Satureja kitaibelii Essential Oil and Extracts: Bioactive Compounds and Pesticide Properties

Abstract

In recent years, the essential oil of Satureja species has been studied as a source of biocidal activity with potential applications in organic farming such as bio-pesticides. The present study aims to determine the potential of essential oil (EO), exudate fraction (EF) and methanolic extract (ME) of Satureja kitaibelii Wierzb. ex Heuff. to inhibit the mycelial growth of phytopathogenic fungi and acetylcholinesterase (AChE). Additionally, ME was tested for inhibitory activity on seed germination and root elongation. Phytochemical analysis was conducted using gas chromatography–mass spectrometry (GC–MS) and thin-layer chromatography (TLC). Biological activities were studied using in vitro methods. p-Cymene, limonene, geraniol, carvacrol and borneol were identified as the main components of EO. Oleanolic and ursolic acid, carvacrol and flavonoid aglycones were determined as the most abundant bioactive compounds of EF, whereas rosmarinic acid and flavonoid glycosides were found in ME. EO reduced the growth of all tested plant pathogens, indicated by 40% to 84% inhibition of mycelial growth (IMG). The growth rates of oomycetes Phytophthora cryptogea Pethybr. & Laff. and Phytophthora nicotianae Breda de Haan were affected to the greatest extent with 84% and 68% IMG. EF showed the most potent AChE inhibitory activity with IC₅₀ value of 0.18 mg/mL. Aqueous solutions of the ME with a concentration above 5 mg/mL were found to inhibit seed germination by more than 90%, whereas a reduction in root elongation was observed at 3 mg/mL. The present study provides for the first time data for the pesticidal properties of EO, EF and ME of S. kitaibelii.

1. Introduction

Pest control by natural remedies is an important characteristic of organic agriculture that is following the world’s desire for an environmentally friendly way of life. Synthetic biocides negatively impact the environment due to their poor biodegradability, which is a precondition for the loss of biodiversity and human and animal health problems [1,2]. Another disadvantage is the development of resistance to synthetic biocides in invertebrates, as observed in over 500 insect and mite species, including cross-resistance (to pesticides from more than one chemical class) [3]. Alternatively, the use of broad-spectrum insecticides leads to a reduction in the populations of beneficial insects [4]. Natural biocides are safer because of a more rapid degradation and the lack of accumulation in the environment [2]. Thus, the need for novel natural sources of biocidal activity is increasing significantly [5].

In recent decades, substantial data have been accumulated on the potential of EOs as pesticidal agents [6,7,8,9]. Essential oil is a complex mixture of volatile compounds—mono- and sesquiterpenes, phenylpropanoids, phenols, etc.—that are localized in the cytoplasm of plant cells, components of various secretory structures—glands, secretory hairs, resin, ducts and secretory cavities. Strong phytotoxic effects and antifungal and insecticidal activities have been previously reported for essential oils [10,11,12,13]. Not all plant species have a high yield of essential oils, and this is a challenge when it comes to the large number of requirements in agriculture.

An exudate is a mixture of lipophilic compounds accumulated on the surface of the plants. Examples of such substances include terpenoids, flavonoid aglycones, lipids and waxes. Such compounds may be of key biological importance. Several studies show their external location to be associated with a protective role against different biotic and abiotic factors, including herbivores and pathogens [14,15,16]. Plant exudate fractions (EFs) are readily and quickly extracted; no special equipment is required. It has been previously reported that EFs of Origanum vulagre ssp. hirtum inhibit the mycelial growth of Phytophthora isolates by about 80% [17]. Furthermore, exudates have been found to exhibit seed germination inhibitory activity and antimicrobial properties [18,19,20].

Compared to EOs and EFs, alcoholic and water extracts often demonstrate weaker biocidal activities [21,22]. Despite that, when satisfactory levels of activity are achieved, extracts do have a high potential for agricultural application because they are obtained in much larger quantities [23].

Species of the genus Satureja are plants traditionally used in the food and pharmaceutical industries. In recent years, the EOs of S. hortensis L. and other species such as S. cuneifolia Ten. have been studied as sources of biocidal activity with possible applications in organic farming such as bio-pesticides. Carvacrol, thymol, p-cymene, γ-terpinene, α-pinene, β-ocimene, camphene and camphor are the dominant EO compounds that are most likely responsible for the pronounced biocidal effects of Satureja spp. [24,25,26]. Great antimicrobial properties have been found for the EO of S. hortensis against 23 bacteria and 15 fungal and yeast species; however, in the same study, the ME did not exhibit any antimicrobial activities [27]. Studies on various Satureja species reported notable inhibitory effects of their EOs on viruses, Leishmania spp., other protozoa, insects, acari, nematodes, helminths, mollusks, etc. [28,29]. In particular, Satureja montana, a notable close relative to S. kitaibelii, possesses insecticidal and nematocidal effects [28].

The genus Satureja is represented in the Bulgarian flora by five native and one introduced species (S. hortensis L.). S. kitaibelii Wierzb. ex Heuff., previously assumed to be a subspecies of S. montana L., is a Balkan endemic plant with wide distribution across the Balkan Peninsula [30]. It is one of the most characteristic species of the petrophyte steppes, which are found in many karst low-hilly areas and canyons in Bulgaria and Eastern Serbia [31,32]. S. kitaibelii is a perennial subshrub, 30–70 cm in height, with a well-established root system. The stems are square in cross section and glabrous or sericeous on two opposite sides, with opposite, coriaceous and narrow lanceolate leaves. The inflorescence is an apical, laxous verticillaster with pink labiate flowers. All aerial parts of the plant emit a strong pleasant aroma [30,33].

Only a few studies have been performed on the biological activities of S. kitaibelii. The ethanolic extract has been found to exhibit significant antioxidant potential and antibacterial properties against Micrococcus luteus and Pseudomonas aeruginosa [34]. The EO of this species has been reported to possess antimicrobial activity against human pathogens such as Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, etc. [35,36,37,38].

Geraniol, p-cymene, limonene and borneol have been determined as the main components of S. kitaibelii’s EO. Their ratio varies greatly according to the origin of the population, developmental stage and the plant parts used for the extraction. Ten chemotypes are distinguished for this species [39,40,41]. The presence of such variability means that researchers need to examine as many origins as possible in the search for the most effective EO profile for the needs of their respective studies. Previous analyses determined phenolic compounds such as rosmarinic acid, clinopodic acid, flavonoids and jasmonic acid derivatives as the main bioactive compounds of the ethanolic extract of S. kitaibelii [34]. Triterpene acids, flavonoid aglycones and phenolic acids have been found in this species’ exudate [42].

The present study aims to determine the potential of essential oil, exudate fraction and methanolic extract of S. kitaibelii to inhibit the mycelial growth of phytopathogenic fungi and acetylcholinesterase. Additionally, the species’ methanolic extract was tested for inhibitory activity on seed germination. Acetylcholinesterase inhibitory activity provides data on the potential repellent (insecticidal) activity.

2. Materials and Methods

2.1. Plant Material

Aerial parts of S. kitaibelii plants in full flowering stage were harvested in October 2022 in the protected area “Kailaka” near the town of Pleven, Middle Danube plain, Bulgaria (Figure 1). The bedrock in this area is limestone, and the altitude is 200 m a.s.l. The sample material was air-dried without exposure to direct sunlight.

2.2. Phytochemical Analysis

2.2.1. Extraction Procedure

Essential oil (EO). EO was obtained by a standard extraction procedure [43]. Air-dried, not ground, aerial parts were subjected to hydrodistillation for 4 h using a conventional glass Clevenger-type apparatus. The EO was dried over anhydrous sodium sulfate and stored at 4 °C until experimental use.

Exudate fraction (EF). Exudate was obtained according to the method introduced by Prof. Eckhart Wollenweber to study exudate flavonoids but without removing substances with a terpenoid structure in the present study [44]. Dry, not ground, aerial parts of S. kitaibelii were dipped into acetone for 4–5 min. Afterwards, the fraction was filtered and evaporated to dryness using a rotary vacuum evaporator at 40 °C. The dry EF was stored at 4 °C before analyses.

Methanolic extract (ME). Air-dried and powdered aerial parts were extracted with methanol by a classic process of maceration for 24 h at room temperature. After filtration, the organic solvent was evaporated, and the dry extract was stored at 4 °C before analyses.

2.2.2. Derivatization of Methanolic Extract and Exudate Fraction

The EF and ME were derivatized before GC–MS analysis as described by Berkov et al. [45]. Fifty milligrams of methanolic extract and exudate fraction were silylated with 50 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in 50 μL of pyridine for 2 h at 70 °C. Compounds of the ME and EF were identified as TMS derivatives using NIST 05 database (NIST Mass Spectral Database, PC-Version 5.0, 2005), Golm Metabolome Database and home-made MS databases. Amounts of all identified compounds are presented as response ratios calculated for each compound relative to the internal standard (3,5-dichloro-4-hydroxybenzoic acid (1 mg/mL)) using the calculated areas for both components. The internal standard was added (50 μL) at the beginning of the extraction process.

2.2.3. Gas Chromatography–Mass Spectrometry

The GC–MS spectra were recorded on a Thermo Scientific Focus GC coupled with Thermo Scientific (Waltham, MA, USA) DSQ mass detector operating in EI mode at 70 eV. ADB-5MS column (30 m × 0.25 mm × 0.25 mm) was used.

The chromatographic conditions used for the EO were as follows: column temperature was 60 °C for 10 min, then programmed at the rate of 3 °C min⁻¹ to 200 °C, and finally, held isothermally for 10 min. The injector temperature was 220 °C. The flow rate of carrier gas (Helium) was 1 mL min⁻¹. The split ratio was 1:50, and 1 μL of the solution was injected. Significant quadrupole MS operating parameters: interface temperature 240 °C; scan mass range of 40 to 400 m/z at a sampling rate of 1.0 scan s⁻¹.

The chromatographic conditions used for the EF and ME were as follows: 100–180 °C at 15 °C min⁻¹, 180–300 °C at 5 °C min⁻¹ and 10 min hold at 300 °C. The injector temperature was 250 °C. The flow rate of the carrier gas (Helium) was 0.8 mL min⁻¹. The split ratio was 1:10 and 1 μL of the solution was injected.

The individual components were identified by their retention times (RTs), retention indices (RIs), relative to C5-C28 n-alkanes, referring to known compounds from the literature, and also by comparison with those of NIST 14 Library [46]. The percentage of composition of the essential oil was computed from the GC peaks areas.

2.2.4. Thin-Layer Chromatography

TLC analyses were performed on 20 × 10 cm silica gel Kiselgel 60 F254 plates (Merck, Rahway, NJ, USA) and DC Alufolien Polyamide 11 F254 (5555) plates (Merck). Five microliters of methanolic extract were applied to the TLC plates. The mobile phases used on silica gel were as follows: S₁ (toluene–dioxin–acetic acid = 90:25:4), S₂ (ethyl acetate–formic acid–acetic acid–methylethylketone–methanol–water = 50:7:3:30:10) and S₃ (ethyl acetate–acetic acid–water = 20:2:1). The mobile phase used on polyamide was S₄ (toluene–methylethylketone–methanol = 60:30:15). Chromatograms were observed under UV light at 336 nm before and after spraying with “Naturstoffreagenz A”, 1% solution of diphenylboric acid ethanolamine complex in methanol. Identification of compounds was carried out by co-chromatography of the EF and ME with authentic standards and comparing their retardation factors (R_f) and fluorescence emission under UV before and after spraying with reagent.

2.3. Antifungal/Anti-Oomycete Bioassay

The three S. kitaibelii samples (EO, EF, ME) were tested against three fungal and two oomycete species to determine their pesticidal properties. These isolates were previously obtained from different ecosystems in Bulgaria, except for Phytophthora nicotianae originating from potted ornamental plants. Their identification was based on morphology and ITS enzyme restriction for P. cryptogea as well as morphology and ITS sequences for the rest. Namely, these are Alternaria alternata (Fr.) Keissl., 1912 (NCBI GenBank accession number PQ803669), Botrytis cinerea Pers. (PQ345538), Fusarium oxysporum Schltdl. (PQ345539), and Phytophthora nicotianae (PQ460002). The two Phytophthora spp. were maintained on V8 Agar Media (16 g agar, 100 mL Campbell’s (Camden, NJ, USA) V8 Juice, and 900 mL distilled water), and fungal isolates on PDA (BD Difco™, Franklin Lakes, NJ, USA). In the conducted bioassay, the antifungal properties of EO, EF and ME derived from S. kitaibelii were assessed through a modified agar disk-diffusion technique [47]. A concentration of 100 mg/mL was achieved for the EF and ME by dissolving the dry samples in DMSO and methanol, respectively. The bioassay consisted of six variants for each isolate: EO, EF, ME, two control treatments with pure solvents (100% DMSO and methanol) and control without treatment. Three replicates were conducted for each variant. One day prior to the introduction of S. kitaibelii samples, the isolates were re-cultured in 9 mm Petri dishes, each containing 20 mL of a suitable nutrient agar medium. To achieve simultaneous growth, they were incubated overnight. The following day, 2 × 15 µL of either EF or ME were dripped directly onto the agar medium in the Petri dish, equidistant from the center. Similarly, the EO was administered using a volume of 2 × 2 µL. The concentrations were selected based on results from previous similar studies [17,48]. The Petri dishes were cultivated in a climatic chamber in darkness at 25 °C. The results were documented when the mycelial colonies in the controls reached the periphery of the Petri dishes. Photographs (Canon, Tokyo, Japan EOS 4000D) of all mycelial colonies were taken, and their mycelial growth areas were measured using the image analysis program ImageJ 1. 54g [49]. Based on the collected information (mean mycelial growth area for each treatment/isolate variant), the inhibition percentage was determined [50] using the following equation:

$$\%\mathrm{I}\mathrm{M}\mathrm{G}=100{\left(\mathrm{C}-\mathrm{T}\right)\mathrm{C}}^{-1},$$

where % IMG is the percentage of inhibition of mycelial growth, C is the area of the fungal colony without treatment (control) and T is the area of the treated fungal colony.

2.4. Acetylcholinesterase (AChE) Inhibition Assay

The microplate assay used for measuring AChE inhibitory activity was performed in 96-well plates using a modified method of Ellman et al. [51], as described by López et al. [52]. Acetylthiocholine iodide (ATCI) in solution with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a substrate for the acetylcholinesterase from Electrophorus electricus (Sigma-Aldrich, Darmstadt, Germany). S. kitaibelii sample solutions with concentrations between 0.08 and 5 mg/mL were tested. Fifty microliters of AChE (0.25 U/mL) dissolved in phosphate buffer (8 mM K₂HPO₄, 2.3 mM NaH₂PO₄, 0.15 M NaCl, pH 7.5) and 50 μL of the samples dissolved in the same buffer were added to the wells. The plates were incubated for 30 min at room temperature before the addition of 100 μL of the substrate solution (0.04 M Na₂HPO₄, 0.2 mM DTNB, 0.24 mM ATCI, pH 7.5). The absorbances were read in a microplate reader (BIOBASE, ELISA-EL10A, Jinan, China) at 405 nm after 5 min. Enzyme activity was calculated as inhibition percentage compared to an assay using a buffer instead of an inhibitor. Galanthamine was used as a positive control. The AChE inhibitory data were then analyzed with the software package Prism 3 (Graph Pad Inc., San Diego, CA, USA). The IC₅₀ values were measured in triplicate, and the results are presented as means ± SD.

2.5. Seed Germination Inhibition Bioassay

A hundred seeds of Lolium perenne L. (a common crop weed) were placed per Petri dish on filter papers moistened with the tested solutions. The methanolic extract, in a water–methanol mixture (99.5:0.5), was assayed at a concentration of 1, 3, 5 and 8 mg/mL. The control consisted only of the water–methanol mixture. The samples were incubated at room temperature for seven days. At the end of the incubation period, the rate of germination inhibition and root length inhibition were calculated using the following equations [53]:

$$\mathrm{G}\mathrm{I}=\left[\right(\mathrm{G}\mathrm{C}-\mathrm{T}\mathrm{G})÷\mathrm{G}\mathrm{C}]\times 100,$$

where GI is the rate of germination inhibition (%); GC is the germination rate of seeds treated with control solutions; TG is the germination rate of seeds treated with ME solution;

$$\mathrm{R}\mathrm{E}\mathrm{I}=\left[\right(\mathrm{R}\mathrm{E}\mathrm{C}-\mathrm{T}\mathrm{R}\mathrm{E})÷\mathrm{R}\mathrm{E}\mathrm{C}]\times 100$$

where REI is the rate of root elongation inhibition (%); REC is the root length of the control; TRE is the root length of the treated plant.

The lengths of the roots were measured using a ruler in millimeters.

Seed germination assays were performed in three independent experiments.

2.6. Data Analysis

Statistical analyses were performed using Microsoft Excel software. Results are presented as means with standard deviation (SD) and standard error (SE). The AChE inhibitory data were analyzed using the software package Prism 3 (Graph Pad Inc., San Diego, CA, USA). The statistical significance of the differences between mean values for the treated and untreated variants from antifungal/anti-oomycete bioassay tests was determined by a one-way ANOVA test, accepting p ≤ 0.05 to be significant.

3. Results

3.1. Phytochemical Analysis

3.1.1. Essential Oil

The EO was obtained with a yield of 0.1% (w/v). The chemical composition of the EO was determined using GC/MS. p-Cymene (23.94%), limonene (8.57%), geraniol (8.63%), carvacrol (7.22%) and endo-borneol (7.15%) were identified as the main components (

Table 1. Identified compounds in EO of the aerial parts of S. kitaibelii.

RT	RI	Compound	Area, %
16.04	927	β-Thujene	0.60 ± 0.1
16.41	935	α-Pinene	3.68 ± 0.4
16.76	952	Camphene	1.20 ± 0.7
18.14	991	β-Myrcene	0.96 ± 0.1
18.50	1017	α-Terpinene	0.38 ± 0.1
19.71	1022	p-Cymene	23.94 ± 1.8
19.82	1031	D-Limonene	8.57 ± 0.9
20.00	1032	Eucalyptol	0.84 ± 0.2
20.14	1042	β-Ocimene	0.67 ± 0.1
20.77	1060	γ-Terpinene	2.70 ± 0.5
21.28	1070	Sabinene hydrate	1.67 ± 0.7
21.98	1097	Linalool	0.53 ± 0.1
24.89	1166	endo-Borneol	7.15 ± 0.6
25.02	1177	Terpinen-4-ol	2.67 ± 0.4
26.51	1244	Carvacrol methyl ether	6.97 ± 0.5
26.75	1253	Geraniol	8.63 ± 0.4
28.22	1299	Carvacrol	7.22 ± 0.6
31.01	1388	(–)-β-Bourbonene	1.45 ± 0.1
32.08	1419	Caryophyllene	1.29 ± 0.3
33.59	1432	β-Copaene	0.53 ± 0.1
33.84	1509	β-Bisabolene	1.28 ± 0.2
35.58	1578	(+)-Spathulenol	1.20 ± 0.2
35.71	1581	Caryophyllene oxide	1.57 ± 0.1

).

3.1.2. Exudate Fraction

The EF is composed mainly of lipophilic compounds dissolved on the surface of the plant tissue. The EF from S. kitaibelii was obtained with a yield of 0.7% (w/w). Ursolic and oleanolic acids, carvacrol and n-alkanes were found as the most abundant components (

Table 2. Components of S. kitaibelii exudate fraction and methanolic extract as determined by GC–MS.

RI	Compound	Amount, μg *
		Exudate Fraction	Methanolic Extract
1220	Borneol	163.9 ± 26	16.5 ± 3
1249	Benzoic Acid	–	2.9 ± 0.7
1289	Glycerol	479.9 ± 31	755.3 ± 24
1321	Succinic acid	19.0 ± 8	79.0 ± 11
1339	Carvacrol	634.7 ± 17	259.8 ± 19
1396	Hydroquinone	3.1 ± 0.9	–
1497	Malic acid	33.7 ± 4	22.1 ± 6
1624	Ribofuranose	–	156.6 ± 10
1635	4-Hydroxybenzoic acid	11.4 ± 0.5	5.2 ± 2
1776	Vanillic acid	1.1 ± 0.6	–
1803	Fructose 1	273.1 ± 33	661.0 ± 25
1811	Fructose 2	450.7 ± 45	1510.4 ± 67
1835	Protocatechuic acid	10.2 ± 3	10.4 ± 8
1842	Quinic acid	18.6 ± 5	167.4 ± 12
1855	Syringic acid	–	8.5 ± 0.9
1890	D-Glucopyranose	130.5 ± 6	1122.2 ± 68
1946	4-Hydroxycinnamic acid	4.01 ± 1.4	2.5 ± 0.7
1996	Methyl caffeate	10.6 ± 1.2	–
2040	Hexadecanoic acid	646.4 ± 49	134.6 ± 28
2080	Catechollactate	16.4 ± 2.9	222.6 ± 31
2104	Ferulic acid	15.1 ± 3.2	0.8 ± 0.1
2129	Myo-Inositol	43.4 ± 9	2876.9 ± 87
2155	Caffeic acid	16.1 ± 2.4	56.1 ± 16
2212	Octadienoic acid	161.5 ± 11	170.9 ± 21
2218	Octatrienoic acid	302.4 ± 24	241.86 ± 28
2246	Stearic acid	40.7 ± 3	29.9 ± 12
2628	Sucrose	410.0 ± 32	6597.0 ± 167
2838	Tetracosanoic acid	69.4 ± 10	–
2872	Naringenin	74.7 ± 12	–
2900	Nonacosane C29H60	486.2 ± 56	–
2942	Taxifolin	8.5 ± 2	–
3100	Hentriacontane C31H64	340.9 ± 44	–
3122	Methylated flavone	50.2 ± 10	–
3194	β-Sitosterol	137.0 ± 13	96.2 ± 33
3335	β-Amyrin	16.2 ± 8	3.8 ± 2
3455	Rosmarinic acid	7.5 ± 2	469.2 ± 43
3540	Uvaol	107.3 ± 17	20.3 ± 8
3570	Oleanolic acid	3505.55 ± 204	128.6 ± 12
3580	Betulinic acid	67.5 ± 21	2.3 ± 0.9
3620	Ursolic acid	4457.9 ± 267	244.5 ± 21
3632	Micromeric acid	47.3 ± 12	–

* amounts (μg) are the mean of three independent experiments and represent the response ratios calculated for each compound relative to the internal standard

). Phenolic acids, monoterpenoids, flavonoids, sterols, fatty acids and organic acid were also identified. Flavonoid aglycones such as scutellarein-6,7,8-trimethyl ether (xanthomicrol) and scutellarein-6,7-dimethyl ether were determined by TLC (

Table 3. Phenolic compounds identified in the exudate fraction and methanolic extract using TLC.

Compounds	Rf Values in Different TLC Conditions *
	SG, S1	SG, S2	SG, S3	PA, S4
Xanthomicrol	0.62			0.95
Scutellarein 6 methyl ether	0.50			0.71
Rosmarinic acid		0.98	0.73
Luteolin-7-glucoronide		0.50
Rutin		0.31	0.06

* SG and PA correspond to silica gel and polyamide; S_1–4 are the mobile phases used as described in Section 2.2.4.

).

3.1.3. Methanolic Extract

The ME was obtained with a yield of 8.5% (w/w). Its composition was examined using GC–MS and TLC. Some of the compounds reported in the EF (carvacrol, borneol, oleanolic acid, ursolic acid, sterols, terpenes) were also found in the ME, but here, they are in much lower quantities. The GC–MS analysis revealed sucrose, fructose and glucose as the most abundant compounds (Table 2). TLC screening with authentic flavonoid compounds showed the presence of flavonoid glycosides (rutin and luteolin-7-glucuronide) (Table 3). Additionally, rosmarinic acid was found to be the most abundant phenolic acid during TLC with S₃ mobile phase.

3.2. Fungal and Oomycete Growth Inhibition Properties

Mycelial colonies in the controls reached the periphery of the Petri dishes in 4–7 days depending on the species. The results from data analysis showed a lack of significant difference between the three types of controls and significant differences (p < 0.05) in the mycelial growth rate inhibition in the EO, EF and ME variants with the Fungi. EF and ME of S. kitaibelii exerted no inhibitory effect on the mycelial growth rate of P. cryptogea and P. nicotianae at the administered doses of 2 × 15 μL of 100 mg/mL stock solution. Contrastingly, the mycelial growth of two of the fungal pathogens investigated was suppressed, albeit to a low degree. For B. cinerea, an IMG between 55% (ME) and 59% (EF) was recorded, and for F. oxysporum, the IMG was between 37% (EF) and 39% (ME) (Figure 1 and Figure 2).

Generally, the essential oil reduced the growth of all tested pathogens, indicated by an IMG between 40 and 84%. Phytophthora spp. were affected the strongest—84% IMG for P. cryptogea and 68% IMG for P. nicotianae. Out of the three fungal species, F. oxysporum and A. alternata were largely inhibited—65% and 50% IMG, respectively. The growth of B. cinerea was reduced by 40% (Figure 2 and Figure 3).

3.3. Acetylcholinesterase (AChE) Inhibitory Activity

EO, EF and ME from S. kitaibelii were studied for inhibitory effects on AChE by an in vitro assay. EO and ME exhibited similar AChE activity with IC₅₀ values of 3.68 ± 0.17 and 3.76 ± 0.25 mg/mL, respectively. EF exhibited the most potent inhibition on AChE with IC₅₀ value of 0.18 ± 0.03 mg/mL. Galanthamine (positive control) achieved an IC₅₀ value of 0.35 ± 0.01 μg/mL (1.22 ± 0.04 μM).

3.4. Seed Germination Inhibition Bioassay

The inhibitory activity of the ME from S. kitaibelii on L. perenne seed germination and root elongation was studied in the concentration range of 1–8 mg/mL. Aqueous solutions of the extract with a concentration of 5 and 8 mg/mL were found to inhibit seed germination and root growth by more than 90% (

Table 4. Inhibition of seed germination and root growth of L. perenne by methanolic extract (ME) of S. kitaibelii.

ME Concentration, mg/mL	Inhibition of Seed Germination, %	Inhibition of Root Growth, %
1	13.4 ± 9	1 ± 1
3	21.8 ± 8	56 ± 6
5	95.8 ± 3	97.5 ± 1
8	99.4 ± 1	100 ± 0

). A substantial reduction in root elongation was also observed at 3 mg/mL.

4. Discussion

4.1. Phytochemical Constituents

The main component of the EO of the studied S. kitaibelii population was determined as p-cymene. Limonene, borneol and carvacrol were found as the next most abundant compounds. The established EO profile corresponds to the previously reported data for the chemical composition of this species [41,54,55] and refers to the p-cymene/limonene chemotype [40]. A variety of biological activities have been already published for the monoterpenes identified in the EO. Antimicrobial, antiparasitic, antiviral, antitumor, anti-inflammatory, antinociceptive, neuroprotective and other activities have been established for p-cymene [56,57]. A lot of pharmacological activities have been reported for the remaining well-represented monoterpenes in the EO—borneol, carvacrol and limonene—defining them as strongly bioactive molecules [58,59]. It is important to emphasize that carvacrol is a lead molecule for pest control [60,61]. In the present study of the EO profile of S. kitaibelii, however, carvacrol content is limited to 7.22% only.

The EF consisting of compounds located on the surface of plant tissues is rich in secondary metabolites with allelopathic potential [14,15]. Ursolic and oleanolic acids were found as the main bioactive compounds in the EF of S. kitaibelii. Various activities have been reported for these acids, but in the context of the present study, the anticholinesterase and antimicrobial properties are of importance [62,63,64]. The composition of S. kitaibelii EF we described follows previously reported data [41,42].

S. kitaibelii’s ME contained mainly polar primary and secondary metabolites. The established metabolite profile is coherent with previous research [34,65]. Rosmarinic and other phenolic acids, as well as flavonoid glycosides, were found as substances with known biological activities [66].

4.2. Fungal and Oomycete Growth Inhibition Properties

Fungi and fungal-like organisms such as oomycetes are causative agents of one of the most devastating diseases in plants. Equally severe harm is caused by such plant pathogens during post-harvest, affecting between 25% and 50% of the total production.

It has been found that essential oils rich in carvacrol demonstrate the most extensive and potent antifungal effects at minimal active doses (0.05–5 µg/mL) [20]. Plant extracts containing caffeic acid and rosmarinic acid inhibited zoospore germination of Phytophthora spp. [67]. The key components identified in the EO from S. kitaibelii (p-cymene, limonene, geraniol, carvacrol, borneol) are known to be effective antimicrobial agents, which is why this species keeps attracting the attention of researchers in medicine, food technology and agriculture. Several studies demonstrated the lack of antifungal activity of p-cymene against pathogenic fungi responsible for human diseases, such as Rhizopus oryzae and Aspergillus niger. However, the evaluation of essential oils rich in this monoterpene against Aspergillus flavus, Saccharomyces cerevisiae and A. niger has shown promising results [56]. Jian et al. demonstrated the ability of limonene to cause significant damage to the mycelium and conidia of Fusarium graminearum [68], revealing the potential of a limonene-formulated product as an alternative to synthetic fungicides. The essential oil obtained from Cinnamomum camphora chvar., rich in endo-borneol, demonstrated significant suppressive effects on five Fusarium species responsible for potato dry rot [69].

As shown in our results, the EO from S. kitaibelii is rich in bioactive components with antifungal and anti-oomycete properties and could find application in biological plant protection.

4.3. Acetylcholinesterase (AChE) Inhibitory Activity

The acetylcholinesterase inhibitory activity test was conducted to assess the possible presence of insecticidal potential because the mechanism of action of organophosphorus insecticides is acetylcholinesterase inhibition [70]. Several previously published articles highlight some of the main components of the EO, EF and ME from S. kitaibelii as AChE inhibitors: oleanolic and ursolic acids [63,64], p-cymene [71] and rosmarinic acid [72,73]. Rosmarinic acid has also been reported as a potent insecticide against important pests like Acyrthosiphon pisum [74]. Since oleanolic and ursolic acids are the most abundant components of the EF and with carvacrol also being present, it is not surprising that this fraction achieved the best AChE inhibition out of all three samples. Regarding the EO and MF, bioactive compounds such as p-cymene, rosmarinic acid and carvacrol appear to be in insufficient quantities in order to demonstrate good results in AChE inhibition.

The IC₅₀ value determined for the EF (0.18 ± 0.03 mg/mL) suggests a good to strong AChE inhibition considering the fact that this is not a pure compound. This value is approximately 500 times larger than that of galanthamine (positive control). An assay including the purified forms of some putatively bioactive components could showcase the most important ones for AChE inhibition.

The IC₅₀ values for the EO and ME from S. kitaibelii are approximately 10 000 times larger than that of galanthamine (positive control), meaning these samples are practically inactive towards the enzyme.

Similar results for the AChE inhibitory potential of extracts and essential oils from the closely related species Satureja montana have been previously reported. Stronger AChE inhibition has been observed probably due to much larger quantities of carvacrol and the presence of its isomer thymol in the samples from S. montana [75,76,77].

4.4. Seed Germination Inhibition Bioassay

When working with alkaloid-bearing plants, the suitable concentration at which the species are screened is 1 mg/mL, while for non-alkaloid-containing plant species, the concentration at which the screening studies are performed is usually higher [78,79,80]. Seed germination inhibition activity was tested within the concentration range of 1–8 mg/mL for the ME of S. kitaibelii. This range is consistent with similar experiments conducted by other research groups [10,81]. In this assay, only ME was used due to low yields of EO and EF.

The achieved inhibition of seed germination by the ME at concentrations 5 and 8 mg/mL is good and promising compared to data already reported in the literature. Caffeic acid and its derivatives showed an inhibitory effect on the growth and germination of Lantana indica seeds [66]. Hernández and Munné-Bosch [82] showed that naringenin, a compound found in our study of the EF, is a strong seed germination inhibitor. Muñoz et al., 2020 considered carvacrol to be a good candidate for bioherbicide formulations [61]. These compounds are present in the ME from S. kitaibelii and could contribute to the phytotoxic properties observed in our study.

Nerium oleander L. flower extract suppressed the growth of Lolium multiflorum Lam. (Italian ryegrass) at 40 g L⁻¹ [78], which is a much higher concentration. In a screening study including six plant extracts, the one from Tamarix mannifera Ehrenb. ex Bunge completely inhibited the seed germination and seedling growth of Phalaris minor Retz., also at 40 g L⁻¹ [79]. Other plant extracts at this concentration showed weaker inhibition. The application of Cardus cardunculus (L.) Baill. crude extract at a concentration of 10 g L⁻¹ has led to the inhibition of roots and hypocotyl growth by 97% and 91%, respectively [80].

5. Conclusions

The present study was aimed at determining the pesticidal potential of the essential oil, exudate fraction and methanolic extract of Satureja kitaibelii. Results showed that the EO has fungicide potential, inhibiting the growth of all studied phytopathogens. Of importance is its inhibitory activity against Phytophthora spp. The ME exhibits weed suppression by inhibiting the seed germination of ryegrass. The EF displayed inhibitory activity against acetylcholinesterase, which is a good base for further analysis of its insecticidal potential. Our results showed that choosing the right approach (extraction method) is crucial to obtaining an extract or a fraction rich in substances with a particular biological activity. Here, we describe for the first time the pesticidal capacity of S. kitaibelii and provide a good direction for further studies.