Five Different Artemisia L. Species Ethanol Extracts’ Phytochemical Composition and Their Antimicrobial and Nematocide Activity

Among the plants that exhibit significant or established pharmacological activity, the genus Artemisia L. deserves special attention. This genus comprises over 500 species belonging to the largest Asteraceae family. Our study aimed at providing a comprehensive evaluation of the phytochemical composition of the ethanol extracts of five different Artemisia L. species (collected from the southwest of the Russian Federation) and their antimicrobial and nematocide activity as follows: A. annua cv. Novichok., A. dracunculus cv. Smaragd, A. santonica cv. Citral, A. abrotanum cv. Euxin, and A. scoparia cv. Tavrida. The study of the ethanol extracts of the five different Artemisia L. species using the methods of gas chromatography–mass spectrometry (GC–MS) and high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC–MS/MS) allowed establishing their phytochemical profile. The obtained data on the of five different Artemisia L. species ethanol extracts’ phytochemical composition were used to predict the antibacterial and antifungal activity against phytopathogenic microorganisms and nematocidal activity against the free-living soil nematode Caenorhabditis elegans. The major compounds found in the composition of the Artemisia L. ethanol extracts were monoterpenes, sesquiterpenes, flavonoids, flavonoid glycosides, coumarins, and phenolic acids. The antibacterial and antifungal activity of the extracts began to manifest at a concentration of 150 µg/mL. The A. dracunculus cv. Smaragd extract had a selective effect against Gram-positive R. iranicus and B. subtilis bacteria, whereas the A. scoparia cv. Tavrida extract had a selective effect against Gram-negative A. tumefaciens and X. arboricola bacteria and A. solani, R. solani and F. graminearum fungi. The A. annua cv. Novichok, A. dracunculus cv. Smaragd, and A. santonica cv. Citral extracts in the concentration range of 31.3–1000 µg/mL caused the death of nematodes. It was established that A. annua cv. Novichok affects the UNC-63 protein, the molecular target of which is the nicotine receptor of the N-subtype.


Introduction
Today, biologically active compounds for preventing and avoiding plant diseases hold great promise for organic farming.It is well known that the chemically made pesticides conventionally used in the integrated agricultural complex are more dangerous for humans and the environment than substances of natural origin used in organic agriculture.The advancement of technological innovations in production and utilization, coupled with the increasing demand for organic goods, encourages the establishment of higher standards for human health and well-being, environmental safeguarding, and biodiversity, thereby enhancing overall food security [1].
At present, the quantity of formulations derived from natural compounds utilized for organic food production is significantly lower in comparison to the formulations employed in conventional agriculture.Therefore, it is essential to search for and develop new bio-based products for organic crop farming.One of the current trends is to utilize environmentally friendly plant extracts or essential oils as they are biodegradable, do not produce any toxic metabolites, and possess a wide range of antibacterial properties.This approach can effectively address the issue of pathogen resistance to plant protection chemicals.
Among the plants that exhibit significant or established pharmacological activity, the genus Artemisia L. deserves special attention.It includes more than 500 species belonging to the largest Asteraceae family [2,3].This genus covers a diverse range of plants, which are widely distributed in the temperate climatic zones of Europe, Asia, North and South Africa, as well as North America.It comprises varieties of perennial, biennial, and annual herbs or small shrubs [4,5].The literature review indicated that various species of Artemisia L. possess an unprecedented range of biological activities and are used in conventional folk and academic medicine [6].The current biological activity studies of numerous Artemisia species do not reveal the full extent of the inherent formidable biochemical potential of this plant.Based on extensive empirical evidence, this plant was initially employed in antimalarial [7,8] and antibacterial therapy [9,10] and, subsequently, as antifungal [9][10][11][12][13] and antihepatotoxic [14,15] agents, with notable anti-spasmatic, anti-inflammatory, and angioprotective properties [6,[16][17][18].Tu Youyou, a Chinese pharmaceutical chemist, was awarded the Noble Prize in Physiology and Medicine [19,20] in 2015 for her efforts in extracting the artemisinin from A. annua L. [21] and synthesizing its derivatives, namely dihydroartemisinin [22], artemeter [23], and artesunate [24].Additionally, she conducted a study on their pharmacological activity against Plasmodium falciparum, demonstrating the high potency of these substances against tropical malaria.
The available research data indicate that the biologically active potential of the various Artemisia species is far from being fully studied, and their conventional applications do not utilize their properties fully.Therefore, the current investigations [48][49][50][51][52][53][54][55] of the chemical compositions and biological activity of the various Artemisia L. species hold particular significance and are experiencing a real surge as they are presently in the focus of bioorganic and bioanalytical chemistry.The chromatography-mass spectrometry technique is widely acknowledged as the most-appropriate and -informative approach for the analysis of extracts or essential oils, specifically their chemical compositions, due to its ability to combine high separation power with reliable mass spectrometry detection [56].However, the volatile compounds of plant extracts or essential oils with no confirmed thermal decomposition at gas transition temperatures should be separated using the method of gas chromatography-mass spectrometry (GC-MS) employing either electron or chemical ionization (EI or CI) techniques [57][58][59][60][61][62].For polar and heavy components' separation, it is advisable to use high-performance liquid chromatography (HPLC), which treats molecules leaving the chromatographic column with electrospray ionization (ESI) and, then, detects the generated particulates by a quadrupole time of flight mass spectrometer (HPLC-MS/MS-ESI) [63][64][65][66][67].
There are currently fourteen species of the Artemisia genus growing in the Aromatic and Medical Plants Collection of the Nikitsky Botanical Garden, having various ecological and geographical origins.Based on the introduction study, ten promising species were allocated for cultivated varieties, and five varieties were obtained for commercial cultivation in Crimea: A. annua cv.Novichok, A. dracunculus cv.Smaragd, A. santonica cv.Citral, A. abrotanum cv.Euxin, and A. scoparia cv.Tavrida.
The purpose of the present work was to study comprehensively the biochemical composition of five extracts of various Artemisia L. species selected by the Nikitsky Botanic Garden, specifically A. annua cv.Novichok, A. dracunculus cv.Smaragd, A. santonica cv.Citral, A. abrotanum cv.Euxin, and A. scoparia cv.Tavrida, utilizing the methods of GC-MS and HPLC-MS/MS-ESI, and to predict the antibacterial, fungicide, and nematocidal properties based on the identified chemical composition of these extracts.

Phytochemical Composition of Certain Species of Artemisia L. Extracts
Presently, a substantial quantity of information on the phytochemical properties has been accumulated for the plant extracts, resulting from the application of chromatography in conjunction with mass spectrometry detection and various methods of ionization [68][69][70].The same applies to extracts and essential oils of various species of Artemisia L. The details of their phytochemical composition, derived from both qualitative and quantitative studies and given in [71][72][73][74][75], demonstrate the extensive array of bioactive compounds found in Artemisia L.
During the chromatographic analysis of five Artemisia L. species, the ethanol extracts were utilized in their native form without being diluted.The conditions of chromatographic fractionation were selected based on the literature data demonstrating that the extracts of various types of Artemisia L. have components that differ in their thermal stability, chromatographic lability, polarity, and boiling points.Hence, the examination of low-polar and volatile constituents of the prepared extracts was conducted by utilizing GC-MS with EI and CI.For the polar constituents, we utilized HPLC MS/MS (ESI+).
The results of GC-MC with different ionization types revealed a diverse range of major and minor constituents in the extracts of A. annua L., A. dracunculus L., A. santonicum L., A. abrotanum L., and A. scoparia L., as presented in Table 1.To identify the constituents, we used the relative match factor (RMF) generated by the NIST MS Search Program, on the automatic deconvolution of the mass spectra and their subsequent comparison with those in the reference digital libraries.The RMF of the compounds was largely within the range of 890-930 of the match factor, which proves the correct identification of the constituents found in the extracts.Only a third of the compounds had an RMF below 890.This is due to a weak peak intensity [M + ] in the mass spectra with EI.In such cases, in order to ensure more-reliable identification [56,76], the peaks of higher intensity [M + H] + were recorded by the CI method.Analyzing the results of the study obtained by GC-MS (Table 1) for the extracts of A. annua L., A. dracunculus L., A. santonicum L., A. abrotanum L., and A. scoparia L. and the quantities of their constituents, we can conclude the following: they are composed of acyclic, monocyclic, and bicyclic monoterpenoids; monocyclic, bicyclic, and tricyclic and eudesmanic sesquiterpenoids; coumarins; phenolic compounds; and other chemical compounds.More detailed results are presented in the Table S1.
The acyclic monoterpenoids group revealed in five Artemisia extracts by the method of GC-MS are distributed as follows: β-myrcene, trans-βand cis-β-ocimene, α-citral, and β-citral-in all tested extracts; Artemisia ketone-in A. annua, A. santonica, and A. scoparia extracts, with its highest content in A. annua; Artemisia alcohol-in A. annua, A. dracunculus, A. santonica, and A. scoparia.Furthermore, the acyclic diterpene alcohol of phytol was detected in the acyclic compounds of A. annua, A. dracunculus, A. abrotanum, and A. scoparia.
Extracted by the GC-MS and HPLC-MS/MS techniques, the collection of chemical compounds constituted a distinct and unique phytochemical profile composed by the five different species (A.annua cv.Novichok, A. dracunculus cv.Smaragd, A. santonica cv.Citral, A. abrotanum cv.Euxin, A. scoparia cv.Tavrida), exhibiting high biological activity and exercising antimicrobial properties attributed to terpenoids and sesquiterpenoids.Furthermore, β-santonin, a constituent of the sesquiterpene lactones group, has a wide spectrum of activity against nematodes and cestodes.

Antibacterial and Antifungal Activity of Ethanol Extracts of Artemisia L. Plants
Based on the literature data, the Artemisia L. essential oils and plant extracts possess high antibacterial activity against a wide spectrum of pathogenic microorganisms.
Thus, the Artemisia L. essential oils effectively inhibit the growth of the following fungal species: Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina, Colletotrichum fragariae, Colletotrichum gloeosporioides, Colletotrichum acutatum, etc., which cause diseases and high crop losses [25,[78][79][80].The A. scoparia L., A. sieberi L., and A. aucheri L. essential oils and extracts were found to be effective against phytopathogens [71,81,82].The antibacterial properties are largely dependent on the time of year when the plants are harvested to produce extracts, essential oils, and BAC; the highest potential was demonstrated by plants prepared in the second half of summer [83].
In a series of laboratory experiments with serial dilution, the antibacterial effectiveness of the Artemisia L. ethanol extracts against the phytopathogenic bacteria (four strains: Rathayibacter iranicus and Bacillus subtilis (Gram-positive) and Agrobacterium tumefaciens and Xanthomonas arboricola (Gram-negative) and phytopathogenic fungi (three strains: Alternaria solani, Fusarium graminearium, and Rhizoctonia solani) was evaluated for the first time.The results of the studies are given in Tables 3 and S3.The antibacterial properties of the extracts varied depending on the species of the plant and pathogenic strain.The extract from A. annua was the most-effective against the Gram-positive bacteria of R. iranicus, with the minimal inhibitory concentration (MIC) being 500 µg/mL and the minimal bactericidal concentration (MBC) being 1000 µg/mL.The antibacterial activity of the A. annua extract was likely mediated by the presence of artemisinic acid, ketone, and alcohol in the Artemisinin extract and its precursors [84].Another Gram-positive B. subtilis strain was more resistant to the components of the A. annua extract.For B. subtilis, the inhibitory concentration was within 2000 µg/mL and above.
The ethanol extract of A. annua demonstrated low antibacterial and antimycotic properties against the studied Gram-negative phytopathogenic bacterial and fungal strains.The concentrations inhibiting the growth of X. arboricola and R. solani were, respectively, 1000 µg/mL and 2000 µg/mL, while 4000 µg/mL and higher for A. tumefaciens, A. solani, and F. graminearum.The antimicrobial properties of the A. annua extract prepared from cv.Novichok (the Nikitsky Botanic Garden) were comparable to those of the A. annua methanol extracts from India, but they were lower than those of the A. annua methanol extracts from Bulgaria [85,86] and significantly higher than those of the ethanol extracts from the plants grown in Romania [87].Among all the species under study, the A. dracunculus ethanol extract demonstrated a selective antimicrobial activity against R. iranicus, requiring the lowest inhibitory and bactericidal concentrations.At an extract concentration of 310 µg/mL, the bacterial cells of R. iranicus were killed.According to the Alvarez-Martínez and Van Vuuren method, which distinguishes plant extracts by their effectiveness against bacteria and fungi, the obtained results correspond to a very high antimicrobial effect [88,89].Such data are likely to be associated with the high estragole content attributed to the extracts of these species, which demonstrates large areas of inhibited Gram-positive bacteria growth [90].Furthermore, estragole is famous for its antimycotic effect against pathogens as such A. niger, A. flavus, T. viride, C. albicans, A. solani, F. graminearum, and R. solani [91,92].As per our research findings, the effective concentration of the A. dracunculus ethanol extract for inhibiting fungal growth is 1120 µg/mL.The obtained findings were similar to the results for the A. santonica ethanol extract.
X. arboricola, a Gram-negative bacterium, was more sensitive to the A. dracunculus and A. santonica extracts compared to B. subtilis and A. tumefaciens, and it ceased growing when the extracts' concentrations reached 625 and 560 µg/mL, respectively.
The antimicrobial activity of the A. dracunculus ethanol extract was lower than that of the aqueous extract and higher than that of the methanol extract and was comparable to that of the A. tarragona ethanol-water extract from Iran, with the exception of the antimicrobial activity against R. iranicus and Gram-negative bacteria.Our experiments had significantly lower results for minimal inhibitory concentrations [82,93,94].
The extract of A. santonica demonstrated significantly higher outcomes compared to those obtained for the anti-phytopathogenic extracts [95] and lower than those obtained for the methanol extracts prepared from Bulgarian plants [86].
The ethanol extract of A. scoparia in comparison to others demonstrated significant effectiveness against Gram-negative bacteria (X.Arboricola and A. Tumefaciens) and fungi (F.graminearum and R. solani).Thus, the MIC values for X. arboricola and R. solani were 150 µg/mL, F. graminearum-310 µg/mL, and A. tumefaciens-625 µg/mL.These values correspond to the very high antimicrobial activity of the extracts [88,89].The minimum bactericide and fungicide concentrations of the A. scoparia L. extract for the X. arboricola, R. solani, and F. graminearium strains were 625 µg/mL, being 2-4-times higher than the MIC values for the same.The values of the antibacterial activity of the ethanol extract of A. scoparia significantly exceed the activity of plants grown in Iran and Palestine [80,96].
The prominent antimicrobial effect of the A. scoparia ethanol extract is probably attributable to the high contents of capillin, capillen, and scoparon.The percentage of their total presence in the extract was 63%.Scoparon is a phytoalectin, a secondary metabolite produced by plants as a reaction to contamination by various pathogens [97].Capillen and capillin are the major components of the essential oils, manifesting their strong antibacterial and antimycotic properties against various pathogens that cause diseases in animals and humans [98][99][100][101].
The extract of A. abrotanum cv.Euxin demonstrated the lowest activity among the studied ethanol extracts.Its MIC, MBC, and MFC values were 2000 µg/mL and higher.The concentrations inhibiting the fungal growth and killing their cells have not been determined for fungal species.According to Alzoreky N.S. [102], the antibacterial concentrations of the A. abrotanum methanol extract from Yemen started from 1320 µg/mL, which is in line with the data obtained in our studies.
Therefore, as per our study, the following antimicrobial sequence can be stated for the ethanol extracts of the Artemisia L. genus plants: A. scoparia > A. dracunculus > A. santonica > A. annua > A. abrotanum.

Nematocidal Activity of cv. Artemisia L. Extracts
Caenorhabditis elegans, a free-living soil nematode, is a suitable model organism for studies in neurotoxicology and ecotoxicology.C. elegans has an advantage over other model organisms due to its easy and inexpensive cultivation in the laboratory, safety for scientists, and high fertility [103][104][105].C. elegans consists of 959 somatic cells including 302 neurons [104].The nervous system of C. elegans has 890 electrical synapses, 1410 neuromuscular synapses, and 6393 chemical synapses using neurotransmitters specific to the vertebrate body (acetylcholine, dopamine, serotonin, glutamate, and γ-aminobutyric acid) [103].At the same time, nematodes lack a circulatory system and an organ of external respiration, thereby significantly facilitating the interpretation of the results of toxicological experiments [104].Ethanol extracts of various raw materials A. annua, A. dracunculus, and A. santonica, were found to cause dose-dependent nematode mortality (Table 4).
(−)-Tetramisole hydrochloride was used as a comparator.At concentrations of 1.875 µg/mL, 3.75 µg/mL, and 7.5 µg/mL, tetramisole caused the death of 14.5 ± 2.5, 26.0 ± 3.1, and 61.5 ± 2.7% of nematodes in 24 h.In order to identify potential molecular targets of the studied extracts' action, we performed experiments to evaluate their toxicity at a concentration of 500 µg/mL for C. elegans of both the wild-type and mutant lines.
Table 5 provides the results of the experiments aimed at examining the effects of the Artemisia extracts on nematodes of the N2 wild-type and the mutant lines of RB896, RB756, and JD217 with null mutations in the genes encoding the muscarin receptors of acetylcholine such as gar-1, gar-2, and gar-3, respectively.The RB918 line is a null mutation of the nicotine receptor gene for N-subtype acetylcholine.The A. annua extract for C. elegans of the RB896, RB756, and RB918 lines was the least toxic extract in these experiments.Three subtypes of the above receptors have been found in C. elegans, GAR-1, GAR-2, and GEAR-3, respectively [106][107][108].The GAR-3 receptor is the best-investigated one; as for the pharmacological profiles of the GAR-1 and GAR-2 receptors, they are understudied.The low toxicity of the A. annua extract for nematodes of the RB896 and RB756 lines indicates that this extract contains substances affecting the GAR-1 and GAR-2 mAChRs of C. elegans (Table 5).The agonists of the acetylcholine nicotine receptor (nAChRs) are represented by a large group of substances and used as pesticides and anthelminthic drugs.Nicotine receptors of both vertebrates and invertebrates are the ion channels formed by five transmembrane proteins.Homomeric and heteromeric agonists of acetylcholine nicotine receptors (nAChRs) are distinguished.The N-type nicotine receptor in C. elegans is homomeric and consists of five identical protein subunits of ACR-16 [109,110].The results of our experiments showed that the extract of A. annua contains substances whose molecular target is the N-type nicotine receptor (Table 6).
Table 6 shows the toxicity of the artemisia extracts for the nematode line of the N2 wild-type and the nematodes of the five mutant lines.Each line lacks one of five subunits of the L-type nicotine receptor sensitive to levamisole.In most cases, the sensitivity of the C. elegans mutant lines to the negative effects of the Artemisia extracts did not exceed the sensitivity of the N2 wild-type line.The exception was the effect of the A. santonica extract on the CB904 nematode line and the effect of the A. annua extract on the CB522 nematode line.The least-toxic extract was A. annua for C. elegans of the VC428 line with the null mutation of the UNC-63 gene.The low sensitivity of C. elegans of the VC428 line to the toxic effect of the extract of A. annua L. suggests that this extract contains substances whose target is one of the subunits of the nAChR L-subtype, namely the UNC-63 protein.Now, the study of nicotine receptors in invertebrates has a strong focus as these receptors are targets of many pesticides and anthelminthics.All nicotine acetylcholine receptors consist of five protein subunits, but there are more genes encoding these receptors.C. elegans has 29 genes of such a type, human-17-and investigated invertebrates-from 7 to 12 [110].Hence, a large number of subtypes of these receptors with selective sensitivity to toxicants can be expected.Levamisole-sensitive L-subtype nicotine receptors are present in nematodes, both free-living and parasitic, but they are missing in warm-blooded animals, allowing the use of these receptor agonists for the treatment of helminthiasis in humans and animals.C. elegans has an L-subtype nicotine receptor consisting of three α-subunits (LEV-8, UNC-38, and UNC-63) and three non-α-subunits (UNC-1 and UNC-29).The α-subunits are thought to be involved in the binding of acetylcholine and other N-cholinergic agonists, and the other subunits form the ion channels in neuronal membranes and locomotor muscles [110][111][112].Our mutant analysis revealed that the A. annua extract has substances affecting the nAChR N-subtype, consisting of five molecules of the ACR-16 protein (Table 4), and also, the α-subunit of the UNC-63 receptor is affected by the substances derived from the A. annua extract (Table 6).

Sampling
For the raw material, we used A. annua L. (Novichok variety), A. dracunculus L. (Smaragd variety), A. santonicum L. (Citral variety), A. abrotanum L. (Euxin variety), and A. scoparia Waldst.& Kit (Tavrida variety) from an experimental area of the Aromatic and Medical Plants Collection in the Nikitsky Botanical Garden, located on the southern coast of Crimea (44 • 31 N, 34 • 15 E, 200 m above sea level).The plants grew in brown carbonate soil with medium humus and low clay content.The climatic conditions of the southern coast of Crimea are attributed to the geographical features, specifically the mountains, which shield the coastline from the northern winds and the warm waters of the Black Sea.All these have resulted in a dry subtropical climate of a Mediterranean type.
The sampling for the study was conducted during the mass flowering of the plants.The raw material was collected between 11 a.m. and 2 p.m., during the hottest hours of the day.The portion above ground was trimmed at the level of the lower leaves of the plant.The collected plants were stored in a well-ventilated location, shielded from direct sunlight, at a temperature range of 30-35 • C.

Extracts' Preparation
A single-stage maceration was used to prepare five types of extracts from A. annua, A. dracunculus, A. santonica, A. abrotanum, and A. scoparia.The dry plant material was ground to a particle size range of 1-2 mm in a LM 202 laboratory mill manufactured by NV-Lab (Moscow, Russia).The ground material test portions, each weighing 15.0 g, were poured with 150 mL of a 70% ethanol solution and macerated at 45 • C under constant stirring for an hour and a half.The prepared extracts were filtered using a first-type Whatman filter (Cat.No. 1001 110).The obtained filtrate materials were concentrated in a LabTex Re 100-Pro rotary evaporator manufactured by DLAB Scientific Co., Ltd., Beijing, China, and stored in a dark location at a temperature of 4 • C.
The Artemisia L. extract composition was determined by recording the mass spectra for EI and CI at 70 eV and 30 eV, respectively, within the range of 50-550 m/z (using CH 4 reagent gas, high purity).In order to process the mass spectra, we used the Chromatec Analytic 3.5 software (Chromatec, Yoshkar-Ola, Russia), the NIST MS Search Program, V.2.3, the NIST'20 mass spectra electronic libraries (NIST, Gaithersburg, MD, USA), and Wiley 12th Edition (Wiley, Hoboken, NJ, USA) [113].
In addition, the retention times and indices were compared with those reported in [114,115] and presented in the databases mentioned above.

High Performance Liquid Chromatography-Mass Spectrometry
The HPLC-MS/MS-ESI study was performed on a Bruker maXis II™ high-resolution ESI-QTOF mass spectrometer (Bruker GmbH, Bremen, Germany) equipped with the ESI source Apollo II (in positive-ion-detection mode, capillary voltage +4 KV, source temperature of 250 • C, voltage shift of 500 V at the end plate, scanning time of 0.2 s) and coupled to the HPLC Dionex UltiMate™ 3000 series (Thermo Fisher Scientific, Waltham, MA, USA), including a gradient pump, a degassing module, an automatic sampler, and a chromatographic oven.The SB-C18 Agilent Zorbax chromatographic column (150 mm × 4.6 mm, 5 µm, Agilent Technologies, Santa Clara, CA, USA) was applied with the guard column (25 mm × 4.6 mm, 5 µm, Agilent Technologies, Santa Clara, CA, USA).
N 2 was used (high purity) as the drying (flow rate of 8 L/min at a temperature of 250 • C), spray (pressure of 4 bar), and collision gas.
The Compass Data Analysis 4.4, Compass Target Analysis 4.4 (Bruker GmbH, Bremen, Germany) software was used to synchronize the work with the HPLC Dionex UltiMate™ 3000 series and control the mass spectral data collection and handling.
Using target analysis, all compounds were detected by applying strict analyzing criteria.Different collision energies were applied to obtain good-intensity parent ions, as well as significant fragment ions.Accurate and reproducible spectra with high mass accuracy helped in the identification process.The acquired data were processed using three acquisition software including Compass Data Analysis 4.4, Compass Target Analysis and Compass Library Editor (Bruker GmbH, Bremen, Germany).The MS intensity threshold was set at >1000, MS 2 intensity > 500, base peak intensity 1000 counts (minimum), ppm error > 5, and mSigma values > 50.
GC-MS and HPLC-MS/MS were run in three replications.Statistical treatment of the data obtained was performed at a significance level of 5%.The results are presented as the average value ± the coverage interval.
The liquid broth containing the spores of the microorganisms was prepared using standard growth media: Potato Extract Glucose broth for B. subtilis EMC B-12, X. arboricola S3, A. Tumefaciens A-47, A. solani K-100054, F. graminearum FG-30, and R. solani EMC F-895 and Corynebacterium Selective Agar for Rathayibacter iranicus EMC Ac-1602.

Antimicrobial Assay In Vitro
In the experiments, the determination of the minimum inhibitory concentration (MIC) of the extracts of the following cultivar plants, A. annua, A. dracunculus, A. santonica, A. abrotanum, and A. scoparia, was performed using the method of two-fold serial dilution [116] with the modification provided in [117,118].Fungistatic activity was defined by the serial dilution method [119] in liquid medium.
The 24 h bacterial cultures a swell as the fungal cultures grown from the 7th to the 14th d were used in the study.The final inoculum contained 10 5 CFU/mL of bacteria and 1.1-1.5 × 10 2 CFU/mL of fungi.The bacteria concentration was determined using a DEN-1B densitometer (Biosan, Riga, Latvia).Tubes containing only culture medium were used as the control.
In order to establish the minimal concentrations of the bactericidal and fungicidal agents (MBC and MFC, respectively), 10 mL of inoculum (or a piece of fungal mycelium) obtained from tubes with no discernible growth was incorporated into Petri dishes containing agarized nutrient medium via an inoculation loop.
The results of the bacterial growth were observed every day for 5 days at 30 • C for R. iranicus VKM Ac-1602, B. subtilis VKM B-12, and A. tumefaciens A-47 and at 25 • C for X. arboricola S3.The fungus was incubated in a thermostat at 26 • C for 7 d.The growth of the microorganisms was visually determined [76].
Norfloxacin, chloramphenicol, and difenoconazole were used as reference compounds in the experiments.
All studies were conducted with a three-fold repetition, and the results of the definitions were handled in accordance with the international standards [120,121].

Conclusions
The ethanol extracts of Artemisia L. cultivated on the southern coast of the Crimean peninsula in the Nikitsky Botanical Garden contain acyclic, monocyclic, and bicyclic monoterpenoids, monocyclic, bicyclic, tricyclic, and eudesmane sesquiterpenoids, coumarins, and phenolic compounds as the main compounds, having antibacterial and antifungal activity against phytopathogenic microorganisms.
The ethanol extract of the A. scoparia of Tavrida variety was the most toxic for microscopic phytopathogenic fungi and Gram-negative bacteria, and the A. dracunculus extract of the Smaragd variety had a toxic effect on Gram-positive bacteria.The minimum inhibitory concentrations of the above extracts were 150 µg/mL due to their high antimicrobial activity.
The nematocidal activity of the ethanol extracts of A. annua of the Novichok variety, A. dracunculus of the Smaragd variety, and A. santonica of the Citral on C. elegans, a soil nematode, characterized by a concentration range of 31.3-1000µg/mL, was established.The mutant analysis revealed the insignificant effects of A. dracunculus and A. santonica extracts on the muscarinic and nicotinic receptors of C. elegans.Moreover, the A. annua extract had compounds acting on both the N-subtype nAChR receptors and levamisole receptors of α-subunit UNC-63.
The total results of the studies on the ethanol extracts obtained from the five varieties of the Artemisia L. species growing in the Nikitsky Botanical Garden collection of aromatic and medical plants indicated that, due to their high content of natural chemical compounds of different classes, showing high antibacterial, antifungal, and nematocidal activity, they are advanced initial raw materials as a basis for designing in the future the most-advanced and selective-goods for organic agriculture using the natural potential of the plants (Tables S1-S3).

Table 4 .
Toxic effects of Artemisia extracts on Caenorhabditis elegans of the N2 wild-type line.

Table 5 .
Toxic effects of Artemisia extracts on Caenorhabditis elegans, both the N2 wild-type and mutant lines of RB896, RB756, JD217, and RB918.
• C; interface temperature of 270 • C; ion source temperature of 250 • C; column oven initial temperature of 60 • C; initial thermostat conditioning time of 1 min; column temperature rate of 5 • /min up to 210 • C; subsequent temperature rate of 12