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Proceeding Paper

Evaluation of the Antifungal Effect of Carvacrol-Rich Essential Oils: In Vitro Study on the Phytopathogenic Fungi Alternaria and Fusarium †

by
Vasileios Papantzikos
*,
Georgios Patakioutas
and
Paraskevi Yfanti
Department of Agriculture, Arta Campus, University of Ioannina, 47100 Arta, Greece
*
Author to whom correspondence should be addressed.
†
Presented at the 3rd International Online Conference on Agriculture (IOCAG 2025), 4–5 September 2025; Available online: https://sciforum.net/event/IOCAG2025.
Biol. Life Sci. Forum 2025, 54(1), 1; https://doi.org/10.3390/blsf2025054001 (registering DOI)
Published: 21 November 2025
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Abstract

Certain essential oils (EOs) from aromatic plants have shown potent antifungal effects. In this work, an in vitro study was conducted to examine the antifungal effect of EOs obtained from Greek flora aromatic plants that belong to the Lamiaceae family on two phytopathogenic fungi. Specifically, Satureja horvatii ssp. macrophylla, Coridothymus capitatus, and Origanum vulgare ssp. hirtum were tested against Alternaria sp., which causes tomato black spot, and Fusarium sp., which causes potato tuber dry rot during storage. The antifungal activity of the EOs was assessed using fumigant assays, and their chemical composition was analyzed using gas chromatography–mass spectrometry (GC–MS). After 8 days of incubation at 26 ± 1 °C, the EOs of O. vulgare ssp. hirtum and C. capitatus completely inhibited mycelial growth at 2 µL plate−1 in the case of Fusarium sp. and at 3 µL plate−1 in the case of Alternaria sp. S. horvatii ssp. macrophylla completely inhibited the mycelial growth of Fusarium sp. at 3 µL plate−1 and that of Alternaria sp. at 4 µL plate−1. All the essential oils used in the experiments were rich in carvacrol (41.4–70.0%), while thymol levels ranged from 0% to 18.9%. This fumigant effect could be further evaluated for the fruits’ postharvest protection from phytopathogenic fungi during storage.

1. Introduction

Phytopathogenic fungi are the main cause of significant economic losses in fruits and vegetables after harvest, during their storage, transportation, and marketing [1]. Due to the high toxicity of synthetic fungicides, their long degradation period, and residuality [2], their use is subject to a set of restrictive measures for postharvest disease control [3]. In addition, the adverse effects of their use possibly pose risks to human health [4]. The use of EOs, especially those defined as generally recognized as safe (GRAS), represents an interesting alternative method for fruit and vegetable protection from postharvest diseases [5], as well as a safer method for the preservation of agricultural products [6,7,8].
Fusarium sp. (Hypocreales: Nectriaceae) infects potato tubers mainly through wounds created during harvest, coupled with the appearance of dark-colored spots, which spread and wrinkle in concentric circles [9,10]. On the affected surface, mycelial hyphae with a white-pink or sub-green color appear. Thus, Fusarium sp. produces trichothecene toxins and fusaric acid [11,12,13] and causes intravascular necrosis, wilting, and brown discoloration, and the tuber then desiccates, shrinks, and mummifies [14,15].
Alternaria sp. is responsible for the common early blight disease or target spot, resulting in brown and necrotic spots arranged in concentric rings on fruits [16]. Alternaria alternata and Alternaria solani (Pleosporales: Pleosporaceae) can cause severe damage to tomatoes and other Solanaceae crops [17]. When Alternaria sp. enters the plant tissue, it synthetizes mycotoxins capable of causing chlorosis and necrosis in plants [18]. One such toxic metabolite is tenuazonic acid [19], which inhibits protein synthesis. In addition, ziniole and altersolanol-A affect membrane permeability [20]. The host-specific toxins (HSTs) produced by A. alternata show unique modes of action in and toxicity to their host plants, such as the AM mycotoxin in pear and apple fruits, affecting chloroplasts [21].
According to the literature data, aromatic plants with pharmaceutical properties belonging to the Lamiaceae family have significant effects on the growth of phytopathogenic fungi in vitro [22,23,24] and in vivo [25,26,27]. Such species endemic to the Mediterranean region are O. vulgare ssp. hirtum, commonly known as Greek oregano; C. capitatus, known as Spanish oregano; and S. horvatii ssp. macrophylla, a savory species grown in the northwest regions of Greece and endemic to the Greek flora [28]. The utilization of EOs from aromatic plants as phytopathogen inhibitors is of great interest; thus, when used as antifungal agents, they permeabilize the fungal cell membranes [29], and this may result in destabilization and eventual rupture of the cell membrane [30,31]. Some EOs can cause extensive damage to the plasma membrane of phytopathogenic fungi [32,33], blockading ergosterol synthesis and finally causing cell membrane dysfunction [34].
Potato and tomato farming is economically significant for rural Greek farmers. The economic damage caused by postharvest diseases as a result of the phytopathogenic fungi Alternaria sp. and Fusarium sp. in stored tomato fruits and potato tubers underscores the relevance of carrying out this study. The EOs of aromatic plants have shown potential in combating the postharvest diseases of economically important crops in Greece, such as tomato and potato. The aim of this work was to conduct an in vitro study to investigate the antifungal effect of EOs from three plants of the Lamiaceae family, S. horvatii ssp. macrophylla, C. capitatus, and O. vulgare ssp. hirtum, on the phytopathogenic fungi Fusarium sp. and Alternaria sp.

2. Methods

2.1. Experimental Design

The antifungal effect of the EOs was investigated in vitro using Petri dishes, evaluating their effect on the growth of two plant pathogenic fungi that cause postharvest diseases: the Fusarium sp. on potato tubers and the Alternaria sp. on tomato fruits. In each of the two phytopathogenic fungi, the effect of carvacrol and the EOs of the aromatic plants C. capitatus, O. vulgare ssp. hirtum, and S. horvatii ssp. macrophylla was assessed. Petri dishes (plates) without EO were used as a control for each of the phytopathogenic fungi (0 μL plate−1). Three replicates were performed for each treatment. The in vitro bioassays were carried out at the Laboratory of Productive Agriculture and Plant Health of the Department of Agriculture, University of Ioannina.

2.2. Essential Oils Used in the Experiment

The EOs used in the experiments were obtained from dried plant material by hydro-distillation (Clevenger apparatus). The aerial part of the selected aromatic plants was collected from wild-grown populations in the Epirus region, Greece. An analysis was performed using GC-MS (Master GC-TOF MS, Dani) equipped with an autosampler, according to previously reported chromatographic conditions [35]. The compounds were identified by comparing the mass spectra with NIST library data. Also, their retention indices relative to n-alkanes were compared with the literature data.

2.3. Preparation of Culture Medium

For the in vitro culture of the phytopathogenic fungi, nutrient medium PDA (Potato Dextrose Agar) with chloramphenicol 0.1% w/v was prepared after sterilization in an autoclave (AVS-N, Raypa, R. Espinar S.L., Barcelona, Spain) at 121 °C for 20 min. Then, the culture medium was dispensed into plastic Petri dishes (Sarstedt AG & Co. KG, Nümbrecht, Germany), 90 mm in diameter (20 mL plate−1), inside an airstream horizontal laminar flow device (AHC-4A1, Esco Micro Pte., Ltd., Singapore) under aseptic conditions, and, after a few minutes, it solidified.

2.4. Isolation of Phytopathogens

Infected potato tubers and tomato fruits showing characteristic symptoms of dry rot and black spot, respectively, were transported to the laboratory (Scheme 1). The isolation of the phytopathogenic fungi took place under laminar airflow in the laboratory using a preparation needle, immersed in a solution of 100% ethanol, flamed over a Bunsen burner. Samples were removed and inoculated in clean Petri dishes with PDA nutrient medium and incubated (FOC 120i, Velp Scientifica Srl, Usmate Velate, Italy) at 26 ± 1 °C. The plates were routinely checked for culture purity. Fungal strains were isolated and identified by microscopic observation under a trifocal microscope (DM1000, Leica GmbH, Wetzlar, Germany), staining the mycelial hyphae with lactophenol blue solution (Merck KGaA, Darmstadt, Germany).

2.5. Fumigant Assay

The antifungal activity of the EOs was evaluated in vitro by monitoring their fumigant effect on mycelial growth, as described by Feng et al. (2011) [36]. A 7 mm diameter mycelium disc (inoculum) was removed from the periphery of the previously single-spored cultures of the Fusarium sp. and Alternaria sp. using a sterile preparation needle (Scheme 1). The inoculum was placed on a clean plate with PDA. The EOs of C. capitatus, O. vulgare ssp. hirtum, and S. horvatii ssp. macrophylla and carvacrol (Merck KGaA, Darmstadt, Germany) were tested in amounts of 1-2-3-4-5 μL plate−1. Each EO or the pure carvacrol was tested separately and injected onto a sterile Whatman No. 1 disc (7 mm diameter), placed on the plate’s inner cover (Scheme 1). The plates were sealed with parafilm in order to avoid volatilization effects, and they were incubated at 26 ± 1 °C for 8 days. In the control (C) treatment, the plates were inoculated with each of the phytopathogens in the absence of the EOs (0 µL plate−1).

2.6. Estimation of Fungal Growth

After 8 days, the mycelial diameter on the Petri dishes was measured using a ruler in two dimensions (horizontally and vertically), and the relative mycelial growth on the Petri dish treatments was calculated as a percentage (% ± SE) compared to the control. The completely inhibited inocula were placed on clean 45 mm plates with PDA and incubated under the same experimental conditions in order to determine the mode of action of the EO (mycostatic or mycotoxic). In the case where mycelial growth was observed after three days, the effect was defined as mycostatic, whereas in the case where zero growth was observed, the effect was regarded as mycotoxic.

2.7. Statistical Analysis

A statistical analysis of the results was performed using IBM SPSS Statistics 25 statistical software, and a one-way analysis of variance (ANOVA) was performed using Fisher’s Least Significant Difference (LSD) with a significance level of 5% (p ≤ 0.05).

3. Results and Discussion

3.1. Composition of Essential Oils

According to the GC-MS results, eighteen (18) compounds were identified in O. vulgare ssp. hirtum EO, seventeen in C. capitatus (17), and twenty-seven (27) in S. horvatii ssp. Macrophylla. The fraction of monoterpenes dominated (78–93%) in all the analyzed EOs. The oxygenated monoterpene carvacrol was dominant in O. vulgare ssp. hirtum (42.5%), C. capitatus (70.0%), and S. horvatii ssp. macrophylla (41.4%). In more detail, the chemical composition of the four EOs used in the experiments is presented in Table 1.

3.2. Effect of EOs on Fusarium sp. Growth

After 8 days of incubation, Fusarium sp. growth was completely inhibited in the presence of carvacrol, as well as in the presence of the C. capitatus and O. vulgare ssp. hirtum EOs at the amount of 2 µL plate−1, with a statistically significant difference from C (100%) (p < 0.001). In the case of S. horvatii ssp. macrophylla (0%), mycelial growth was completely inhibited at 3 µL plate−1, with a statistically significant difference (p ≤ 0.001) from C. The minimum amount of the C. capitatus EO (1 µL plate−1) appeared to be more effective in inhibiting the mycelial growth of Fusarium sp. (31%) than the rest of the EOs, as presented in Figure 1.

3.3. Effect of EOs on Alternaria sp. Growth

After 8 days of incubation, the mycelial growth of the Alternaria sp. was completely inhibited by carvacrol, as well as by the EOs of C. capitatus and O. vulgare ssp. hirtum at the amount of 3 µL plate−1 (0%), presenting a statistically significant difference from C (100%) (p < 0.001), according to Figure 2. In the case of S. horvatii ssp. macrophylla, mycelial growth was completely inhibited at the amount of 5 µL plate−1 (0%), with a statistically significant difference from the control (p < 0.001). At the minimum amount (1 µL plate−1), C. capitatus (72%) and O. vulgare ssp. hirtum (71%) were more effective than S. horvatii ssp. macrophylla in inhibiting the mycelial growth of the Alternaria sp., also showing a statistically significant difference.

3.4. Mode of EOs’ Effect on Mycelial Growth

The C. capitatus and O. vulgare ssp. hirtum EOs showed a mycostatic effect on Fusarium sp. at 3–5 μL plate−1. The S. horvatii ssp. macrophylla EO presented a mycostatic effect on Fusarium sp. at 5 μL plate−1. Carvacrol demonstrated a mycostatic effect on Fusarium sp. at 2–4 μL plate−1 and a mycotoxic effect at 5 μL plate−1. Carvacrol had a mycostatic effect on Alternaria sp. at 3–5 μL plate−1.
The inhibitory effect of the EOs on the mycelial growth of Fusarium sp. was greater than that on Alternaria sp. An EO is a mixture of many different components, and the antifungal action observed is the result of the synergistic or antagonistic action of its components. The GC-MS analysis showed that the EOs used in the bioassays were rich in carvacrol (70.0–41.4%) and contained either a small percentage of thymol or none at all (0.0–18.9%). Both monoterpenic phenols have been mentioned for their antifungal properties against a wide range of phytopathogenic fungi [37,38].
Carvacrol was the main component in the S. horvatii EO used in the experiment (41.4%), while thymol constituted a small portion (6.2%). These two compounds are mainly responsible for the antifungal activity of Satureja genus EOs [39,40]. The results of this study are in agreement with those of the study by Yfanti et al. (2021) [35], where the EO of S. horvatii ssp. macrophylla (Lamiales: Lamiaceae) was found to inhibit the mycelial growth of the F. oxysporum f.sp. lycopersici.
The terpenic phenols carvacrol and/or thymol are usually the main compounds in O. vulgare ssp. hirtum EO [41]. The high carvacrol and thymol content (61.4%) in the O. vulgare ssp. hirtum EO used in our experiment may account for its great antifungal activity, as this has been found in several studies [42,43]. A similar antifungal effect of O. vulgare EO was observed on Fusarium sp. and Penicillium sp. (Eurotialles: Aspergillaceae) in the work of Kocić-Tanackov et al. (2012) [44].
The EO of C. capitatus significantly affected the mycelial growth of the phytopathogens until the end of the experiment. The two dominant components, carvacrol and thymol, have also been recognized as the main chemotypes of C. capitatus EO [45,46], which has demonstrated antimicrobial effects [47]. Usually, the EO of Greek C. capitatus has a carvacrol content of around 81.5% [48]. The carvacrol content in the C. capitatus EO used in the experiment amounted to 70.0%, while thymol was not identified. The p-cymene and γ-terpinene content was 7%. These components may enhance the antifungal activity of the EO on phytopathogenic fungi such as F. oxysporum and A. solani, according to the study by Alam et al. (2014) [47].
The EOs were effective, and this may be attributed to their high content of the terpenic phenols carvacrol and thymol. Carvacrol is a naturally oxygenated monoterpene phenol, is isomeric with thymol, and is usually present as a component of aromatic plants with an oregano odor [49]. According to the literature, both carvacrol and thymol act on the cell membrane and affect its structure and functionality [50]. Carvacrol can inhibit ergosterol synthesis in a dose-dependent manner in the phytopathogenic fungus Rhizopus stolonifer (Mucorales: Mucoraceae), which causes postharvest rot in peach fruits [51]. Zhao et al. (2023) [52] suggested that carvacrol inhibits membrane-associated poly-carbohydrates such as chitinase and β-1.3-glucanase. Wang et al. (2024) [53] observed an inhibitory effect of carvacrol on Alternaria sp., as well as the destruction of plasma membrane integrity, cytoplasmic leakage, and intracellular oxidative damage. Findings regarding the antifungal impact of carvacrol, when examined as either the dominant component in EOs of aromatic plants [29,40,46,54] or as a single agent in experiments [55,56,57,58], are in agreement with the results of this work, showing that it is highly effective against Fusarium sp., which causes potato tuber dry rot. Carvacrol has been reported to exhibit a similar mode of action in Fusarium moniliforme (Hypocreales: Nectriaceae), Rhizoctonia solani (Cantharellales: Ceratobasidiaceae), Sclerotinia sclerotiorum (Helotiales: Sclerotiniaceae), and Phytophthora capsici (Peronosporales: Peronosporaceae) [59]. As the carvacrol content (or the terpene phenol content) increases, the minimum EO quantity that inhibits fungal growth decreases. The minimum EO quantity that inhibits fungal growth was lower in Fusarium sp. than in Alternaria sp., and this could be attributed to the different growth rates of the fungi, according to Schiro et al. (2018) [60].

4. Conclusions

Τhe carvacrol- and thymol-rich EOs of the aromatic plants C. capitatus, O. vulgare ssp. hirtum, and S. horvatii ssp. macrophylla strongly inhibited the mycelial growth of Fusarium sp. and Alternaria sp., which cause potato tuber dry rot and black spot of tomato fruits, respectively, during storage. The biological action of the carvacrol-based EOs in the quantities used was mycostatic for both phytopathogens, demonstrating higher efficacy on Fusarium sp. than on Alternaria sp. The fumigant effect shown by these EOs seems to be proportional to their carvacrol and thymol content, while other components present in small quantities may play a synergistic role. With the proper standardization and formulation, these EOs could be used for postharvest applications and the disinfection of storage warehouses against Fusarium sp. and Alternaria sp., as compared to synthetic agrochemicals, EO utilization has the advantages of low toxicity to humans and the environment.

Author Contributions

Conceptualization, V.P., G.P. and P.Y.; methodology, P.Y. and V.P.; software, V.P. and P.Y.; validation, V.P., G.P. and P.Y.; formal analysis, V.P.; investigation, V.P.; resources, V.P.; data curation, V.P. and P.Y.; writing—original draft preparation, V.P.; writing—review and editing, V.P., P.Y. and G.P.; visualization, V.P. and P.Y.; supervision, G.P. and P.Y.; project administration, G.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author V.P.

Conflicts of Interest

The authors declare no conflicts of interest.

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Blsf 54 00001 sch001
Scheme 1. Schematic presentation of the fungi isolation, EO preparation, and fumigant assay method used in the experiment.
Scheme 1. Schematic presentation of the fungi isolation, EO preparation, and fumigant assay method used in the experiment.
Blsf 54 00001 sch001
Blsf 54 00001 g001
Figure 1. Fumigant effect of the EOs of S. horvatii ssp. macrophylla (S.h.), O. vulgare ssp. hirtum (O.v.), and C. capitatus (C.c.) and carvacrol (Carv.) in multiple quantities (1–5 μL plate−1) on the radical growth (% ± SE) of Fusarium sp. on PDA Petri plates after 8 days of exposure. C = control (0 μL plate−1). Different letters between treatments represent a statistically significant difference (LSD p ≤ 0.05).
Figure 1. Fumigant effect of the EOs of S. horvatii ssp. macrophylla (S.h.), O. vulgare ssp. hirtum (O.v.), and C. capitatus (C.c.) and carvacrol (Carv.) in multiple quantities (1–5 μL plate−1) on the radical growth (% ± SE) of Fusarium sp. on PDA Petri plates after 8 days of exposure. C = control (0 μL plate−1). Different letters between treatments represent a statistically significant difference (LSD p ≤ 0.05).
Blsf 54 00001 g001
Blsf 54 00001 g002
Figure 2. Fumigant effect of the EOs of S. horvatii ssp. macrophylla (S.h.), O. vulgare ssp. hirtum (O.v.), and C. capitatus (C.c.) and carvacrol (Carv.) in multiple quantities (1-2-3-4-5 μL plate−1) on the radical growth (% ± SE) of Alternaria sp. on PDA Petri plates after 8 days of exposure. C = control (0 μL plate−1). Different letters between treatments represent a statistically significant difference (LSD p ≤ 0.05).
Figure 2. Fumigant effect of the EOs of S. horvatii ssp. macrophylla (S.h.), O. vulgare ssp. hirtum (O.v.), and C. capitatus (C.c.) and carvacrol (Carv.) in multiple quantities (1-2-3-4-5 μL plate−1) on the radical growth (% ± SE) of Alternaria sp. on PDA Petri plates after 8 days of exposure. C = control (0 μL plate−1). Different letters between treatments represent a statistically significant difference (LSD p ≤ 0.05).
Blsf 54 00001 g002
Table 1. Chemical composition (GC-MS) of the three EOs used in the experiment.
Table 1. Chemical composition (GC-MS) of the three EOs used in the experiment.
NoRTRICompoundsArea (%)
O. vulgare ssp. hirtumC. capitatusS. horvatii ssp. macrophylla
16.09928α-Thujene2.30.91.3
26.36937α-Pinene1.10.71.4
36.89955Camphene 1.8
47.769861-Octen-3-ol1.20.81.7
57.98993α-Myrcene3.01.91.1
68.681012α-Phellandrene0.60.40.2
79.031023α-Terpinene2.92.41.6
89.401033p-Cymene10.26.212.9
99.531035Limonene0.40.40.6
106.191038b-Phellandrene0.60.5
119.7610391.8-cineole 1.4
1210.591065γ-Terpinene9.25.75.5
1311.131180cis sabinene hydrate1.4 1.1
1412.251107Linalool 1.41.6
1515.551185Borneol0.81.85.0
1615.841192Terpinene-4-ol0.81.11.3
1716.241201p-Cymen-8-ol 0.3
1816.551207α-Terpineol 0.4
1918.411242Thymol methyl ether 3.2
2021.861304Thymol18.9 6.2
2122.681313Carvacrol42.570.041.4
2231.4171424Caryophyllene2.74.94.5
2332.721444Aromadendrene 0.6
2433.851461α-Humulene 0.3
2536.061494Viridiflorene 0.3
2636.3471498γ-Elemene 1.5
2737.101514β-Bisabolene1.00.3
2840.631591Spathulenol 0.9
2940.751594Caryophyllene oxide0.40.82.0
Monoterpene hydrocarbons30.319.026.5
Oxygenated monoterpenes62.974.257.6
Sesquiterpene hydrocarbons3.85.27.0
Oxygenated sesquiterpenes0.40.82.9
Others2.60.85.9
RI = retention index relative to n-alkanes on BPX-5 capillary column; RT = retention time.
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Papantzikos, V.; Patakioutas, G.; Yfanti, P. Evaluation of the Antifungal Effect of Carvacrol-Rich Essential Oils: In Vitro Study on the Phytopathogenic Fungi Alternaria and Fusarium. Biol. Life Sci. Forum 2025, 54, 1. https://doi.org/10.3390/blsf2025054001

AMA Style

Papantzikos V, Patakioutas G, Yfanti P. Evaluation of the Antifungal Effect of Carvacrol-Rich Essential Oils: In Vitro Study on the Phytopathogenic Fungi Alternaria and Fusarium. Biology and Life Sciences Forum. 2025; 54(1):1. https://doi.org/10.3390/blsf2025054001

Chicago/Turabian Style

Papantzikos, Vasileios, Georgios Patakioutas, and Paraskevi Yfanti. 2025. "Evaluation of the Antifungal Effect of Carvacrol-Rich Essential Oils: In Vitro Study on the Phytopathogenic Fungi Alternaria and Fusarium" Biology and Life Sciences Forum 54, no. 1: 1. https://doi.org/10.3390/blsf2025054001

APA Style

Papantzikos, V., Patakioutas, G., & Yfanti, P. (2025). Evaluation of the Antifungal Effect of Carvacrol-Rich Essential Oils: In Vitro Study on the Phytopathogenic Fungi Alternaria and Fusarium. Biology and Life Sciences Forum, 54(1), 1. https://doi.org/10.3390/blsf2025054001

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