.				O. sanctum
			2021		2022		2021		2022		2021		2022
			I	NI	I	NI	I	NI	I	NI	I	NI	I	NI
ß-pinene	6.73	981	0.1				5.4	5.4	5.7	4.0	0.1
limonene	8.19	1028	0.6	0.1	0.1	0.1	16.2	16.1	16.1	14.1	0.2
1,8-cineole	8.38	1034	1.9	0.7	2.0	0.1	35.4	32.1	38.0	36.2	0.3	0.2	0.3	0.2
linalool	10.76	1097	12.5	22.6	22.1	9.4	0.1	0.2	0.6	0.4	0.7	0.5	0.4	0.3
camphor	12.68	1144	1.2	0.2		0.8	14.3	9.9	8.0	24.5	0.1		0.1
α-terpineol	14.55	1189	1.0	0.6	1.0	0.9	4.0	4.8	4.0	2.8
nerol	16.15	1227	11.6	9.3	10.1	10.6
neral	16.58	1238	15.2	10.9	12.8	16.4
geraniol	17.20	1252	2.2	2.5	2.1	1.3
geranial	17.86	1268	20.3	15.3	16.8	21.7
eugenol	21.44	1361	0.1	0.1							49.3	50.3	36.4	43.1
ß-elemene	22.55	1391	1.3	2.1	1.1	1.1	0.1	0.2	0.2	0.1	5.7	6.0	5.6	4.4
methyl eugenol	23.31	1411									7.2	3.8	5.6	6.0
(E)-ß-caryophyllene	23.68	1419	4.5	4.2	2.9	4.0	1.6	2.1	1.8	1.4	26.4	30.6	38.5	31.0
trans-α-bergamotene	24.36	1437	1.7	1.8	1.7	2.5		0.1					0.1
α-humulene	25.07	1454	1.6	1.4	0.6	0.9	6.4	7.6	6.9	5.7	2.0	1.8	2.3	2.1
germacrene D	26.18	1482	1.6	2.3	0.7	1.0	1.1	1.7	1.6	1.2			0.1
α-bulnesene	27.23	1508			0.3								1.1
caryophyllene oxide	30.20	1590	3.0	2.1	5.0	7.0					3.2	2.7	4.7	7.0
tau-cadinol	32.26	1644			1.9	0.1							0.1
Others (<1%)			9.7	15.4	11.5	10.8	14.4	17.9	15.6	14.7	3.7	2.9	3.8	1.0
Total identified (%)			94.4	95.8	94.2	93.1	99.0	98.2	98.5	98.9	98.9	99.0	99.1	95.1
Monoterpenes			1.8	1.5	1.0	0.9	26.0	25.7	24.5	27.1	0.4	0.2	0.1	0.1
Oxygenated Monoterpenes			69.9	65.9	70.0	65.7	59.9	54.4	63.8	67.2	1.3	0.8	0.9	0.7
Sesquiterpenes			16.9	20.2	12.5	16.3	13.1	18.0	14.3	11.6	36.9	41.8	49.9	41.3
Oxygenated Sesquiterpenes			5.7	7.7	9.2	9.4		0.1	0.3	0.1	3.7	3.2	6.2	9.0
Phenylpropanes			0.1	0.5	1.5	0.8					56.6	54.2	42.0	44

RT is retention time. LRI is the linear retention index relative to C9–C23 n-alkanes on an HP-5MS capillary column. I is for irrigated, and NI is for non-irrigated. Different letters indicate significantly different groups.

). On average, more than 40 compounds were identified in all cases except for O. sanctum, where a total of 20 compounds were identified. In O. basilicum cultivars, linalool emerged as the predominant compound, while camphor and 1,8-cineole were the primary constituents in O. × africanum Lour. Ocimum americanum plants demonstrated elevated proportions of nerol, neral (citral-b), and geranial (citral-a). Ocimum sanctum, on the other hand, featured eugenol and trans-ß-caryophyllene as its main compounds. It was observed that the impact of water supply on the volatile oil composition varied, with taxa-specific responses. Notably, irrigation had a positive influence on linalool ratios.

3.4. Effect of Water Supply on Total Phenol Content and Antioxidant Capacity

Drought stress is known to modify secondary compound accumulation in plants including total phenolic content and their antioxidant activities. The effects of these drought-induced changes on Ocimum species were demonstrated through a multiyear experiment conducted in open-field settings. Thus, the two-year open-field study showed significant drought-induced changes in TPC and AOC (

Table 7. Effect of water supply on total phenolic content and antioxidant capacity of Ocimum species.

Species	TRT	Total Phenolic Content (mg GAE g−1 DM)		Antioxidant Capacity (mg AAE g−1 DM)
		2021	2022	2021	2022
O. basilicum ‘Ohře’	I	217.8 ± 21.8 Abc	299.5 ± 80.6 Aa	73.3 ± 10.7 Ab	79.9 ± 6.3 Aa
	NI	200.5 ± 43.4 Bb	247.8 ± 21.3 Aa	70.0 ± 13.2 Ab	76.7 ± 5.0 Aa
O. basilicum ‘Genovese’	I	252.6 ± 24.7 Ab	241.8 ± 54.1 Aab	83.9 ± 9.70 Aab	80.2 ± 12.6 Aa
	NI	232.4 ± 21.8 Bb	200.0 ± 20.7 Bbc	110.9 ± 66.1 Aab	72.1 ± 6.9 Ba
O. × africanum	I	208.0 ± 30.9 Ac	189.1 ± 39.9 Bbc	66.2 ± 16.7 Ab	83.3 ± 7.2 Aa
	NI	187.9 ± 20.7 Ab	217.6 ± 36.5 Aab	79.1 ± 12.9 Aab	62.6 ± 8.6 Bb
O. americanum	I	211.5 ± 35.8 Abc	166.0 ± 5.0 Ac	70.8 ± 7.9 Ab	53.2 ± 6.3 Ab
	NI	189.6 ± 40.5 Ab	157.3 ± 8.7 Ad	72.4 ± 23.7 Aab	48.4 ± 2.2 Bc
O. sanctum	I	295.8 ± 45.4 Aa	143.3 ± 28.6 Bc	100.7 ± 27.7 Aa	45.0 ± 9.1 Ab
	NI	341.4 ± 84.1 Aa	170.4 ± 18.8 Acd	122.9 ± 12.6 Aa	49.6 ± 8.3 Ac

Values are expressed as mean ± SD, where TRT represents treatment, I denotes the irrigated condition, and NI denotes the non-irrigated condition. Different letters indicate significantly different groups. Capital letters differentiate between drought stress under fixed species and small letters differentiate between species under fixed drought stress.

). Depending on the species, soil moisture level, and year of production, the effect could be negative, positive, or non-existent. The sweet basil cultivars ‘Ohře’ and ‘Genovese’ demonstrated a positive response to irrigation in terms of TPC in each consecutive year. Irrigation resulted in a yearly average TPC increase of 31.0 mg GAE g⁻¹ DM (15%) and 34.6 mg GAE g⁻¹ DM (14.8%) for ‘Ohře’ and ‘Genovese’ cultivars, respectively. Conversely, drought stress in the second year led to a 15.1% increase in TPC for O. × africanum and a 19% increase in O. sanctum. However, no significant changes were observed in TPC for O. americanum across all years. A similar pattern was noted for AOC. Consequently, in the initial year of cultivation, soil moisture level had no significant on the AOC of tested basil species. However, in the second year, irrigation affected the AOC of ‘Genovese’, O. × africanum, and O. americanum plants. Nonetheless, no significant changes in AOC were detected for the remaining species.

4. Discussion

Drought stress, a common environmental factor, can affect the physiological, growth, and biochemical responses of medicinal plants and their secondary metabolism [30,31,32]. These two-year experiments on five Ocimum genotypes demonstrated that a shortage of water supply (lack of irrigation) significantly influenced growth, development, and volatile oil accumulation. In response to water supply, basil plants undergo physiological and biochemical changes that can ultimately impact their growth and biomass production. Physiological changes in response to drought stress involve the closure of stomata to reduce water loss through transpiration. While this response conserves water, it also limits the uptake of carbon dioxide for photosynthesis [33]. As a result, non-irrigated Ocimum genotypes had shorter plants and a slender canopy, ultimately reducing both fresh and dry herb yield. Taxa-specific responses were also observed under drought stress conditions. As a result, O. × africanum suffered significant losses in non-irrigated plots. On the other hand, O. sanctum suffered less. The negative effects of drought stress on the growth and herb production of medicinal and aromatic plants are widely recognized and extensively studied phenomena that have been consistently documented by numerous scientific investigations. The constrained growth under drought stress has been previously reported in O. basilicum cultivars [17,34,35], O. × africanum [20], O. americanum [36,37], and O. sanctum [18]. It is important to note that the extent of reduction depends on the severity of the drought stress and the specific basil species. Moreover, a decrease in soil water content to 40% led to a substantial reduction in biomass production across four Lamiaceae species: lemon balm, thyme, peppermint, and marjoram [38].

The synthesis and accumulation of secondary compounds is an intricate and dynamic process governed by various biotic and abiotic factors [16]. Our two-year experiments have demonstrated significant heterogeneity and inconsistency in this process. Consequently, factors such as drought intensity, specific taxa, and year of production play a crucial role in influencing secondary compound production (EOC, EOY, TPC, and AOC). Consistent with our findings, various authors have also noted taxa and drought intensity-specific differences in the EOC of medicinal plants. Consequently, positive impacts of drought stress on EOC were observed in Ocimum basilicum cultivars [19,36,39], Ocimum × africanum [20], Thymus citriodorus [40], and Salvia officinalis [41]. Conversely, negative effects were documented for Salvia officinalis [42], Melissa officinalis L. [43], and Mentha piperita L. [44]. Furthermore, no discernible changes were reported in Ocimum gratissimum L. [45], Hyssopus officinalis [46], and Thymus vulgaris [46]. Our experiments additionally demonstrated the negative effect of drought stress on the EOY of the species. This adverse effect can be attributed to the significant constraint imposed by drought stress on biomass production, a factor directly associated with EOY. The extent of EOY reduction varies based on the specific Ocimum species and the growth environment. Furthermore, alterations in biochemical composition resulting from water availability can also negatively affect the plant’s biological activities.

Moreover, the impact of drought stress on the essential oil composition of the various Ocimum species was heterogeneous as well. In addition, a wider variability in essential oil composition was observed. This diversity highlights the dynamic nature of essential oil composition in basil species, shaped by both genetic factors and cultivation conditions. Similarly, other researchers have reported the presence of the same dominant compounds across various genotypes, though their proportions vary based on factors like geographic location, cultivation practices, and processing methods. Our study also showed variability due to irrigation and production years. Carović-Stanko et al. [47] found linalool to be the major compound in sweet basil genotypes like ‘Genovese’, while Zeljković et al. [13] identified linalool, 1,8-cineole, and geraniol as key compounds in ‘Ohře’. Scholars have also identified limonene, 1,8-cineole, and camphor as the main constituents in O. × africanum [6,18]. Additionally, Ocimum americanum was found to contain citral chemotypes (geranial and neral) and linalool [15,47]. In another study, Raina et al. [48] identified two chemotypes in thirty-two Indian holy basil accessions, high in eugenol and methyl eugenol, with significant levels of β-caryophyllene and β-elemene.

In addition to the observed variation in biomass production and secondary metabolite accumulation resulting from differences in water availability and genotypic variability, substantial disparities in biomass yield were also recorded between the two experimental years (2021 and 2022). These inter-annual variations can most likely be attributed to differences in soil physicochemical properties, as presented in Table 1, and to variations in microclimatic conditions—including air temperature, relative humidity, and precipitation—during the respective experimental periods (Figure 1). Such environmental and edaphic influences on plant growth and metabolite synthesis are well-documented phenomena in the scientific literature. Several studies have demonstrated that fluctuations in soil fertility, temperature, and moisture regimes can markedly alter plant physiological responses, biomass accumulation, and secondary metabolite profiles [15,49,50].

5. Conclusions

In conclusion, this study highlights the significant impact of water supply on biomass production, essential oil yield, and phenol content in basil species. Drought stress reduced plant height, canopy diameter, and biomass yield, while irrigation boosted fresh and dry herb production. The effect of water supply on essential oil production varied by species and year, with some species showing increased oil content under drought, while others, like O. sanctum, experienced reductions. Drought also influenced total phenolic content and antioxidant capacity, with species-specific responses. Based on production-related parameters, O. sanctum was the least affected by drought stress. In contrast, drought significantly altered the productivity of O. basilicum ‘Ohře’; however, its essential oil content, total phenolic content (TPC), and antioxidant capacity (AOC) remained relatively stable. Overall, these findings emphasize the critical role of water management in optimizing basil growth and quality.