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Article

Artemisia santonicum L. and Artemisia lerchiana Web. Essential Oils and Exudates as Sources of Compounds with Pesticidal Action

by
Milena Nikolova
1,*,
Aneta Lyubenova
2,
Elina Yankova-Tsvetkova
1,
Borislav Georgiev
1,
Strahil Berkov
1,
Ina Aneva
1 and
Antoaneta Trendafilova
3
1
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
Department of Agrobiotechnology, AgroBioInstitute, Agricultural Academy, 1164 Sofia, Bulgaria
3
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Plants 2023, 12(19), 3491; https://doi.org/10.3390/plants12193491
Submission received: 29 August 2023 / Revised: 19 September 2023 / Accepted: 5 October 2023 / Published: 6 October 2023
(This article belongs to the Special Issue Chemical Characteristics and Bioactivity of Plant Natural Products)
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Versions Notes

Abstract

:
The application of natural products for pest control is important in modern farming. In the present study, Artemisia santonicum L. and Artemisia lerchiana Weber essential oil and exudate profiles were determined, and their potential as inhibitors of seed germination, acetylcholinesterase, and phytopathogenic mycelium growth were evaluated. Essential oils (EO) were obtained via hydrodistillation and exudates (AE) by washing aerial parts of the species with acetone. EO and AE’s composition was identified using GC/MS. Eucalyptol (1,8-cineole) and camphor were found to be the main components of A. lerchiana EO, while β-pinene, trans-pinocarveol, α-pinene, α-terpineol, and spathulenol were established as major compounds of A. santonicum EO. Strong inhibition on Lolium perenne seed germination was found at 2 µL/mL and 5 mg/mL using aqueous solutions of EO and AE, respectively. An inhibitory effect on acetylcholinesterase was established, with an IC50 value of 64.42 and 14.60 μg/mL for EO and 0.961, >1 mg/mL for the AE of A. lerchiana and A. santonicum, respectively. The low inhibition on the mycelium growth of studied phytopathogenic fungi was established by applying 2 µL of EO and 15 µL of 100 mg/mL of AE, with the exception of A. lerchiana AE against Botrytis cinerea. These results show that the studied EO and AE exhibited strong phytotoxic and AChE inhibitory activities, providing new data for these species.

1. Introduction

Environmental pollution caused by synthetic pesticides, as well as pest resistance to them, requires a continuous search for new natural sources of biocidal activity [1,2,3,4,5]. In recent decades, the interest in studying essential oils and plant extracts as new natural remedies for pest control has increased [4,6,7,8,9]. A lot of research points out essential oils as promising agents for the control of crop pests—weeds, insects, and phytopathogens [10,11,12,13,14]. As exudates are composed of substances located on the surface of plant tissues, they have chemoecological importance as protectors against pests, diseases, and UV radiation. Available data point out that exudates possess plant-growing inhibitory, antimicrobial, antiplasmodial, antifeedant, and insecticidal activities [15,16,17,18,19,20].
The Asteraceae family has been found to be a good source of compounds with biocidal activity [21,22,23,24,25]. Flowers from Tanacetum cinerariifolium (Trevir.) Sch. Bip. (syn. Chrysanthemum cinerariaefolium (Trevir.) Vis.; Pyrethrum cinerariifolium Trevir.) are the best-known example of commercially available plant insecticide [26]. Phytotoxic, antifungal, and insecticidal activities have been reported for essential oils and extracts from several Artemisia species [24,25,27,28]. Based on these facts, we directed our interest to study the pesticide properties of two closely related species with limited distribution in Bulgaria—Artemisia santonicum L. and Artemisia lerchiana Weber. The essential oils of these species are of various origins and have been studied before [29,30,31,32,33], while their biological activities have been insufficiently studied. Insecticidal properties against Sitophilus granarius adults and antifungal activity against Aspergillus sp., Penicillium sp., and Trichoderma viride, as well as antibacterial activity, have been reported for Artemisia santonicum EO [33,34,35]. The antifungal activity of Artemisia lerchiana EO on Sclerotinia sclerotiorum has been reported [36]. Data concerning the composition of exudates on both species have been reported in our previous work [37]. Still, regarding biological action, there is only information on their antibacterial activity [38].
The aim of the present study is to determine A. santonicum and A. lerchiana essential oil and exudate profiles and to evaluate their potential as inhibitors of seed germination, acetylcholinesterase, and the mycelium growth of phytopathogenic fungi. The inhibition of acetylcholinesterase exists at the base of the mechanism of action of highly toxic organophosphate insecticides [39,40]. We consider this effect as a measure of the insecticidal activity of the investigated fractions and an important indicator of their effectiveness.

2. Results

2.1. Phytochemical Analysis

The GC/MS analysis of essential oils obtained from the aerial parts of Artemisia santonicum and A. lerchiana led to the identification of 83 compounds accounting for 98.1% and 97.1% of the total oil, respectively (Table 1). Out of them, 79 compounds were unambiguously identified, and 4 were determined as 3 sesquiterpene alcohols and a sesquiterpene lactone based on their mass-spectral fragmentation. Oxygenated monoterpenes (62.8%) were the predominant class of compounds in A. lerchiana essential oil, followed by oxygenated sesquiterpenes (24.4%) and monoterpene hydrocarbons (8.2%). A. santonicum essential oil contained almost equal amounts of monoterpene hydrocarbons (34.0%) and oxygenated monoterpenes (35.9%), followed by O-containing sesquiterpenes (19.5%). Both essential oils were poor in sesquiterpene hydrocarbons. The essential oils differed significantly in the content of individual components too. Thus, eucalyptol (22.1%) and camphor (14.0%) were the main components of A. lerchiana essential oil, while β-pinene (15.2%), trans-pinocarveol (9.6%), α-pinene (9.3%), α-terpineol (9.2%), and spathulenol (8.5%) were the major compounds of A. santonicum essential oil.
The identified compounds of exudates using GC/MS are presented in Table 2. Primary and secondary metabolites were established. Monoterpenes, phenolic acids, sugar alcohols, triterpenes, and flavonoid aglycones were determined as the main bioactive compounds. Acetone exudate of A. lerchiana was found to be rich in monoterpenes—eucalyptol (6.45%), camphor (4.58%), borneol (3.69%), carvacrol (1.86%) and ascaridole (1.49%). 10-Undecenoic acid (11.45%) was identified as a fatty acid in large amounts. The exudate profile of A. santonicum organic acids—malic (5.72%) and fumaric (4.25%)—were found to be the most abundant. Pinitol (3.61%), erythronic acid (2.45%), hydroquinone (1.90%), capillene (1.32%), cinnamic acid (0.95%), and chlorogenic acid (0.68%) were also identified as the main bioactive compounds in the exudate.

2.2. Inhibition of Seed Germination

The inhibitory activity of aqueous solutions of A. lerchiana and A. santonicum EO and AE on the germination of Lolium perenne seeds was assessed. The results are presented in Figure 1. The strong inhibition of seed germination was achieved by applying aqueous solutions of essential oils at a 2 µL/mL concentration. Artemisia lerchiana EO showed slightly higher phytotoxicity compared to that of A. santonicum. Applying exudates at 5 mg/mL determined the strong inhibition of seed germination. Again, A. lerchiana AE exhibited stronger phytotoxicity.

2.3. Inhibition of Acetylcholinesterase (AChE)

The essential oils and exudates of the studied species were evaluated for their AchE inhibitory activity. The results are shown in Table 3. None of the tested samples were proven to be a stronger AchE inhibitor than galanthamine. Both Eos exhibited potent enzyme inhibition, but A. santonicum EO showed a lower IC50 value, indicating a higher AchE inhibitory activity than A. lerchiana. The exudates of both species showed similar activity, with IC50 values much higher than Eos. Therefore, Aes can be considered weak AchE inhibitors.

2.4. Inhibition of Phytopathogenic Mycelium Growth

The inhibitory effect of essential oils and acetone exudates of A. lerchiana and A. santonicum on the mycelium growth of Phytophthora cryptogea, Botrytis cinerea, and Fusarium oxysporum was assessed. The results are presented in Figure 2. The slightly inhibiting effect on F. oxysporum and P. cryptogea mycelium growth (34% and 25% IMG, respectively) was established for A. santonicum EO, while an opposite effect toward B. cinerea was found. These fungi formed a larger and denser colony than the control (Figure 2). The essential oil of the second plant species, A. lerchiana, had a stronger inhibitory activity on F. oxysporum (33% IMG) and P. cryptogea (31% IMG) mycelium growth than A. santonicum EO. The stimulating effect on B. cinerea growth was found once again with A. lerchiana EO, even more pronounced.
The acetone exudate of A. Lerchiana had the strongest inhibitory effect on the growth of colonies of B. cinerea (60% IMG) and was weaker on the growth of F. oxyisporum and P. cryptogea (20% IMG for both).
The A. santonicum AE exhibited a minor inhibitory effect on F. oxysporum and B. cinerea growth (21% and 28% IMG, respectively), whereas in the P. cryptogea variant, the stimulating effect on mycelium growth was observed again, but much weaker compared to the Eos against B. cinerea.
The studied A. santonicum and A. lerchiana EO and AE showed low antifungal activity against the tested phytopathogenic isolates at the applied concentrations of 2 µL and 15 µL from 100 mg/mL for EO and AE, respectively.

3. Discussion

3.1. Phytochemical Analysis

The established composition of the essential oil A. lerchiana was in accordance with previously reported data for Bulgarian, Romanian, and Russian populations of this species [29,30,31]. The literature survey on the essential oil profile of A. santonicum showed significant variability in major components depending on the origin. Thus, eucalyptol (1,8-cineole), chrysanthenone, and cis-thujone were reported as the main components in the oil from a Serbian population [41]. At the same time, camphor was found to be the principal compound in the plant material from Turkish populations [32,35,42]. The main components of the A. santonicum profile established in the present study (β-pinene, trans-pinocarveol, α-pinene, α-terpineol, and spathulenol) were different from those reported for other populations [32,35,41,42]. This great variability in the essential oil profile of this species could be related to the fact that this species is considered a very variable taxon from the A. maritima group [43].
Data on the exudate composition of the studied species have been previously reported by our research group only [37]. In the present study, information on the composition of the exudates was added with data from several newly identified bioactive compounds such as carvacrol, hydroquinone, capillene, pinitol in A. santonicum exudate and eucalyptol, camphor, carvacrol, borneol and 10-undecenoic acid in A. lerchiana exudate. Based on TLC analysis in our previous report [37], the exudates of both species are rich in flavonoid aglycones. Luteolin, apigenin, 6-hydroxyluteolin 6-methyl ether, 6-hydroxyluteolin 6,3′-dimethyl ether, scutelarein 6-methyl ether, and scutelarein 6,4′-dimethyl ether have been reported as common flavonoid aglycones for both species. Quercetin, quercetagetin 6-methyl ether, and apigenin 4-methyl ether have been found in A. santhonicum exudate, while quercetagetin 3,6,4′-trimethyl ether has been found in A. lerchiana exudate [37]. GC/MS analysis with the derivatization method used in the present study did not allow the identification of a large part of the flavonoid aglycones.

3.2. Inhibition on Seed Germination

The observed strong inhibition of seed germination with aqueous solutions of studied essential oils at 2 µL/mL indicates significant inhibitory activity. This value is comparable to the values reported for essential oils considered as promising for application as bioherbicides, such as Satureja hortensis, S. montana, Mentha piperita, Peumus boldus, Origanum vulgare ssp. hirtum, Artemisia annua, Artemisia scoparia and Cymbopogon citratus. The total inhibitory activity in the 4–5 µL/mL range has been determined as strong [24,28,44,45,46]. Artemisia lerchiana EO is slightly more active compared to A. santonicum EO. Both main compounds (1,8-cineole for A. lerchiana EO and β-pinene for A. santonicum EO) are considered to possess strong phytotoxic properties [24,47,48]. Fagodia et al. [49] reported that 1,8-cineole shows a greater inhibitory activity on seed germination than β-pinene. This is a probable reason for the difference observed in the inhibitory activity of studied essential oils.
More than 90% inhibition of seed germination was achieved by the application of aqueous solutions of AE at 5 mg/mL. This result shows the strong inhibitory activity of the studied exudates because such inhibition has been reported by applying plant extracts at much higher concentrations [7]. Over 90% inhibition after applying the Cardus cardunculus (Asteraceae) crude extract has been observed at a concentration of 10 g L−1 [50]. The herbicide potential of A. lerchiana and A. santonicum EO and AE were evaluated for the first time in the present study.

3.3. Inhibition of Acetylcholinesterase (AChE)

Obtained data for the AChE inhibitory activity of studied oils has shown high potential comparable to that of A. dracunculus essential oil [51]. The stronger AChE inhibition caused by A. santonicum EO compared to A. lerchiana EO can be explained by the higher β-pinene content. It has been reported that monoterpene β-pinene displays better AChE activity than 1,8-cineole, which is the main component of A. lerchiana EO [52,53].
A. santonicum EO has been previously investigated for inhibitory activity on acetyl- and butyrylcholinesterase, but the IC50 value has not been determined [42]. Thus, here we report for the first time the IC50 values for acetylcholinesterase inhibitory activity of A. lerchiana and A. santonicum EO and AE.

3.4. Inhibition of Phytopathogenic Mycelium Growth

The essential oils of both species showed minor inhibitory activity on the mycelium growth of the tested phytopathogens at applied doses: 2 μL and 15 μL from 100 mg/mL stock solution for EO and AE, respectively. Kordali et al. [54] reported the inhibitory activity of A. santonicum EO against fungi, but the EO applied was 10 μL and 40 μL per Petri dish. The concentration selected for screening in the present study was based on data in the literature that determined the presence of antifungal activity in the oil at a concentration range (0.05–5 μg/mL) as strong [55,56]. It has been reported that essential oils from Cinnamomum zeylanicum, Cananga odorata, Ocimum basilicum, Cymbopogon citratus, Boswellia thurifera, Majorana hortensis at a concentration of 1 μL/mL inhibit the growth of Aspergillus niger, which is a common spoilage fungus [55]. The essential oil of Origanum vulgare ssp. hirtum at concentrations of 0.2–0.8 μL/mL was reported to inhibit the growth of Fusarium solani, F. oxysporum, Alternaria solani, A. Alternata, and Botrytis cinerea [57].
Several recent studies have investigated the antimicrobial properties of different Artemisia spp. Trifan and colleagues reported that Artemisia root extracts exhibit strong anti-Mycobacterium effects. Chloroform and methanol extracts obtained from the roots and aerial parts of five Artemisia species displayed good antibacterial effects against Mycobacterium tuberculosis H37Ra, with MIC values of 64–256 mg/L [58]. In another study, the antibacterial effects of leaf and stem ethanolic extracts of Artemisia absinthium L. and Artemisia annua L. on clinically important pathogenic bacteria were assessed. Artemisia extracts exhibited potent antibacterial activity against E. coli, S. aureus, L. monocytogenes, S. enteritidis, and Klebsiella sp. [59]. The essential oil of Artemisia negrei L., a species that is widespread in Morocco, Africa, also demonstrated potent antibacterial activity toward several multidrug-resistant bacteria. In the same study, antifungal activity with a percentage inhibition of 32%, 33%, and 33% was demonstrated against the fungal pathogens F. oxysporum, A. niger, and C. albicans at a dosage of 10 μL of essential oils on a Petri dish, which is at least twice that demonstrated in the current study [60].
The observed stimulating effect on the phytopathogenic mycelium growth is not unusual. Slavov et al. [61] previously reported that the acetone exudate and aqueous-methanolic extract of Tagetes patula, as well as the aqueous-methanolic extract of Tanacetum vulgare, enlarge the size of the mycelium colonies of Phytophthora cambivora when applied in vitro. This reaction could be attributed to the presence of sugars in the extracts and exudates, which were used in the cited study.

4. Materials and Methods

4.1. Plant Material

Aerial parts of the studied species were collected from the Bulgarian Black Sea coast in the summer of 2020 in the flowering phenological stage. A. lerchiana was collected from Byalata laguna locality, close to Balchik (43°24′24.92″ N/28°14′31.21″ E, 31 m a.s.l.). A. santonicum was collected from a locality close to Primorsko (42°14′42.49″ N/27°45′22.82″ E, 13 m a.s.l.). Plant species were identified by Dr. Ina Aneva. Voucher specimens—CO 1435 (A. santonicum) and CO 1436 (A. lerchiana)—were deposited at the Herbarium, Institute of Biodiversity and Ecosystem Research (SOM), Bulgarian Academy of Sciences, Bulgaria. The collected plant material was dried at room temperature (around 25֩C) without direct sunlight.

4.2. Extraction Procedures

The essential oils were extracted from dried aerial parts of the studied species on a Clevenger apparatus via water distillation for 2 h. The extraction process was repeated several times to obtain the necessary amount of essential oil for the experiments. The oils were dried over anhydrous sodium sulfate and stored at −4 °C in a sealed vial until required. Exudates were 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 [62,63]. Dry rather than ground aerial parts of both species were dipped into acetone for 5 min to dissolve the material accumulated on the surface of plant tissues and secretory structures. The obtained exudates were filtered, and the acetone was removed using a rotary vacuum evaporator.

4.3. Derivatization of the Exudates

In total, 100 µL of pyridine and 100 µL of N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) were added to the dried samples of AE, and they were heated at 70 °C for 2 h. After cooling, 300 μL of chloroform was added, and the samples were analysed using GC/MS.

4.4. GC/MS Analysis

GC/MS spectra were recorded on a Thermo Scientific Focus GC coupled with a Thermo Scientific DSQ mass detector operating in the EI mode at 70 eV (Thermo Fisher Scientific, Waltham, MA, USA). The ADB-5MS column (30 m × 0.25 mm × 0.25 μm) was used. The chromatographic conditions were as follows: helium was the carrier gas at a flow rate of 1 mL/min; the injection volume was 1 μL; and the split ratio was 1:50. Column temperature was 60 °C for 10 min, programmed at the rate of 3 °C/min to 200 °C, and finally, held isothermally for 10 min. The injection port was set at 220 °C. The significant quadrupole MS operating parameters were as follows: interface temperature of 240 °C; electron impact ionization at 70 eV with a scan mass range of 40 to 400 m/z at a sampling rate of 1.0 scan/s [64]. The temperature program for the analysis of AE samples was: 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 carrier gas (Helium) was 0.8 mL × min−1. The split ratio was 1:10. In total, 1 mL of the solution was injected [65].
Relative percentage amounts of the compounds were calculated from TIC. Relative retention indices (RRI) of the compounds were calculated using retention times of C8–C25 n-alkanes under the same chromatographic conditions. The individual components were identified by their MS and RRI values, referring to known compounds from the literature [66,67] and also by comparison with those of the NIST 14 Library and homemade MS databases.

4.5. Preparation of Fractions (EO and AE) before Bioassays

Table 4 summarizes the solvents and concentrations of EO and AE used in the different bioassays. Details of fraction preparation are included in the descriptions of the individual bioassay.

4.6. Inhibition on Seed Germination

The seed germination experiment was performed in laboratory conditions. A hundred seeds of Lolium perenne (a common crop weed) were placed per Petri dish on filter papers moistened with the tested solutions. Aqueous solutions of the essential oils at a concentration of 0.5, 1, 2, and 3.0 μL/mL were prepared using 0.1% Tween 40 (Sigma) as an emulsifier. The solutions were processed on Vortex. The exudates were dissolved in a water–acetone mixture (99.5:0.5) and were assayed at concentrations of 1, 3, 5, and 8 mg/mL. The two control solutions consisted only of 0.1% Tween 40 or a water–acetone mixture for EO and AE, respectively. The samples were incubated at room temperature for seven days. At the end of the week, the rate of germination inhibition [%] was calculated, as described by Atak et al. [68]:
GI = [(GC − TG)/GC] × 100,
where GI is the rate of germination inhibition (%); GC is the germination rate of seeds treated with control solutions; and TG is the germination rate of seeds treated with an EO or AE solution. Experiments were performed in three independent replicates.

4.7. Inhibition of Acetylcholinesterase (AChE)

Acetylcholinesterase inhibitory activity was determined using Ellman’s colorimetric method, as modified by López et al. [69]. First, all samples were dissolved in 10% MeOH at a concentration of 10 mg/mL. Next, they were serially diluted 10-fold using a phosphate buffer (PBS) (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, pH 7.5) to provide the concentration range needed. AE and EO solutions with seven different concentrations from 0.001 to 1000 µg/mL were tested. Acetylthiocholine iodide (ATCI) in a solution with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a substrate for AChE from Electrophorus electricus (Sigma-Aldrich, Taufkirchen, Germany) (0.04 M Na2HPO4, 0.2 mM DTNB, 0.24 mM ATCI, pH 7.5).
Then, 50 µL of AChE (0.25 U/mL) dissolved in PBS and 50 µL of the tested sample solution were added to the wells. The incubation of the plates was performed at room temperature for 30 min. Then, 100 µL of the substrate solution was added to start the enzymatic reaction. The absorbances were read in a microplate reader (BIOBASE, ELISA-EL10A, Jinan, China) at 405 nm after 3 min. Enzyme activity was calculated as an inhibition percentage compared to an assay including a buffer instead of an inhibitor. Galanthamine was used as a positive control. All data were analyzed with the software package Prism version 3.00 (Graph Pad Inc., San Diego, CA, USA). The IC50 values (half maximal inhibitory concentration) for AEs and EOs were measured in triplicate, and the results are presented as means ± SD.

4.8. Inhibition of Phytopathogenic Mycelium Growth

We investigated the impact of A. santonicum and A. lerchiana essentials oils and acetone exudates on the mycelium growth of three economically important plant pathogens—Botrytis cinerea, Fusarium oxisporum and Phytophthora cryptogea—which were obtained from agricultural ecosystems in Bulgaria and deposited in the fungal collection of AgroBioInstitute, Agricultural Academy. The plant pathogens were identified using classical methods and molecular techniques based on the sequencing of the ITS region of rRNA genes [70]. P. cryptogea was obtained by baiting, according to Jung and Blaschle [71], of the rhizosphere soil of Rubus idaeus. F. oxysporum and B. cinerea were previously isolated from diseased raspberry plants.
The agar disk-diffusion method was used for the bioassay [72]. Small agar blocks (5 × 5 mm) with the mycelium of the corresponding isolate were cultured in the center of 90 mm Petri dishes. We grew the oomycete Phytophthora cryptogea on V8 Agar (16 g agar, 100 mL V8 Juice, and 900 mL distilled water) and the fungi Botrytis cinerea and Fusarium oxysporum on PDA (BD Difco™, BD, Franklin Lakes, NJ, USA). The Petri dishes were incubated overnight for a synchronized onset of growth before the application of the essential oils and acetone exudates. Four variants were performed with all the isolates: A. santonicum EO, A. santonicum AE, A. lerchiana EO, and A. lerchiana AE. The acetone exudates of each plant species were dissolved in DMSO at a concentration of 100 mg/mL, and two drops with a volume of 15 µL were dripped at equal distances from its center. In the same way, the essential oils were applied with a volume of 2 µL in pure form without dilution. Two control variants—one without treatment and one with DMSO—were also included in the experiment. Four replicates were made for each variant. The Petri dishes were cultivated in a climatic chamber at 25 °C in darkness. The results were documented after 6 days when fungi from the control variants of the three species reached the periphery of the Petri dishes. Photographs (Canon EOS 4000D, Canon, Tokyo, Japan) of all mycelial colonies were taken, and their mycelial growth areas were measured using the image analysis program ImageJ 1.54d [73]. Based on the obtained data (average mycelial growth area for each variant), the inhibition percentage was calculated using the following equation [21]:
IMG = 100 (C − T) 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 fungal colony with treatment. Four replicates were performed for each variant.

4.9. Data Analysis

Statistical analyses were performed using Microsoft Excel software 2016. The AChE inhibitory data were analyzed using the software package Prism 3 (Graph Pad Inc., San Diego, USA). IC50 values were calculated using the same software.

5. Conclusions

The present study represents preliminary research on the pesticidal properties of A. lerchiana and A. santonicum essential oils and exudates. Both species are clearly distinguished by their essential oil profiles. The strong inhibitory activity on L. perenne seed germination and acetylcholinesterase was found by studying essential oils. However, essential oils showed weak inhibitory activity against the examined phytopathogens in the studied doses. The exudates of both species displayed phytotoxicity against L. perenne seed germination and weak AChE inhibitory activity. A. lerchaiana AE exhibited strong inhibitory activity against the mycelium growth of Botrytis cinerea. Essential oils and exudates of A. lerchiana and A. santonicum showed a phytotoxic potential that provides reasons for more detailed studies on their herbicidal activity. Artemisia santonicum EO is recommended to be examined for insecticidal activity and A. lerchiana AE for more extended studies on its inhibitory activity against B. cinerea and other pathogens. According to our knowledge, this is the first report of inhibitory activity on seed germination and acetylcholinesterase of A. lerchiana and A. santonicum essential oils and exudates.

Author Contributions

Conceptualization, M.N.; collection and identification of plant material, I.A.; formal analysis, M.N., A.L, A.T., B.G. and E.Y.-T.; methodology and investigation, M.N., A.L., A.T., B.G. and S.B.; writing—original draft preparation, M.N.; writing—review and editing, M.N., A.L., A.T., B.G., E.Y.-T. and S.B.; supervision and project administration, A.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grant BG05M2OP001-1.002-0012 from Operational Program Science and Education for Smart Growth 2014–2020 of Bulgaria, co-financed by the European Union through the European Structural and Investment Funds. This study undertook the sustainable utilization of bioresources and waste of medicinal and aromatic plants for innovative bioactive products.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are grateful for the financial support provided by the Operational Program Science and Education for Smart Growth 2014–2020 of Bulgaria, co-financed by the European Union through the European Structural and Investment Funds (grant BG05M2OP001-1.002-0012). This study undertook the sustainable utilization of bioresources and waste of medicinal and aromatic plants for innovative bioactive products.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 12 03491 g001
Figure 1. Inhibition on L. perenne seed germination using EO (a) and AE (b) of the studied species.
Figure 1. Inhibition on L. perenne seed germination using EO (a) and AE (b) of the studied species.
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Plants 12 03491 g002
Figure 2. Antifungal activity of A. santonicum and A. lerchiana essential oils (EO) and acetone exudates (AE) in vitro, after 6 days at 25 °C (a–c), against Botrytis cinerea (a), Fusarium oxysporum (b) and Phytophthora cryptogea (c). Error bars represent the standard error of the mean (n = 4). %IMG—Inhibition of mycelial growth.
Figure 2. Antifungal activity of A. santonicum and A. lerchiana essential oils (EO) and acetone exudates (AE) in vitro, after 6 days at 25 °C (a–c), against Botrytis cinerea (a), Fusarium oxysporum (b) and Phytophthora cryptogea (c). Error bars represent the standard error of the mean (n = 4). %IMG—Inhibition of mycelial growth.
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Table 1. Essential oil composition [area %] from the aerial parts of the studied Artemisia species.
Table 1. Essential oil composition [area %] from the aerial parts of the studied Artemisia species.
RRI *CompoundA. lerchianaA. santonicum
9142-Methyl-2,5-divinyltetrahydrofuran0.8-
929α-Thujene0.5-
937α-Pinene0.39.3
952Camphene4.8-
979β-Pinene0.315.2
9912,3-Dehydro-1,8-cineole0.3-
1017α-Terpinene-0.1
1024p-Cymene-1.25.3
1028Limonene-2.7
1032Eucalyptol (1,8-Cineole)22.1-
1052cis-Arbusculone0.1-
1060γ-Terpinene0.50.4
1065Acetophenone-0.2
1070trans-Arbusculone0.1-
1070cis-Sabinene hydrate0.6-
1088Terpinolene0.40.9
1090p-Cymenene0.10.1
1098trans-Sabinene hydrate0.2-
1125α-Campholenal-0.8
1139trans-Pinocarveol-9.6
1140trans-p-2-Menthen-1-ol0.1-
1142cis-Verbenol-0.1
1145Camphor14.0-
1157Isoborneol0.12.1
1163Pinocarvone-3.0
1165Borneol6.6-
1178Terpinen-4-ol2.10.1
1189α-Terpineol1.39.2
1195Myrtenol1.00.1
1205Verbenone-0.1
1208cis-Piperitol1.4-
1217trans-Carveol1.40.6
1233cis-Carveol0.1-
1242Carvone0.60.8
1254Piperitone0.6-
1258Carvenone0.6-
1283Lavandulyl acetate0.1-
1285Bornyl acetate1.12.7
1297trans-Pinocarvyl acetate-0.1
1292Thymol0.20.1
1302Carvacrol6.13.8
1306Isoascaridole1.5-
1327Myrtenyl acetate-0.2
1347Silphinene-0.1
1350α-Terpinyl acetate0.1-
1354Citronellol acetate-0.3
1357Eugenol0.3-
1364Neryl acetate-0.2
1379Silphiperfol-6-ene-0.1
1382Geranyl acetate-0.8
1382Sabinyl propionate-0.9
1392(E)-Jasmone0.5-
1402Methyleugenol0.10.1
1412cis, threo-Davanafuran0.2-
1414Sabinyl isobutanoate-0.2
1483α-Curcumene-1.0
1485β-Selinene0.5-
1490Davana ether1.0-
1501Capillene-4.2
1505C15H22O (MW 220)-0.1
1510Cameroonan-7α-ol-0.8
1515Davana ether isomer4.3-
1530Artedouglasia oxide C1.8-
1535Artedouglasia oxide A3.3-
1544Italicene ether-0.3
1566Artedouglasia oxide D1.1-
15731,5-Epoxysalvial-4(14)-ene-1.4
1578Artedouglasia oxide B1.8-
1580Spathulenol1.88.5
1595Viridiflorol0.9-
1608β-Antlantol-2.1
1639Capillin-3.2
1655C15H24O (MW 220)2.8-
1660Neointermedeol3.3-
1677Valeranone-0.7
1688Eudesma-4(15),7-dien-1β-ol-1.1
1692C15H26O (MW 222)1.0-
1752γ-Costol0.2-
1776α-Costol0.4-
1820(E)-Artemidin-3.1
1835C15H20O3 (MW 248)0.4-
1844Hexahydrofarnesyl acetone0.1-
1880(Z)-Artemidin-1.5
Monoterpene hydrocarbons8.234.0
Oxygenated monoterpenes62.735.9
Sesquiterpene hydrocarbons0.51.2
Oxygenated sesquiterpenes24.419.5
Other1.37.6
Total97.198.1
* Relative retention index (RRI) determined relative to a homologous series of n-alkanes (C8–C25) on the HP-5MS column.
Table 2. Exudate composition [area %] from the aerial parts of the studied Artemisia species.
Table 2. Exudate composition [area %] from the aerial parts of the studied Artemisia species.
RRI *CompoundA. lerchianaA. santonicum
1022Eucalyptol6.45-
1070Glycolic acid0.34-
1150Camphor4.58-
1161Borneol3.69-
1177Terpinen-4-ol0.2-
1195Myrtenol1.95-
1253Succinic acid 0.10.39
1255Ascaridole1.49-
1260Glycerol0.862.1
1265Octanoic acid-2.27
1289Bornyl acetate0.37-
12944-Methoxyphenol-0.95
1337Carvacrol1.860.18
1340Glyceric acid-0.45
1345Fumaric acid-4.25
1393Hydroquinone-1.9
1436trans-Cinnamic acid-0.95
14614′-Hydroxyacetophenone0.210.42
148010-Undecenoic acid11.45-
1481β-Selinene0.63-
1498Malic acid-5.72
1500Capillene-1.32
1503meso-Erythritol-0.1
1515Pyroglutamic acid-1.63
1536Artedouglasia oxide A0.45-
1562Methyl p-coumarate0.19-
1578Erythronic acid-2.45
16404-Hydroxybenzoic acid0.20.1
1660Neointermedeol1.23-
1700Methyl 3,4-dihydroxybenzoate-0.31
1774Costol0.76-
1776Vanillic acid0.20.25
17901,2-Longidione-0.83
1811Protocatechuic acid0.330.3
1813Pinitol-3.61
18162-Ethylhexyl salicylate0.94-
1845Fructose 0.210.41
1849Quinic acid0.510.96
1880Syringic acid0.1-
1940Hydroxycinnamic acid0.10.1
1972Glucose0.150.21
2042Hexadecanoic acid (palmitic acid)0.340.47
2097Ferulic acid0.21
2126Myo-Inositol 0.310.28
2137Caffeic acid-0.3
2162Octadecanoic acid (stearic acid)-0.14
22019,12-Octadecadienoic acid (linoleic acid)-0.22
2568rac-Glycerol 1-palmitate-0.34
2709Sucrose0.801.45
2757Glycerol monostearate-0.20
3100Chlorogenic acid-0.68
31496-O-Methylapigenin (hispidulin)0.1-
31986-O-Hydroxyluteolin 4′,6-dimethl ether (jaceosidin)1.05-
3325β-Amyrin0.10.2
3355α-Amyrin0.871.27
34026-O-Hydroxyluteolin 4′,6,7-trimethyl ether (eupatorin)0.94-
* Relative retention index (RRI) determined relative to a homologous series of n-alkanes (C8–C25) on the HP-5MS column.
Table 3. Acetylcholinesterase inhibitory activity of A. lerchiana and A. santonicum EO and AE.
Table 3. Acetylcholinesterase inhibitory activity of A. lerchiana and A. santonicum EO and AE.
Studied FractionIC50, [μg/mL]
A. lerchianaA. santonicum
Essential oils64.42 ± 2.6914.60 ± 0.58
Acetone exudates961 ± 0.17>1000
Galanthamine (positive control)0.21 ± 0.02
Table 4. Used solutions and concentrations of EO and AE in the studied bioassays.
Table 4. Used solutions and concentrations of EO and AE in the studied bioassays.
BioassayStudied FractionSolventTested Dose/Concentration
Inhibition of seed germinationEO0.1% solution Tween 400.5 to 3.0 μL/mL
AEwater:acetone 99.5:0.5 (v/v)1 to 8 mg/mL
Inhibition of acetylcholinesteraseEOMethanol0.001 to 1000 µg/mL
AEMethanol0.001 to 1000 µg/mL
Inhibition of phytopathogenic mycelium growthEOWithout0.2 µL
AEDMSO15 µL (100 mg/mL)
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Nikolova, M.; Lyubenova, A.; Yankova-Tsvetkova, E.; Georgiev, B.; Berkov, S.; Aneva, I.; Trendafilova, A. Artemisia santonicum L. and Artemisia lerchiana Web. Essential Oils and Exudates as Sources of Compounds with Pesticidal Action. Plants 2023, 12, 3491. https://doi.org/10.3390/plants12193491

AMA Style

Nikolova M, Lyubenova A, Yankova-Tsvetkova E, Georgiev B, Berkov S, Aneva I, Trendafilova A. Artemisia santonicum L. and Artemisia lerchiana Web. Essential Oils and Exudates as Sources of Compounds with Pesticidal Action. Plants. 2023; 12(19):3491. https://doi.org/10.3390/plants12193491

Chicago/Turabian Style

Nikolova, Milena, Aneta Lyubenova, Elina Yankova-Tsvetkova, Borislav Georgiev, Strahil Berkov, Ina Aneva, and Antoaneta Trendafilova. 2023. "Artemisia santonicum L. and Artemisia lerchiana Web. Essential Oils and Exudates as Sources of Compounds with Pesticidal Action" Plants 12, no. 19: 3491. https://doi.org/10.3390/plants12193491

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