Essential Oil Composition of Ambrosia artemisiifolia and Its Antibacterial Activity against Phytopathogens ^†

Abstract

The composition of essential oil from aerial parts of Ambrosia artemisiifolia L. from Bor (Serbia) was analyzed. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography (GC-FID, GC-MS). In total, 45 compounds were detected (98.49% of the total). The essential oil was dominated by monoterpene (45%) and sesquiterpene (38.51%) hydrocarbons. The principal constituents were germacrene D (25.3%), limonene (21.6%), and α-pinene (15.7%). The microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oil against five Gram-negative phytopathogenic strains. Essential oil exhibited strong antimicrobial activity against two Xanthomonas campestris strains and one referent and one natural isolate of Ervinia amylovora, causative agents of black rot and fire blight.

1. Introduction

Ambrosia L. (Asteraceae, Heliantheae, Ambrosiinae) includes nearly 35–40 species [1,2] distributed mainly in America [1]. The genus comprises annual or perennial [3] anemophilous plants [4].

Ambrosia artemiisifolia L. is an annual herb native to North America [5,6], but it is widespread in many parts of the world [7]. Ragweed was introduced from North America into Europe in the 19th century [5], and it grows in the central and southern parts of the continent, usually in wasteland near urban areas [8]. Nowadays, it is known in most European countries [9]. This plant is also widespread in Serbia, especially in the northern part of the country [10].

Ragweed has a strong reproductive capacity. Each plant can produce a large number of seeds and pollen, causing numerous allergic reactions [11].

Only a few previous studies focused on the analysis of the composition of the essential oil of A. artemisiifolia and showed that different classes of specialized metabolites are present in the essential oil. Monoterpenes and sesquiterpenes were dominant compounds [6,12].

To the best of our knowledge, no data exist regarding the antibacterial activity of A. artemisiifolia essential oil.

The objectives of the present study were to determine the composition of the EOs of ragweed and investigate its potential use as a biological control agent.

2. Material and Methods

2.1. Plant Material

Plant material of A. artemisiifolia was collected in October 2020, during the flowering period near the town Bor, in Eastern Serbia. Plants were identified using flora of Serbia and Europe [3,10]. Voucher specimens were deposited at the Herbarium of the University of Belgrade—Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac” (BEOU 17821). Standard herbarium acronym follows Index herbariorum [13].

2.2. Isolation of Essential Oil

Dried flowering aerial parts (200 g) were chopped and placed in a round-bottomed flask, and then 2 l of cold distilled water was added. Hydrodistillation was performed 3 times for 3 h using the Clevenger-type apparatus, according to the procedure described in Ph. Eur. 6 [14]. The obtained oils were stored at 4 °C before the GC analyses.

The extraction yield of oil was calculated according to the equation given: y = V/W × 100 where y is the oil yield (%, w/w), V is the mass of extracted plant oil (g), and W is the mass of dried plant material (g).

2.3. GC-FID and GC/MS Analyses

The GC-FID and GC/MS analyses were conducted according to the procedure described in [15].

2.4. Antibacterial Activity

2.4.1. Bacterial Strains and Growth Conditions

Antibacterial activity was tested using five Gram-negative phytopathogenic strains Pseudomonas syringae pv. syringae, Xanthomonas pv. campestris, X. arboricola pv. juglandis, Erwinia amylovora, E. amylovora. The bacterial strains were cultured in TY medium (composition g/L: tryptone 5, yeast extract 5, CaCl₂ × 2 H₂O 0.9) for 48 h at 30 °C. Suspensions were prepared in phosphate saline buffer (1 × PBS, Sigma Aldrich, St. Louis, MO, USA) in the final concentration of 10⁶ CFU/mL.

2.4.2. MIC Assay

The microdilution method [16] was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the A. artemisiifolia essential oil. Two-fold serial dilutions with TY medium in 96-well microtiter plates were performed. Except for the sterility control, each well was inoculated with 20 µL of bacterial suspensions (1 × 10⁶ CFU/mL), reaching a final volume of 200 µL. Final essential oil concentrations in the first well ranged from 0.008–2 mg/mL. Besides a negative control, a sterility control, and control for the solvent (DMSO), the antibiotic streptomycin (Sigma-Aldrich, USA) was tested as a positive control in a concentration range of 0.003–0.2 mg/mL. The final concentration of dimethyl sulfoxide (DMSO) as a solvent was 10%. All dilutions were performed in duplicate, and the results are expressed in mg/mL. After reaching the final volume, 22 µL of resazurin as an indicator in the final concentration of 0.675 mg/mL was added, and the 96-well microtiter plates were incubated for 48 h at 30 °C. According to the resazurin reaction, the lowest concentration, which showed no change in color was defined as the MIC. The lowest concentration that after sub-culturing did not show bacterial growth overnight was defined as the MBC value.

3. Results

3.1. A. Artemisiifolia EO Composition

The yield of essential oil was 0.03%. The oil was transparent yellow, with a sharp and strong smell. The conducted GC-FID, and GC-MS analyses resulted in the detection of 45 compounds (41 identified, 4 unidentified), making on average 98.49% of the total oil. All identified compounds are listed in

Table 1. Chemical constituents of the essential oil of investigated A. artemisiifolia.

No.	RI 1	Compound	[%] 2
1	903	Santolina Triene	0.10
2	929	α-Pinene	15.75
3	944	Camphene	0.33
4	969	β-Phellanderene	0.94
5	973	β-Pinene	1.77
6	987	Myrcene	4.54
7	1027	Limonene	21.59
8	1139	trans-Pinocarveol	0.21
9	1144	1,3,8-p-Menthatriene	0.31
10	1164	Borneol	0.63
11	1166	Ni (109,69,93,81,41)	0.30
12	1285	Bornyl Acetate	1.81
13	1290	Lavandulyl Acetate	0.46
14	1337	δ-Elemene	0.29
15	1375	α-Copaene	0.22
16	1384	β-Bourbonene	0.58
17	1390	β-Cubebene	0.27
18	1391	β-Elemene	0.33
19	1419	(E)-Caryophyllene	3.22
20	1429	β-Copaene	0.47
21	1435	trans-α-Bergamotene	0.53
22	1453	α-Humulene	1.09
23	1457	(E)- β-Farnesene	0.28
24	1475	β-Chamigrene	0.48
25	1482	Germacrene D	25.26
26	1484	β-Selinene	1.35
27	1496	Bicyclogermacrene	1.92
28	1508	β-Bisabolene	0.91
29	1511	Lavandulyl isovalerate	0.51
30	1515	δ-Amorphene	0.39
31	1523	δ-Cadinene	0.75
32	1556	Germacrene B	0.18
33	1559	Ni sesquiterpene (159,177,135,41,91)	0.44
34	1576	Spathulenol	1.98
35	1582	Caryophyllene oxide	3.06
36	1608	Humulene epoxide II	0.64
37	1610	β-Atlantol	0.39
38	1617	Junenol	0.92
39	1622	1,10-di-epi-Cubenol	0.25
40	1631	Muurola-4,10(14)-dien-1- β-ol	0.29
41	1634	Ni (246,119,105,91,187)	0.35
42	1653	α-Cadinol	0.66
43	1658	Valerianol	1.59
44	1686	Germacra-4(15),5,10(14)-trien-1-α-ol	0.81
45	1766	Ni (81,93,107,123,147)	0.35
Total monoterpenes			48.43
Monoterpene hydrocarbons			45.01
Oxygenated monoterpenes			3.42
Total sesquiterpenes			50.05
Sesquiterpene hydrocarbons			38.51
Oxygenated sesquiterpenes			11.54
Unknown			1.00
TOTAL			98.49

¹ The retention indices (RI) were experimentally determined using the standard method involving retention times (tR) of n-alkanes, which were injected under the same chromatographic conditions. ² Contents are given as percentages of the total essential oil composition; Ni = not identified.

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The results showed that monoterpene hydrocarbons and sesquiterpene hydrocarbons were the dominant constituents in the EO (45% and 38.51%, respectively). Oxygenated monoterpenes and oxygenated sesquiterpenes were also present, but in smaller quantities (3.42% and 11.54%, respectively). The most dominant constituents were germacrene D (25.3%), limonene (21.6%), and α-pinene (15.7%).

3.2. Antibacterial Activity of A. artemisiifolia EO

Tested essential oil exhibited strong antimicrobial activity against both X. campestris strains, and against one referent and one natural isolate of E. amylovora. Strains were inhibited by lower concentrations which could be designated as similar detected in the positive control of streptomycin. Moderate activity was detected against P. syringae pv. syringae, while X. arboricola pv. juglandis was the most resistant strain tested. All inhibitory and bactericidal activities of EO were below the detected inhibitory concentrations of DMSO as solvent. Results of tests on A. artemisiifolia oil antibacterial activity are given in

Table 2. Antibacterial activity of A. artemisiifolia EO.

Phytopathogenic Strains	Ambrosia EO (mg/mL)		DMSO (%)		Streptomycin (mg/mL)
	MIC	MBC	MIC	MBC	MIC	MBC
Xanthomonas pv. campestris	0.004	0.008	1.875	2.500	0.025	0.100
Xanthomonas pv. campestris	0.063	0.125	0.469	0.625	0.050	0.100
Erwinia amylovora	0.016	0.031	7.500	10.000	0.006	0.100
Erwinia amylovora	0.047	0.063	7.500	10.000	0.012	0.100
Pseudomonas syringae pv. syringae	0.500	2.000	>10.000	-	0.006	>0.200
Xanthomonas arboricola pv. juglandis	1.500	2.000	>10.000	-	0.003	0.013

- not detected in the range of tested concentrations.

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4. Discussion

In the present study, the most abundant compounds were germacrene D (25.3%), limonene (21.6%), and α-pinene (15.7%). These results are congruent with the scarce literature data [6,12]. There are some differences in the relative amounts of major classes of compounds between EOs of A. artemisiifolia and related species. The oil of A. artemisiifolia is much more abundant in monoterpene hydrocarbons and sesquiterpene hydrocarbons, in contrast with A. trifida [17].

It was shown that significant bactericidal activity of A. artemisiifolia essential oil was effective even in very dilute solutions against a broad range of human opportunistic bacterial strains, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus subtilis, Sarcina lutea, Shigella flexneri and Salmonella enteritidis [12]. In general, plant extracts and essential oils contain numerous compounds such as sesquiterpenoids, which can exhibit antimicrobial activity [18]. Although many sesquiterpene lactones have been related to allergenic effects, some previous studies showed how isabelin, the lactone isolated from A. artemisiifolia, was able to inhibit soil-borne bacteria [19]. That might imply the potential of these molecules to modify the surrounding soil microbiota and associated pathogens eventually. In the present study, A. artemisiifolia essential oil exhibited strong antimicrobial activity against X. campestris and E. amylovora strains, causative agents of black rot and fire blight. Thus, our results indicate that essential oil produced by invasive plant A. artemisiifolia could be a valuable source of compounds with great potential for biological control of economically important phytopathogens.