In Vitro Determination of the Antifungal Activity of Artemisia campestris Essential Oil from Algeria

Abstract

The chemical composition of the essential oil isolated from the aerial parts of Artemisia campestris from Algeria and its antifungal activity against 10 filamentous fungal strains were investigated. The A. campestris essential oil was obtained in a yield of 0.71% (v/w). The major constituents of the oil were α-pinene (18.65%), β-pinene (16.78%), β-myrcene (17.34%), and germacrene D (10.34%). Our study showed that A. campestris essential oil was a potent antifungal agent against some pathogenic fungal species. Fusarium graminearum was the most sensitive strain to A. campestris essential oil with minimal inhibitory concentration and minimal fungicidal concentration values of 1.25 µL/mL (v/v). The essential oil also exhibited a strong fungicidal activity against the tested fungi, except for Penicillium citrinum, P. viridicatum, and Aspergillus niger (MFC >20 µL/mL). Our findings suggested the application of A. campestris essential oil as a biofungicide in order to reduce the dependence on synthetic fungicides and ensure food safety and quality.

Keywords:

• Antifungal activity
• Artemisia campestris
• Chemical composition
• Essential oil

INTRODUCTION

Many cereals and other crops are susceptible to fungal attack either in the field or during storage.^[1] This infection not only results in reducing crop yield and quality with significant economic losses, but also in contaminating grains with poisonous fungal secondary metabolites called mycotoxins.^[2] In order to protect food quality and the environment, low persistent synthetic fungicides are still relevant at present to prevent diseases of food crops.^[3] Though, synthetic fungicides improve plant protection, most of them result in environmental pollution, health hazards, and affect the natural ecological balance.^[4] Today, there are strict regulations on chemical pesticide use, and there is political pressure to remove the most hazardous chemicals from the market,^[5] which necessitates finding alternatives or complements to synthetic fungicide.

In recent years, the need to develop fungal disease control measures using phytochemicals as alternatives to synthetic chemicals has become a priority of scientists worldwide.^[6] Therefore, researchers have focused on the potentiality of plants and their metabolites to inhibit toxigenic fungus growth and/or toxin production as a useful tool for controlling mycotoxin contamination of crops and agricultural commodities.^[7] Plant products, especially essential oils, are recognized as one of the most promising groups of natural compounds for the development of safer antifungal agents.^[8] The antimicrobial compounds in plant materials are commonly contained in the essential oil fraction of leaves (rosemary, sage), flowers and flower buds (clove), bulbs (garlic, onion), rhizomes (asafoetida), fruit (pepper, cardamom), or other parts of the plant.^[9,^10] Essential oils consist of a mixture of bioactive compounds such as esters, aldehydes, ketones, and terpenes. These compounds may be lethal to microbial cells or they may simply inhibit the production of a metabolite (e.g., mycotoxins).^[11,12] Numerous reports clearly indicated that essential oils should find practical application in the inhibition of mycotoxin production by mycotoxigenic fungi.^[13]

Several studies have been reported on the use of plant essential oils to control toxigenic fungi and their toxins. From the Algerian flora, 11 species of Artemisia are recorded.^[14] Among them, Artemisia campestris which belongs to the Asteraceae family, widespread in the south of Algeria, commonly known as “dgouft.” Many reports are available on the analysis of the essential oil compositions and their antimicrobial activities from many Artemisia sp. However, none of them have studied the antifungal activity of A. campestris essential oils against phytopathogenic and post-harvest fungal species. Therefore, the aims of the present study are to characterize the chemical composition of the essential oil isolated from the aerial parts of Artemisia campestris from Algeria and to determinate in vitro its antifungal activity against 10 filamentous fungal strains within the genera of Aspergillus, Fusarium, and Penicillium.

MATERIALS AND METHODS

Plant Material

Aerial parts of Artemisia campestris were collected during the flowering stage from Oued Moura, Laghouat region of Algeria. The botanical identification of the plant was done at Laboratory of Plant Ecology, Department of Biology, Laghouat University, Algeria. The plant material was air dried in the dark at room temperature (<30°C) and then collected in paper bags until analysis.

Isolation and Analysis of Essential Oils

The essential oil was isolated by hydrodistillation for 4 h from the dried aerial parts using a Clavenger-type apparatus, according to the method recommended in the British Pharmacopoeia.^[15] An analysis of the oil was carried out by gas chromatography coupled to mass spectrometry (GC–MS) using a Perkin Elmer Clarus 500 instrument, equipped with SGE capillary column (60 m 0.25 mm ID; BPX5, 0.25 µm film thickness, USA). Column oven temperature programmed at 60–250°C at 4°C/min, 250°C (10 min); and injector temperature was 240°C. Helium was used as carrier gas at a flow rate of 1.5 mL/min. A sample was diluted in n-hexane (1%, v/v) and the injection volume was 1 μL. Mass spectra were taken over m/z 35–425, using an ionization voltage 70 ev with a split/split-less mode. The identification of components was made by computer matching with the NIST and Wiley libraries. The relative percentage of the oil constituents were calculated from peak area of the chromatogram.

Antifungal Activity Strains

The antifungal activity of Artemisia campestris essential oil was evaluated against 10 filamentous fungal strains: two type strains from the Agricultural Research Service (ARS) culture collection (Aspergillus ochraceus, NRRL 3174, Aspergillus flavus NRRL 3251), four type strains from the Belgian Coordinated Collections of Micro-organisms (Fusarium graminearum MUCL 53452, Fusarium moniliforme MUCL 53645, Penicillium citrinum MUCL 31475, Penicillium expansum MUCL 29192) and one type strain from the Central Bureau of Fungal Cultures (CBS) culture collections of micro-organisms (Aspergillus parasiticus CBS 100926). The other strains (Penicillium viridicatum, Aspergillus niger, and Fusarium culmorum) were from the culture collection of the Department of Agriculture, Faculty of Science, Laghouat University-Algeria. Prior to antifungal susceptibility testing, each isolate was cultured on potato dextrose agar (PDA) for 7–14 days at 25°C to ensure rapid sporulation and purity.

Antifungal Activity

A broth macrodilution method was used to determine the minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC), according to the Clinical and Laboratory Standards Institute^[16] M38-A for filamentous fungi. For the assay, the essential oil was two-fold diluted in dimethyl sulfoxide (DMSO; Sigma 34943, USA), with concentrations ranging from 0.04 to 20 μL/mL. The final concentration of DMSO was ≤1%. Recent cultures of each strain were used to prepare the cell suspension adjusted to 0.4–5×10⁴ colony-forming unit (CFU)/mL for filamentous fungi using a spectrophotometer (Jenway, 6405 ultraviolet-visible [UV/VIS], UK) at 530 nm. All tests were performed in RPMI-1640 medium (Sigma R6504, USA) buffered to a pH 7.0 with 3-Morpholinopropane-1-sulfonic acid (MOPS) buffer (Sigma M3183, USA) at a concentration of 0.164 mol/L. After inoculation, the test tubes were incubated aerobically at 35°C for 48–72 h, including two control tubes per strain and then the MICs were determined. The MIC was defined as the lowest essential oil concentration preventing any fungal growth as detected visually. After the MIC reading, aliquots (20 μL) of broth from each negative tube were taken and cultured in Sabouraud dextrose agar (Eur-Pharm, 1024.00, Spain) plates in order to evaluate MFCs. The plates were then incubated for 72 h at 35°C. The MFC was defined as the lowest essential oil concentration showing either no growth or fewer than three colonies to obtain an approximately 99–99.5% killing activity. All experiments were performed in triplicate and repeated if the results differed.

RESULTS AND DISCUSSION

The Artemisia campestris essential oil was obtained in a yield of 0.71% (v/w; Table 1). A similar yield (0.7%) was obtained by Baykan Erel et al.^[17] from A. campestris of western and southwestern Turkey. Belhattab et al.^[18] obtained a yield of 0.66% (v/w) from A. campestris grown in Algeria. However, Dob et al.^[19] reported that this plant is poor in essential oil (yield = 0.1% w/w). A noticeable variation has been found in the oil yield of A. campestris, it ranged 0.65–1.20% (v/w). This variation could be due either to chemotypes or to differences in the climatic and geographical parameters (temperature, rainfall, altitude, wind direction, number of sunshine hours, etc.), as well as the periods of the collect (growth stage).^[20]

TABLE 1 Chemical composition of Artemisia campestris essential oil

N°	Components^a	Retention time (min)	% Composition
			Artemisia campestris
1	α-Pinene	15.50	18.65^b
2	β-Pinene	18.35	16.78
3	β-Myrcene	18.76	17.34
4	Limonene	21.05	5.90
5	β-Ocimene Y	21.74	3.22
6	γ-Terpinene	22.49	3.83
7	α-Terpinolene	23.70	0.71
8	Alloocimene	25.62	0.41
9	α-Campholene aldehyde	26.34	0.17
10	Trans-pinocarveol	27.16	0.32
11	Verbenol	27.39	0.16
12	Pinocarvone	28.15	0.19
13	4-Terpineol	28.58	0.36
14	α-Terpineol	29.34	0.51
15	Myrtenal	29.60	0.28
16	Carvone	31.52	1.65
17	Bornyl acetate	32.34	0.28
18	Thymol	33.24	0.24
19	Citronellyl acetate	34.28	0.17
20	Copaene	35.18	0.32
21	Neryl acetate	35.43	0.44
22	Aromadendrene	35.60	0.21
23	Caryophyllene	36.93	0.54
24	β-Farnesene	37.54	0.65
25	Germacrene D	39.13	10.34
26	γ-Elemene	39.56	1.54
27	δ-Cadinene	40.08	0.77
28	Spathulenol	42.71	3.65
29	Photonerol	43.16	0.35
30	Epiglobulol	44.10	0.73
31	T-Cadinol	44.47	0.07
32	α-Cadinol	44.96	1.12
33	β-Eudesmol	45.15	1.05
	Total identified		92.95
	Oil yield (%)		0.71

^aComponents listed in order to their elution on the SGE column.

^bIdentification method: GC–MS.

The qualitative and quantitative compositions of A. campestris oil are presented in Table 1. A total of 33 components were identified using GC–MS, representing more than 92.95% of the total sample. The major constituents of A. campestris oil were α-pinene (18.65%), β-pinene (16.78%), β-myrcene (17.34%), and germacrene D (10.34%). Other compounds were also present in A. campestris oil with minor concentrations. Akrout et al.^[20] found that β-pinene was the most abundant component (24.0–49.8%) for all samples of A. campestris collected from four areas of the southern Tunisia. α-Pinene (23.9, 23.0, and 29.2%) and spathulenol (23.9, 15.8, and 29.2%) were the main constituents in the flower, leaf, and stem oils, respectively.^[21] Belhattab et al.^[18] reported that the main components of A. campestris grown in Algeria were α-terpenyl acetate and α-pinene (19 and 18%, respectively). However, Bakchiche et al.^[22] found that the main constituents of this oil were β-pinene 25.6% and sabinene (17%). In our study, the oil is dominated by the presence of α-pinene, β-pinene, β-myrcene, and germacrene D which is noticeably different from the oils of the same plant from the other studies. The extracted product can vary in quality, quantity, and in composition according to climate, soil composition, plant organ, age, and vegetative cycle stage.^[23] It is assumed that the specific climatic factors of the various growing sites influence the composition of the essential oils.^[24] Sefidkon et al.^[25] confirmed the effect of climatic factors and distillation methods on the changes in the chemical composition of essential oil of the same plant.

The results of the antifungal activity of A. campestris essential oil are shown in Table 2. Fusarium graminearum was the most sensitive strain to A. campestris essential oil with MIC and MFC values of 1.25 µL/mL (v/v). This oil showed a potent inhibitory effect against F. moniliforme, F. culmorum, P. expansum, A. flavus, A. ochraceus, and A. parasitcus with MIC value of 2.5 μL/mL (v/v). Penicillium viridicatum and Aspergillus niger were the most resistant fungi to A. campestris essential oil (MIC = 10 µL/mL) while the growth of P. citrinum was effectively inhibited at MIC value of 5 μL/mL (v/v). It must be noted that MIC values of A. campestris essential oil showed a variability among all the tested fungi (1.25–10 µL/mL) which could be related mainly to the fungal specie. The essential oil also exhibited strong fungicidal activity against the tested fungi, except for P. citrinum, P. viridicatum, and A. niger (MFC >20 µL/mL). The oil concentration of 2.5 µL/mL (v/v) exhibited fungicidal activity against F. moniliforme, F. culmorum, P. expansum, and A. flavus, whereas 5 μL/mL (v/v) was enough to exert a fungicidal effect against A. parasitcus and A. ochraceus. Few studies have investigated the antifungal effect of essential oils from different Artemisia sp. against agricultural pathogenic fungal species. In one study,^[26] the 20 µL doses of the oils of Artemisia santonicum, A. spicigera, and A. absinthium, as well as A. dracunculus were found to be fungitoxic against 34 fungal species tested. Wenqiang et al.^[27] reported that the essential oil of Artemisia argyi Lévl. et Vant inflorescence exhibited antifungal activity against Botrytis cinerea and Alternaria alternate, two common storage pathogens of fruits and vegetables. Furthermore, the highest level of antifungal activity is observed against A. niger for all the investigated oils of Artemisia absinthium (diameter of inhibition: 14–16 mm, MIC: 12.5%, MBC: 25%).^[28] However, the effects A. campestris essential oils against phytopathogenic and post-harvest fungal species have not been studied. In our study, A. campestris essential oil at 5 μL/mL (v/v) was found to be the fungitoxic concentration against the tested fungal strains, except for P. citrinum, P. viridicatum, and A. niger. So, this fungicidal activity could be related to the presence of high concentrations of the main terpenes previously presented. Kordali et al.^[26] reported that the essential oils rich in oxygenated monoterpenes have a relatively higher antifungal activity. In addition, the essential oils consist in complex mixtures of numerous constituents. Other major or minor compound(s) might cause the antifungal activity exhibited. Possible synergistic and antagonistic effects of compound(s) in the essential oil should also be taken into consideration.^[26] Our oil had also weak antifungal activity against P. citrinum, P. viridicatum, and A. niger (MFC >20 µL/mL). Some essential oils had no effect on mycelial growth and/or aflatoxin production.^[29,30] Aspergillus niger was used in hydroxylation reactions with terpenes^[31] and Demyttenaere and De Kimpe^[32] reported on the biotransformation of geraniol, nerol, and citral by spores of Penicillium digitatum. Enzymes and extracts from bacteria, cyanobacteria, yeasts, microalgae, fungi, plants, and animal cells have been used for the production and/or bioconversion of terpenes.^[33] It can be concluded that some fungal strains may consume terpenes as a carbon source, degrade or transform them, which may explain the ineffectiveness of some essential oils against certain fungal species. Shan et al.^[34] reported that the action mode of antimicrobial agents essentially depends on the type of the treated microorganism in relationships with their cell wall structure and the outer membrane arrangement. Several authors^[35–37] have observed that essential oils can cause morphological changes as lack of sporulation, loss of pigmentation, aberrant development of conidiophores, and distortion of hyphae in the Aspergillus species. In addition, it is important to recognize that there are complex interactions with environmental factors, such as water availability and efficiency of essential oils.^[13] Considering the different mechanisms of action and/or targets in the cell of essential oils, the application of a combination of two or more essential oils as biofungicides needs to be explored by further studies.

TABLE 2 Antifungal activity (MIC, MFC) of Artemisia campestris essential oil

Strains	Artemisia campestris
	MIC^a	MFC^a
Fusarium graminearum MUCL 53452	1.25	1.25
Fusarium moniliforme MUCL 53645	2.5	2.5
Aspergillus parasiticus CBS 100926	2.5	5
Aspergillus ochraceus NRRL 3174	2.5	5
Aspergillus flavus NRRL 3251	2.5	2.5
Penicillium expansum MUCL 29192	2.5	2.5
Penicillium citrinum MUCL 31475	5	>20
Aspergillus niger	10	>20
Fusarium culmorum	2.5	2.5
Penicillium viridicatum	10	>20

^aMIC and MFC were determined by a macrodilution broth method and expressed in µL/mL (v/v).

CONCLUSIONS

In conclusion, the present findings showed that A. campestris essential oil was a potent antifungal agent against some pathogenic fungal species which could be attributable to the presence of high concentrations of α-pinene, β-pinene, β-myrcene, and germacrene D in this oil. In order to reduce the dependence on synthetic fungicides and ensure food safety and quality, our study suggested the application of A. campestris essential oil as a biofungicide in greenhouse crops or in storage silos taking into consideration the volatile nature of essential oils. The impact on toxicology and safety of these compounds require further investigations before considering their use in phytoprotection or in food preservation.