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Frontiers in Tropical Diseases ·

Volume 7 - 2026 | https://doi.org/10.3389/fitd.2026.1924531

Comparative evaluation of in vitro antifungal activity of major essential oil terpenes against non-dermatophyte fungal strains causing onychomycosis

  • José Pablo Saborío-Zúñiga1
  • Luis Alejandro Guido-Martínez1
  • Luis Felipe Fernández-Rojas1
  • Mary Paz Cruz-González1
  • Rebeca Jiménez-Castro1
  • Allan Valverde-Vindas1
  • Daniela Jaikel-Víquez1,2
  • Stefany Lozada-Alvarado3*corresponding author
  1. 1Mycology Section, Department of Microbiology and Immunology, School of Microbiology, Universidad de Costa Rica, San Pedro, San José, Costa Rica
  2. 2Centro de Investigación en Enfermedades Tropicales (CIET), Universidad de Costa Rica, San Pedro, San José, Costa Rica
  3. 3Clinical Laboratory and Blood Bank, Universidad de Costa Rica, San José, Costa Rica
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Abstract

Onychomycosis caused by non-dermatophyte filamentous fungi has increased in frequency. Among the most relevant etiological agents are Fusarium spp., Neoscytalidium dimidiatum, Aspergillus section Versicolores, Scopulariopsis spp. and Paecilomyces spp. Their treatment represents a clinical challenge because these fungi are resistant to conventional antifungals. In this context, the main components of essential oils represent a novel alternative due to their antifungal activity. Thus, the aim of this study was to evaluate the in vitro inhibitory activity of major components of essential oil terpenes on the growth of non-dermatophyte filamentous fungi, isolated in Costa Rica, that cause onychomycosis. Thus, an experimental study was carried out using 73 clinical isolates preserved in the Fungal Collection of the School of Microbiology of the Universidad de Costa Rica; obtained between 2007 and 2022. Isolates of N. dimidiatum (n=20), A. s ection Versicolores (n=18), F. solani (n=15), Scopulariopsis brevecaulis (n=10), and Paecilomyces spp. (n=10) were included. The inhibitory activity of eucalyptol, limonene, linalool, pinene, and terpineol was evaluated by the broth microdilution method according to CLSI M38-A2. Minimum inhibitory concentrations (MICs) were determined, and statistical analysis was performed using Kruskal-Wallis test with a Dunn’s Post Hoc test. Statistically significant differences were observed between the compounds evaluated for all the genera studied (p < 0.05). Terpineol and linalool showed the highest inhibitory activity against most isolates and pinene the least. Therefore, the results show the in vitro antifungal potential of various terpene components of essential oils against clinically relevant non-dermatophyte filamentous fungi which could represent promising therapeutic alternatives in the face of growing antifungal resistance.

Keywords

1 Introduction

The increasing incidence of infections caused by non-dermatophyte filamentous fungi has become a significant challenge in medical mycology, because of diagnostic challenges, scarce therapeutic options, and the growing prevalence of antifungal resistance. Within this group, species belonging to the genera Fusarium, Aspergillus, Paecilomyces, Scopulariopsis and Neoscytalidium stand out (1). These fungi are ubiquitous in the environment and were traditionally considered environmental contaminants or simply phytopathogens; however, they are currently recognized as etiological agents of onychomycosis and, in some cases, even invasive human infections (2–6). In Costa Rica, several studies have shown a high prevalence of non-dermatophyte filamentous fungi as etiological agents of onychomycosis. For example, Salas-Campos and Gross-Martínez (2012) reported isolation percentages of up to 23.8% in toenails and 47.6% in fingernails (7); figures higher than those described in other countries (8, 9).

The treatment of these infections continues to be complicated due to a progressive increase in antifungal resistance (1). In the case of Fusarium spp., therapeutic failures have also been documented in systemic conditions (10–12). Similarly, A. versicolor, Paecilomyces and N. dimidiatum are resistant to multiple antifungals of conventional use, such as fluconazole, itraconazole, terbinafine and griseofulvin, which considerably reduces the therapeutic options available (13–19). This scenario has driven the search for alternative compounds with antifungal potential.

Within these alternatives, plant-based essential oils have developed great interest due to their antifungal, antibacterial and anti-inflammatory properties. These compounds correspond to volatile liquid fractions, rich mainly in terpenes and other secondary metabolites capable of altering cell membranes, inducing morphological changes and compromising the viability of different pathogenic fungi (20, 21). Several studies have described the inhibitory activity of essential oils against dermatophyte and non-dermatophyte fungi (22–24). However, most research to date has evaluated whole essential oils, which are complex mixtures whose components can interact synergistically or antagonistically. For this reason, it is necessary to study the main components of these oils individually. Therefore, this study aimed to evaluate the in vitro inhibitory activity of the main components of essential oils (MCEO) on the growth of non-dermatophyte filamentous fungi that cause onychomycosis to suggest a compound as a possible candidate for further therapeutic studies.

2 Materials and methods

2.1 Fungal isolates

An experimental study was carried out to evaluate the inhibitory activity of the MCEO of lavender, rosemary, lemon, cinnamon and eucalyptus on Costa Rican isolates of N. dimidiatum (n=20), Aspergillus section Versicolores (n=18, A. amoenus and A. protoberus), F. solani (n=15), Scopulariopsis brevicaulis (n=10) and Paecilomyces spp. (n=10, P. variotii and P. lilacinum) obtained from clinical samples (nails, skin, bones and abscess aspirate). These fungal isolates were donated and deposited in the Fungal Collection of the School of Microbiology of the Universidad de Costa Rica (VI-B7732), between 2007 and 2022. After donation and before proper deposition in the collection, the protocol requires corroborating their presumed identity. Thus, different techniques were applied, depending on the fungal type. For all isolates, morphological characteristics were corroborated, both macroscopic and microscopic. For filamentous strains, further identification was performed by sequencing specific markers such as the internal transcribed spacer (ITS), the calmodulin gene, or the β-tubulin gene, or by mass spectrometry (25). The F. solani isolates were identified by sequencing of the ribosomal gene 28S (25). The other isolates were identified by mass spectrometry using the Maldi Biotyper® (Bruker Daltonics, Billerica, MA, USA). The spectra were analyzed using the Bruker Library and the MSI (Mass Spectra Identification; https://msi.happy-dev.fr) platform developed by the Assistance Publique-Hôpitaux of Paris, the Sorbonne University of Paris and the Belgian Coordinated Collections of Microorganisms (BCCM).

Before the susceptibility tests were performed, the purity of the cultures was once again determined by analyzing their morphological characteristics. The macroscopic appearance of each culture was checked by observing the obverse and reverse of the colony on APD. The microscopic characteristics were determined using blue cotton lactophenol stain (hyaline molds) and clear lactophenol stain (black molds). The wet mounts were made by placing a drop of the reagent on a slide on which a small part of the fungus was placed; then a coverslip was placed, and the slide was heated slightly in a flame. Finally, it was observed under a light microscope at 400×. The isolates were kept in tubes with potato dextrose agar (APD) at room temperature [ca. 20-30] °C).

2.2 In vitro susceptibility assay for the major components of essential oils

For the inoculum preparation, a seven-day subculture in APD, incubated at room temperature, was used. A spore suspension (conidia or arthrospores) was made in sterile 0.85% saline solution. The spore concentration was determined using a Neubauer-type cell counting chamber (Hausser Scientific, Horsham, PA, USA) and standardized to (1-5) × 106 spores/mL. Subsequently, a 1/50 dilution was performed in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA).

C. albicans American Type Culture Collection (ATCC) 90028 was used as a control strain for susceptibility studies. This strain was used in all the experiments to corroborate that the concentrations of the MCEO deposited in the plates were the correct ones. Therefore, each time an experiment was performed, a plate with this yeast was mounted, and its minimum inhibitory concentration (MIC) was determined. The MICs obtained for this strain were: 1%v/v for eucalyptol and limonene, 0.1-0.2% v/v for linalool, 10%v/v for pinene, and 0.2%v/v for terpineol. In the case of the control strain, a blastospore suspension, adjusted to an optical density of 0.5 McFarland ([1-5] x 106 blastospores/mL), was made in sterile saline solution (0.85%). The strain used to prepare the inoculum was subcultured 24 hours before in glucose Sabouraud medium. Finally, a 1:1000 dilution with RPMI of the blastospore suspension was performed.

The MIC for the filamentous fungi was determined according to the Clinical and Laboratory Standards Institute (CLSI) “Microdilution Method in M38-A Broth for filamentous fungi” (CLSI, 2017). Dilutions of 1/5, 1/50, 1/125, 1/250, 1/500, 1/1000, 1/2000 and 1/4000 were performed in RPMI 1640 medium of the following, commercially obtained, main components of essential oils: eucalyptol (99%), (R)-(+)-limonene (97%), linalool (97%), (+)-α-pinene (98%) and α-terpineol (90%) (Sigma Chemicals Co. St. Louis, Mo, USA). One hundred microliters of each dilution were plated in a 96-hole microtiter plate. It is worth noting that all treatments were analyzed in different microtiter plates to ensure that the growth inhibition was caused by the reagent tested and not by fumes from another one. One hundred microliters of each spore suspension were inoculated into the wells with major components and in the growth control wells (100 µL of RPMI 1640). The plates were incubated at 30 °C without shaking for 48 hours, or until growth was observed in the growth control well. The final concentrations of the major components of essential oils were: 1/10 (10% v/v), 1/100 (1% v/v), 1/250 (0.4% v/v), 1/500 (0.2% v/v), 1/1000 (0.1% v/v), 1/200 (0.05% v/v), 1/4000 (0.03% v/v) and 1/8000 (0.01% v/v). The susceptibility test was performed in duplicate for each isolate.

The results were read by direct visual observation of fungal growth in the wells. The MIC was defined as the lowest concentration of the MCEO that completely inhibited fungal growth when compared to the growth control wells.

2.3 Statistical analysis

The statistical analysis of the results was obtained by using the SPSS program, version 20 (SPSS Inc., Chicago, Ill, USA). The geometric mean with standard deviation, range, and the 50th and 90th MICs (50th and 90th percentiles) were calculated for each antifungal substance tested for each fungal genus. The normality of the MIC distributions was evaluated with the Kolmogorov-Smirnov test for all isolates (n =73) and with the Shapiro-Wilk test for the different genera (n < 50). Statistical differences between the MICs obtained for each component, for each genus were evaluated using a Kruskal-Wallis test with a Dunn’s Post Hoc test (p ≤ 0.05).

3 Results

The macroscopic and microscopic morphology each isolate was evaluated to confirm the purity of the cultures. The 18 isolates of Aspergillus section Versicolores presented, macroscopically, cottony mycelium of whitish coloration with yellow, green and brown variations on the obverse, while the reverse showed red, yellow and orange pigment tones. Microscopically, septate, hyaline hyphae and complex, biseriate conidiophores (Figure 1A) were observed. In the case of F. solani, the isolates showed the typical morphological characteristics of the genus, corresponding to fast-growing hyaline filamentous fungi, with the production of pink or purple pigments. Microscopically, septate, hyaline hyphae with conidiophores that produce micro and macroconidia (Figure 1B) were seen in the wet mounts. The 20 N. dimiatum isolates developed cottony filamentous colonies with a dark gray color on the obverse and a dark brown to black coloration on the reverse. Microscopic examination revealed septate, fuliginous mycelium with the presence of arthrospore chains (Figure 1C). The isolates of S. brevicaulis presented initially white and cottony colonies, which later acquired a light brown coloration with greater intensity in the periphery. Microscopically, septate hyaline mycelium, branched conidiophores and anellidic conidia arranged in chains were observed (Figure 1D). Finally, Paecilomyces spp. developed cottony colonies of white to cream color during the first days of incubation, which progressively acquired yellow-green or olive tones and a velvety texture. Microscopic examination revealed septate, hyaline mycelium, long and branched conidiophores and divergent phialides with ellipsoidal or fusiform conidia chains (Figure 1E).

Microscopical morphology of non-dermatophyte molds that cause onychomycosis. **(A)** *Aspergillus* section *Versicolores*; hyaline mycelium with complex conidiophores. **(B)** *Fusarium solani*; hyaline mycelium with macroconidia. **(C)** *Neoscytalidium dimidiatum*; fuliginous mycelium with arthrospores. **(D)** *Paecilomyces* sp.; hyaline mycelium with branched conidiophores. **(E)** *Scopulariopsis* sp.; hyaline mycelium with branched conidiophores.
Figure 1. Microscopical morphology of non-dermatophyte molds that cause onychomycosis. (A) Aspergillus section Versicolores; hyaline mycelium with complex conidiophores. (B) Fusarium solani; hyaline mycelium with macroconidia. (C) Neoscytalidium dimidiatum; fuliginous mycelium with arthrospores. (D) Paecilomyces sp.; hyaline mycelium with branched conidiophores. (E) Scopulariopsis sp.; hyaline mycelium with branched conidiophores.

The antifungal susceptibility patterns to MCEO of non-dermatophyte agents of onychomycosis were determined by the CLSI M38 guideline. The mean MIC (for all isolates; n=73) for eucalyptol was 3.51 ± 4.55%v/v, for limonene 4.35 ± 4.60%v/v, for linalool 2.31 ± 3.76%v/v, for terpineol 1.52 ± 3.00%v/v and for pineol 6.99 ± 4.44%v/v. Statistical differences were found among the MICs of the different treatments (p ≤0.05), with terpineol and linalool being the compounds with the highest inhibitory activity against these fungi and pineol the one with the least.

The MICs for each genus are presented in Table 1. In the case of Aspergillus section Versicolores and F. solani, the Post Hoc test grouped terpineol and linalool as the treatments with the highest activity and the other three in a second group with the least activity (p ≤0.0001). For N. dimidiatum the Post Hoc test grouped the treatments into three groups: terpineol, linalool, and eucalyptol in the first group as the most active, limonene in the second group, and pinene in a third group as the least active (p ≤0.0001). The MICs obtained for the ten S. brevicaulis isolates among the compounds showed significant differences between the inhibition patterns observed (p ≤0.0001); eucalyptol, limonene, linalool, and pinene showed similar activity against them, while terpineol showed the least activity. Finally, in the case of Paecilomyces spp., the Post Hoc test grouped the MCEOs into three groups: group one with pinene and limonene, group two with terpineol and eucalyptol, and group three with linalool (p =0.004).

Table 1. Susceptibility patterns of non-dermatophyte agents of onychomycosis to major components of essential oils (MCEO) (n = 73).

SpeciesNumberMCEOMinimal inhibitory concentration (MIC) %v/vMIC rangeMIC50MIC90Mean ± SD*
0.010.030.050.100.200.41.0010.00
Aspergillus section Versicoloresa(n=18)Eucalyptol3690.20-10.005.5010.005.37 ± 4.64 b
Limonene6121.00-10.0010.0010.007.00 ± 4.24 b
Linalool47250.10-10.000.2010.002.00 ± 4.36 a
Pinene18NA10.0010.0010.00 ± 0.00 b
Terpineol9810.20-10.000.601.001.10 ± 2.19 a
Fusarium solani(n=15)Eucalyptol3121.00-10.0010.0010.008.20 ± 3.60 b
Limonene15NA10.0010.0010.00 ± 0.00 b
Linalool3120.20-1.001.001.000.84 ± 0.32 a
Pinene15NA10.0010.0010.00 ± 0.00 b
Terpineol4110.20-1.001.001.000.79 ± 0.35 a
Neoscytalidium dimidiatum(n=20)Eucalyptol20NA0.200.200.20 ± 0.00 a
Limonene1821.00-10.001.001.901.90 ± 2.70 b
Linalool20NA0.200.200.20 ± 0.00 a
Pinene1.00-10.0010.0010.008.65 ± 3.30 c
Terpineol2180.10-0.200.200.200.19 ± 0.03 a
Scopulariopsis spp.(n=10)Eucalyptol190.10-0.200.200.200.19 ± 0.03 a
Limonene10NA0.200.200.20 ± 0.00 a
Linalool280.20-1.001.001.000.84 ± 0.34 a
Pinene1270.03-0.200.200.200.16 ± 0.06 a
Terpineol1540.20-10.001.0010.004.52 ± 4.48 b
Paecilomyces spp.(n=10)Eucalyptol42130.05-10.000,1010,003.06 ± 4.54 b
Limonene7120.10-0.400.100.400.17 ± 0.12 a
Linalool190.05-10.0010.0010.009.01 ± 2.99 c
Pinene3340.20-1.000.401.000.58 ± 0.35 a
Terpineol11530.03-10.000.1010.003.06 ± 4.54 b

NA, Not applicable; *The data is presented as the geometric mean and the standard deviation. The different letters within the column represent statistically different groups (p ≤ 0.05), according to Dunn’s test. The treatments group in a are the most active compounds inhibiting fungal growth and those in c the least active.

Thus, in general terms, the results obtained show that terpene compounds such as terpineol and linalool presented the highest inhibitory activity against most of the non-dermatophyte filamentous fungi evaluated, while compounds such as pinene and limonene showed lower inhibitory efficacy depending on the genus studied. Likewise, important variations in susceptibility patterns were observed between the different fungal species analyzed.

4 Discussion

Onychomycosis and other infections caused by non-dermatophyte filamentous fungi represent a growing problem in medical mycology due to their high frequency of isolation, diagnostic difficulties and limited response to conventional antifungal treatments. Fungi such as F. solani, Aspergillus section Versicolores, N. dimidiatum, S. brevicaulis and Paecilomyces spp. have been described as emerging opportunistic agents capable of producing superficial and even systemic infections, particularly in immunocompromised patients, although they can also affect immunocompetent individuals (16, 17, 26). In Costa Rica, for example, the MICs to azoles, allylamines, and polyenes of these fungi have been previously determined revealing low in vitro susceptibility to these treatments (1, 13, 18, 19, 27–32). One of the possible explanation for this low susceptibility is that these fungi are phytopathogens and are exposed to antifungal treatments in the field that have similar structures to the azoles used for human treatments, thus developing secondary resistance to these substances (27). Therefore, the intrinsic or acquired resistance that many of these microorganisms have against commonly used antifungals, such as fluconazole, itraconazole, or terbinafine (1, 33), has driven the search for new therapeutic alternatives based on bioactive natural products, including essential oils and their main components.

Essential oils are composed of a mixture of volatile compounds, mainly terpenes and phenolic compounds, to which antimicrobial, antioxidant, and insecticidal properties are attributed (34). Several studies have shown that terpenes exert antifungal activity through multiple mechanisms, including alterations of cell membrane integrity and permeability, inhibition of ergosterol biosynthesis, mitochondrial alterations, and structural damage to mycelium (34–37). In this study, the in vitro inhibitory activity of individual MCEOs was evaluated on clinical isolates of five genera of non-dermatophyte filamentous fungi to determine which was the most effective at inhibiting growth.

In general, the results obtained showed that terpineol and linalool exhibited the highest inhibitory activity against most of the fungi tested. These findings are consistent with multiple studies that describe an important antifungal activity of these terpenes on both anthropophilic and environmental fungi. For example, linalool has been described to have negative effects on cell membrane and wall integrity and inhibit biofilm formation (36, 38, 39). Finally, de Oliveira et al. (2017) described that linalool inhibited mycelial growth and reported leakage of intracellular material in Trichophyton rubrum isolates exposed to this treatment (38). As for terpineol’s effect on fungal growth, An et al. (2019) reported that terpineol inhibited mycelial growth and spore germination of Aspergillus niger (40), and Nazzaro et al. (2017) reported that it inhibits biofilm formation (36). Park et al. (2009) described that terpineol caused Trichophyton mentagrophytes’ mycelium to collapse by analyzing electron microscopy images (41). Also, Zhou et al. (2014) studied the antifungal effect of citral, octanal, and terpineol against Geotrichum citri-aurantii and described that the volatile compounds induced a decrease in the total lipid content, indicating destruction of cell membrane structures (42).

Eucalyptol showed significant inhibitory activity over N. dimidiatum, S. brevicaulis and Paecilomyces spp. Several authors have reported the effectiveness of eucalyptol-rich oils against fungi such as Aspergillus flavus (43, 44), A. niger, Aspergillus terreus, Fusarium culmorum (44), Sporothrix brasiliensis (45) and Trichophyton indotineae (46). This compound has been proposed to inhibit biofilm development, inhibit efflux pumps, and have a negative effect on cell growth and morphology (36).

The results obtained for Paecilomyces spp. showed a different behavior than those observed for the other genera evaluated. In this case, limonene and pinene showed the highest inhibitory activity, while linalool showed poor antifungal capability. Although there are few specific studies on Paecilomyces, previous research has shown that limonene can alter membrane permeability, induce oxidative stress, and affect ATP production in Candida parapsilosis (47). Likewise, α-pinene has shown activity against Candida albicans (48).

The different behaviors observed between the different fungal genera evaluated show that susceptibility to MCEO depends considerably on the physiological and structural characteristics of each fungus. It also shows that the results obtained with complete essential oils cannot always be extrapolated to their individual components due to phenomena of synergism, antagonism, and chemical variability of the oils (36, 37). This study presents preliminary but promising results in the use of terpineol and linalool as potential compounds used in developing new antifungal treatments. Both MCEOs contain oxygen-bearing hydroxyl groups that increase water solubility and cell damage (49).

Finally, this study constitutes one of the first efforts to comparatively evaluate the in vitro susceptibility of multiple clinically relevant non-dermatophyte filamentous fungi against individual MCEOs. The results obtained show the antifungal potential of compounds such as terpineol and linalool, particularly against fungi that are resistant to conventional treatments. However, this study presents two possible candidates (terpineol and linalool) as potential and promising antifungal compounds. It is worth noting that there is a limitation: this study it only presents in vitro assays designed to determine the MIC of terpenes on fungal growth. To choose one of these terpenes as a potential antifungal compound, further studies are needed, such as determining toxicity profiles, validating in vivo efficacy, evaluating nail penetration and formulation feasibility.

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Footnotes

Edited by: María Guadalupe Frías De León, Hospital Regional de Alta Especialidad de Ixtapaluca, Mexico

Reviewed by: Suryani Hutomo, Duta Wacana Christian University, Indonesia

Deepak Khobragade, Datta Meghe Institute of Medical Sciences, India

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This research was supported by Vicerrectoría de Investigación, Universidad de Costa Rica (grant B7732).

Data availability

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s.

Author contributions

SJ-P: Investigation, Methodology, Writing – review & editing. LG-M: Investigation, Methodology, Writing – review & editing. LF-R: Investigation, Methodology, Writing – review & editing. MC-G: Investigation, Methodology, Writing – review & editing. RJ-C: Investigation, Methodology, Writing – review & editing. AV-V: Supervision, Writing – original draft. DJ-V: Conceptualization, Data curation, Formal analysis, Funding acquisition, Software, Supervision, Visualization, Writing – original draft. SL-A: Conceptualization, Supervision, Visualization, Writing – review & editing.

Acknowledgements

The authors thank Alejandra Gómez Arrieta (Medical Mycology Section, School of Microbiology, Universidad de Costa Rica) for her technical contributions to this work.

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Citation

Saborío-Zúñiga, J. P., et al. (2026). Comparative evaluation of in vitro antifungal activity of major essential oil terpenes against non-dermatophyte fungal strains causing onychomycosis. Frontiers in Tropical Diseases, Volume 7. https://doi.org/
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7 - 2026

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Copyright © 2026 Saborío-Zúñiga, Guido-Martínez, Fernández-Rojas, Cruz-González, Jiménez-Castro, Valverde-Vindas, Jaikel-Víquez and Lozada-Alvarado.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Stefany Lozada-Alvarado

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