Mode of Antifungal Action of Daito-Gettou (Alpinia zerumbet var. exelsa) Essential Oil against Aspergillus brasiliensis

Plant-derived essential oils (EOs) are used in medicines, disinfectants, and aromatherapy products. Information on the antifungal activity of EO of Alpinia zerumbet var. exelsa (known as Daito-gettou) found in Kitadaito Island, Okinawa, is limited. Therefore, we aimed to evaluate the antifungal activity of EOs obtained via steam distillation of leaves of Daito-gettou, which is a hybrid of A. zerumbet and A. uraiensis. Daito-gettou EO showed antifungal activity (minimum inhibitory concentration = 0.4%) against Aspergillus brasiliensis NBRC 9455, which was comparable to that of A. zerumbet found in the Okinawa main island. Gas chromatography/mass spectrometry revealed that the main components of Daito-gettou EOs are γ-terpinene, terpinen-4-ol, 1,8-cineole, 3-carene, and p-cymene. Terpinen-4-ol content (MIC = 0.075%) was 17.24%, suggesting that the antifungal activity of Daito-gettou EO was mainly attributable to this component. Daito-gettou EO and terpinen-4-ol inhibited mycelial growth. Moreover, calorimetric observations of fungal growth in the presence of Daito-gettou EO showed a characteristic pattern with no change in the initial growth rate and only a delay in growth. As this pattern is similar to that of amphotericin B, it implies that the action mode of Daito-gettou EO and terpinen-4-ol may be fungicidal. Further studies on the molecular mechanisms of action are needed for validation.


Introduction
Essential oils (EOs) derived from plants contain various components with antimicrobial activity [1][2][3][4][5][6][7], and they have long been used as disinfectants and medicinal agents. These EOs have also been used in food products to control spoilage by microorganisms and extend shelf life. In recent years, the emergence of bacteria resistant to chemical preservatives and the consumer preference for natural products have increased the demand for plant-derived EOs [8]. EOs derived from herbs or spices such as Melaleuca alternifolia (tea tree) [9,10], Lavandula angustifolia (lavender) [11], Angelica major [12], Curcuma longa L. [13], Thymus capitatus [14], Thymus vulgaris L. [15], and Alpinia calcarata Roscoe [12] have been reported to have antibacterial and antifungal activities. They are anticipated to inhibit the growth of food-spoilage microorganisms and maintain the quality and safety of food products.

Minimum Inhibitory Concentration
Aspergillus brasiliensis NBRC 9455 was purchased from the National Institute of Technology and Evaluation Biological Resource Center (NBRC, Tokyo, Japan) and used for antifungal tests. It was inoculated at three points on a Sabouraud dextrose agar plate containing 4.0% (w/v) glucose, 1.0% (w/v) peptone, and 1.5% (w/v) agar (Becton Dickinson, Sparks, MD, USA). After being incubated for 7 d at 25 • C, spore suspension was prepared by adding sterile saline (15 mL) containing 0.1% (w/v) polypeptone (Becton Dickinson) and 0.05% (w/v) Tween-80 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to the plate and gently rubbing the surface of the fungal colonies with a sterile loop. The spore suspension was filtered through a coarse cloth to remove mycelia, and the spores were counted using a hemocytometer. The concentration of the spore suspension was adjusted to 2 × 10 5 spores/mL using Sabouraud dextrose broth containing 4.0% (w/v) glucose and 1.0% (w/v) peptone for the broth dilution method.
The minimum inhibitory concentration (MIC) of EOs and antifungal agents for A. brasiliensis was determined using the broth dilution method. EOs and antifungal agents were dissolved in 20% ethyl alcohol solution. Ethyl alcohol was selected as the solvent in consideration of the future application of Daito-gettou to foods. Amphotericin B, which is insoluble in ethyl alcohol, was dissolved in DMSO (FUJIFILM Wako Pure Chemical Corporation). These solutions were serially diluted with Sabouraud dextrose broth, and 1 mL of the diluted solutions was pipetted into sterile test tubes containing 1 mL of the spore suspensions. The MIC was determined via visual inspection after incubating the mixtures at 25 • C for 3 d.

Gas Chromatography/Mass Spectrometry Analysis
The components of EOs were analyzed using gas chromatography/mass spectrometry (GC/MS). The instrument (JMS-T100GCV; JEOL Ltd., Tokyo, Japan) was equipped with an Agilent DB-5MS capillary column (30 m × 0.25 mm i.d., 0.25-µm film thickness, Agilent Technologies, Santa Clara, CA, USA). The oven was programmed as follows: initial temperature of 40 • C (held for 5 min) and heated at a rate of 10 • C /min to 320 • C (held for 3 min). The injector temperature was maintained at 250 • C. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The ionization voltage was 70 eV, and the mass range was 35-650 m/z. Analysis was performed using two methods. First, the mass spectrum of each EO component was determined by comparison with the mass spectrum from the NIST 11 spectrum library. The percentage composition of each component was calculated based on the respective peak areas. Second, some major components of the EO were quantitatively analyzed. The amount of each component was obtained from a calibration curve prepared using the corresponding standard product.

Effects on Spore Germination and Mycelial Growth
The inhibitory effects of Daito-gettou EO, Shima-gettou 1 EO, terpinen-4-ol, p-cymene, and 1,8-cineole on the mycelial growth of A. brasiliensis were determined using the modified "agar dilution method (2007)" for fungi [23]. Each antifungal agent was adjusted to concentrations at 25-, 50-, and 100-fold of the MIC with ethyl alcohol. The ethyl alcohol solutions (0.15 mL) and the dissolved Sabouraud dextrose agar medium (15 mL) were mixed in a Petri dish (ϕ90 mm) and solidified. The final concentrations of antifungal agents in agar media were adjusted to 1/4, 1/2, and 1/1 MIC, respectively. A sterile paper disk (Toyo Roshi Kaisha, Ltd. (Tokyo, Japan): ϕ13-mm thick) was placed on an agar plate, and 0.1 mL of the spore suspension (1 × 10 5 spores/mL) was inoculated on the disk. After culturing at 25 • C for 3 d, the radius of the mycelia grown (excluding the radius of the paper disc) was measured, and the difference from the radius of the mycelia grown on an agar plate without antifungal agents (control) was used to estimate the inhibition of mycelial growth. The inhibitory rate was calculated as follows: Mycelial growth inhibitory rate (%) = (1 − mycelial growth distance on agar medium containing antifungal agent/mycelial growth distance on control agar medium) × 100.
Furthermore, the inhibition of mycelial growth of A. brasiliensis by Daito-gettou EO and terpinen-4-ol was evaluated based on the weight of mycelia. Each solution of EO and terpinen-4-ol was serially diluted with Sabouraud dextrose broth, and 7.5 mL of the diluted solution was pipetted into a sterile Petri dish (ϕ90 mm) containing 7.5 mL of the spore suspension (2 × 10 5 spores/mL with Sabouraud dextrose broth). The concentration of EO and terpinen-4-ol in the broth was adjusted to 0-0.4% or 0-0.075%, respectively. After incubating at 25 • C for 5 d, the mycelia in the cultures were filtered through a membrane filter (cellulose acetate, pore size: 0.8 µm) and washed with distilled water. Mycelia on the membrane filter were weighed after removing excess moisture with filter paper.

Calorimetric Measurements
A multiplex batch calorimeter, Leonis (ADVANCE RIKO, Inc., Yokohama, Japan), with 25 calorimetric units was used. The apparatus was developed by Japan Science and Technology, a government agency, and it is now sold as a commercial product termed "Nondestructive and Non-invasive Analytical Instrument," which can quantitatively determine microbial growth activity [24][25][26]. As a sensor, semiconducting thermopile plates were employed and placed in an aluminum heat sink to detect the thermal change in the sample vessels (Petri dishes) set in each calorimetric unit. The calorimetric signals obtained were analyzed according to a method reported previously [25].
Petri dishes (ϕ60 mm) were placed on units as calorimetric vessels. When temperature changes occurred in the sample vessels, the sensor detected them, and the differentially generated voltage was proportional to the temperature changes. A mixed solution of 4.9 mL of the spore suspension (1 × 10 5 spores/mL) prepared in Sabouraud dextrose broth and 0.1 mL of antifungal agent diluted stepwise with ethanol was poured into Petri dishes. The sample dishes were then placed in a calorimetric unit and maintained at 25 • C. Calorimetric output signals associated with fungal growth were monitored for incubation periods of 3-5 d.

Statistical Analysis
The results were statistically analyzed using a two-tailed unpaired Student's t-test (Microsoft Excel 365; Microsoft Corporation, Redmond, WA, USA). Results with p < 0.05 were considered statistically significant.

Antifungal Activity
The susceptibility of A. brasiliensis used in this study to various antifungal agents was measured before evaluating the antifungal activity of gettou EOs. The MICs of the antifungal agents are shown in Table 1. Three azol antifungal agents (MCZ, ITCZ, and VRCZ) and amphotericin B (AMPH-B), a polyene drug, showed high antifungal activities. Fluconazole (FLCZ) and flucytosine (5-FC), a pyrimidine-analog drug, presented lower activities. The antifungal agent TBZ (2-(4-thiazolyl) benzimidazole), which is widely used as a pesticide and food additive, had an MIC of 50 ppm (250 µmol/L). Table 1 also shows the MICs of EOs of various types of gettou and tea tree. Daito-gettou EO exhibited the same level of antifungal activity (MIC = 0.40%) as Shima-gettou 1-3 EOs, but it was less active than tea tree oil. The GC/MS analysis of each EO component revealed that Daito-gettou contains γterpinene, terpinen-4-ol, 1,8-cineole, 3-carene, and p-cymene (Table 2, Figure 1). In contrast, the composition of Shima-gettou EO was quite different from that of Daito-gettou, and it was dependent on the product. Shima-gettou 1 and 2 EOs contained p-cymene, limonene, and α-pinene as the main components, with no traces of terpinen-4-ol. Tea tree EO contained γ-terpinene and terpinen-4-ol, but the peak area of terpinen-4-ol was considerably larger than that of the other EOs. These results are in agreement with those reported by Ninomiya et al. [10]. The antifungal activities of five compounds, which showed large peak areas in Daitogettou EO, and three compounds (limonene, α-pinene, and camphene) detected in Shimagettou 1 and 2 EOs were measured. As shown in Table 3, terpinen-4-ol showed the highest activity (MIC = 0.075%, 4.9 mmol/L) among the tested components. Therefore, terpinen-4ol was considered as the major substance responsible for the antifungal activity of Daitogettou EO. This result is consistent with those of Terzi et al. [27] and Roana et al. [28], who investigated the antifungal activity of tea tree oil. Terzi et al. described terpinen-4-ol as the  The antifungal activities of five compounds, which showed large peak areas in Daitogettou EO, and three compounds (limonene, α-pinene, and camphene) detected in Shimagettou 1 and 2 EOs were measured. As shown in Table 3, terpinen-4-ol showed the highest activity (MIC = 0.075%, 4.9 mmol/L) among the tested components. Therefore, terpinen-4ol was considered as the major substance responsible for the antifungal activity of Daitogettou EO. This result is consistent with those of Terzi et al. [27] and Roana et al. [28], who investigated the antifungal activity of tea tree oil. Terzi et al. described terpinen-4-ol as the most active component of tea tree oil against fungi. 1,8-Cineole also showed antifungal activity, with an MIC of 0.50% (32 mmol/L).  Table 4 shows the terpinene-4-ol and 1,8-cineol concentration/level of each EO. The concentration/level of p-cymene, which is a common component of all gettou EOs, is also shown. These components were quantified using the respective standards. The composition of Shima-gettou 1 and 2 EOs was significantly different from that of Daito-gettou. Table 4. Chemical components of EOs of gettou and tea tree obtained using gas chromatography/mass spectrometry analysis.    Table 4 shows the terpinene-4-ol and 1,8-cineol concentration/level of each EO. The concentration/level of p-cymene, which is a common component of all gettou EOs, is also shown. These components were quantified using the respective standards. The composition of Shima-gettou 1 and 2 EOs was significantly different from that of Daito-gettou.

Effects of Daito-Gettou EO on Mycelial Growth
To further understand the mechanism underlying the antifungal action of Daito-gettou EO, its effect on the mycelial growth of A. brasiliensis was measured. Shima-gettou 1 EO, terpinen-4-ol, p-cymene, and 1,8-cineole were used for comparison. The inhibitory rate increased with the increasing concentration (1/4 to 1/1 MIC) of all antifungal agents ( Figure 2). When treated at 1/1 MIC, the inhibitory rate was 63.8% for Daito-gettou EO, 59.7% for Shima-gettou 1 EO, and 100% for terpinen-4-ol. Mycelial growth was observed despite treatment at the MIC, which might mainly be attributed to the obtained MIC value (Table 3), which was determined using the broth dilution method in this study. As mycelial growth inhibition was measured using the agar medium dilution method in which the antifungal agent was added to the agar medium and the MICs measured using the broth medium dilution method and agar medium dilution method may differ, the experimental results were believed to be inconsistent. In contrast, treatment at 1/4 and 1/2 MIC of p-cymene and 1/4 MIC of 1,8-cineole resulted in negative inhibition rates. In the presence of these compounds at MIC, mycelial growth was inhibited, whereas at low concentrations, growth was promoted.
EO, terpinen-4-ol, p-cymene, and 1,8-cineole were used for comparison. The inhibitory rate increased with the increasing concentration (1/4 to 1/1 MIC) of all antifungal agents ( Figure 2). When treated at 1/1 MIC, the inhibitory rate was 63.8% for Daito-gettou EO, 59.7% for Shima-gettou 1 EO, and 100% for terpinen-4-ol. Mycelial growth was observed despite treatment at the MIC, which might mainly be attributed to the obtained MIC value (Table 3), which was determined using the broth dilution method in this study. As mycelial growth inhibition was measured using the agar medium dilution method in which the antifungal agent was added to the agar medium and the MICs measured using the broth medium dilution method and agar medium dilution method may differ, the experimental results were believed to be inconsistent. In contrast, treatment at 1/4 and 1/2 MIC of pcymene and 1/4 MIC of 1,8-cineole resulted in negative inhibition rates. In the presence of these compounds at MIC, mycelial growth was inhibited, whereas at low concentrations, growth was promoted. The effects of Daito-gettou EO and terpinen-4-ol on mycelial weight were measured in Sabouraud dextrose broth medium. The addition of 0.2% EO to the medium increased the dry mycelial mass of the tested fungi, but the mass decreased as the EO concentration increased (Figure 3a). Furthermore, when 0.4% EO (equal to the MIC) was added, some amount of mycelium growth was observed. Additionally, only a few EOs have been shown to inhibit fungal spore germination and mycelial growth [15]. Pereira et al. [29] have also reported that the EO of Cymbopogon winterianus inhibits the mycelial growth of Trichophyton rubrum. It has also been shown that the vaporous phase of the EO of Thymus vulgaris L. (thyme) strongly suppresses the sporulation of fungi in glass chambers [30]. In The effects of Daito-gettou EO and terpinen-4-ol on mycelial weight were measured in Sabouraud dextrose broth medium. The addition of 0.2% EO to the medium increased the dry mycelial mass of the tested fungi, but the mass decreased as the EO concentration increased (Figure 3a). Furthermore, when 0.4% EO (equal to the MIC) was added, some amount of mycelium growth was observed. Additionally, only a few EOs have been shown to inhibit fungal spore germination and mycelial growth [15]. Pereira et al. [29] have also reported that the EO of Cymbopogon winterianus inhibits the mycelial growth of Trichophyton rubrum. It has also been shown that the vaporous phase of the EO of Thymus vulgaris L. (thyme) strongly suppresses the sporulation of fungi in glass chambers [30]. In this study, terpinen-4-ol, the main component of Daito-gettou EO, was measured in the same way (Figure 3b). Terpinen-4-ol at 0.075% (equal to the MIC) completely inhibited mycelial growth. this study, terpinen-4-ol, the main component of Daito-gettou EO, was measured in the same way (Figure 3b). Terpinen-4-ol at 0.075% (equal to the MIC) completely inhibited mycelial growth.

Growth Thermogram of A. brasiliensis in the Presence of Daito-Gettou EO
Figure 4a-d show the growth thermograms for cultures of A. brasiliensis in Sabouraud dextrose broth containing various concentrations of TBZ, AMPH-B, Daito-gettou EO, and terpinen-4-ol, respectively. These figures are termed "g(t) curves." The vertical axis of each figure represents the thermoelectromotive force indicated by the heat detector (µ V), and the horizontal axis represents the incubation time (min). The heat generated in the broth containing each sample is considered to be the metabolic heat associated with the process of fungal spore germination and the subsequent mycelial growth [31]. When spores of A. brasiliensis and TBZ at concentrations below the MIC were mixed in Sabouraud dextrose broth and cultured, the pattern of the growth thermogram changed as the TBZ concentration increased, and the slope of the g(t) curve decreased. Moreover, it was observed that the apparent peak time shifted to the longer side of the culture. Additionally, a delay in growth was observed. Thus, changes in the initial rate and delay in growth time occurred, and TBZ was found to suppress fungal growth in a concentration-dependent manner. In contrast, the growth of A. brasiliensis in the presence of AMPH-B was delayed, but no apparent change was observed in the initial growth rate. As shown in Figure 4c,d, the growth thermogram of Daito-gettou EO was similar to that of terpinen-4-ol. Our results revealed that there were time delays in the fungal growth as the concentrations of Daito-gettou EO or terpinen-4-ol increased; however, their initial growth rates did not change. The thermogram pattern of both Daito-gettou EO and terpinen-4-ol was closer to that of AMPH-B than to the pattern of TBZ.  terpinen-4-ol, respectively. These figures are termed "g(t) curves." The vertical axis of each figure represents the thermoelectromotive force indicated by the heat detector (µV), and the horizontal axis represents the incubation time (min). The heat generated in the broth containing each sample is considered to be the metabolic heat associated with the process of fungal spore germination and the subsequent mycelial growth [31]. When spores of A. brasiliensis and TBZ at concentrations below the MIC were mixed in Sabouraud dextrose broth and cultured, the pattern of the growth thermogram changed as the TBZ concentration increased, and the slope of the g(t) curve decreased. Moreover, it was observed that the apparent peak time shifted to the longer side of the culture. Additionally, a delay in growth was observed. Thus, changes in the initial rate and delay in growth time occurred, and TBZ was found to suppress fungal growth in a concentration-dependent manner. In contrast, the growth of A. brasiliensis in the presence of AMPH-B was delayed, but no apparent change was observed in the initial growth rate. As shown in Figure 4c,d, the growth thermogram of Daito-gettou EO was similar to that of terpinen-4-ol. Our results revealed that there were time delays in the fungal growth as the concentrations of Daito-gettou EO or terpinen-4-ol increased; however, their initial growth rates did not change. The thermogram pattern of both Daito-gettou EO and terpinen-4-ol was closer to that of AMPH-B than to the pattern of TBZ.

Growth Thermogram of A. brasiliensis in the Presence of Daito-Gettou EO
An increase in microbial growth activity can be observed when growth thermograms are transformed into calorimetrically defined growth curves [24,[32][33][34]. The growth curves described as "f (t) curves" were obtained by computation based on Equation (1): where t is the growth time and K is the heat conduction constant (Newton's cooling constant) of each calorific value measuring unit including the Petri dish containing the culture solution [24][25][26]35]. The obtained f (t) curves are shown in Figure 5, which correspond to the growth curves of microorganisms in the presence of different agents. As shown in Figure 5a, we observed that the slope of the curve decreased and the growth rate slowed as the TBZ concentration increased. In contrast, the slope of the curve in Figure 5b did not change even when the concentration of AMPH-B increased, and only a delay in fungal growth was observed. Moreover, when the concentrations of Daito-gettou EO were 0.025% and 0.05%, the slope of the curves was almost similar to that of the curve without EO. However, at concentrations above 0.1%, the slope of the curves was greater than that of the curve without EO. The f (t) curve of Daito-gettou EO exhibited a unique shape, but it Foods 2023, 12, 1298 9 of 14 more closely resembled the characteristics of the AMPH-B curve than those of the TBZ curve. In addition, as shown in Figure 5d, the slope for terpinen-4-ol increased with the drug concentration, indicating a time lag. The f (t) curve of Daito-gettou EO showed the characteristics of an AMPH-B curve and was notably similar to that of the terpinen-4-ol. An increase in microbial growth activity can be observed when growth thermograms are transformed into calorimetrically defined growth curves [24,[32][33][34]. The growth curves described as "f(t) curves" were obtained by computation based on Equation (1): where t is the growth time and K is the heat conduction constant (Newton's cooling constant) of each calorific value measuring unit including the Petri dish containing the culture solution [24][25][26]35]. The obtained f(t) curves are shown in Figure 5, which correspond to the growth curves of microorganisms in the presence of different agents. As shown in Figure 5a, we observed that the slope of the curve decreased and the growth rate slowed as the TBZ concentration increased. In contrast, the slope of the curve in Figure 5b did not change even when the concentration of AMPH-B increased, and only a delay in fungal growth was observed. Moreover, when the concentrations of Daito-gettou EO were 0.025% and 0.05%, the slope of the curves was almost similar to that of the curve without EO. However, at concentrations above 0.1%, the slope of the curves was greater than that of the curve without EO. The f(t) curve of Daito-gettou EO exhibited a unique shape, but it more closely resembled the characteristics of the AMPH-B curve than those of the TBZ curve. In addition, as shown in Figure 5d, the slope for terpinen-4-ol increased with the drug concentration, indicating a time lag. The f(t) curve of Daito-gettou EO showed the characteristics of an AMPH-B curve and was notably similar to that of the terpinen-4-ol.

Discussion
Aspergillus brasiliensis, a common fungus in the soil, often contaminates food. commonly used fungal strain for the preservative efficacy test of ISO11930 (Interna Organization for Standardization, 2012), the Japanese Pharmacopoeia (2021), an Standards for Food and Food Additives (Ministry of Health and Welfare Notificatio 370, 1959, Japan). In this study, we evaluated the activity of Daito-gettou EO aga brasiliensis. The susceptibility of A. brasiliensis used in this study was first confirmed evaluating the antifungal activity of gettou. The trends of the MICs of antifungal

Discussion
Aspergillus brasiliensis, a common fungus in the soil, often contaminates food. It is a commonly used fungal strain for the preservative efficacy test of ISO11930 (International Organization for Standardization, 2012), the Japanese Pharmacopoeia (2021), and the Standards for Food and Food Additives (Ministry of Health and Welfare Notification No. 370, 1959, Japan). In this study, we evaluated the activity of Daito-gettou EO against A. brasiliensis. The susceptibility of A. brasiliensis used in this study was first confirmed before evaluating the antifungal activity of gettou. The trends of the MICs of antifungal agents were consistent with those reported previously [36,37].
The antifungal activity of Daito-gettou EO against A. brasiliensis was found to be similar to that of Shima-gettou EOs (MIC = 0.40%), but it was lower than the activity of tea tree EO (Table 1). In addition, the GC/MS analysis revealed that the composition of Daito-gettou EO was different from that of Shima-gettou EOs ( Table 2). Similar to tea tree oil, Daito-gettou EO contained a large amount of terpinen-4-ol (Table 3). Generally, external effects cause changes in the ratios of the constituents of EOs [38]. The composition of gettou EOs is known to fluctuate under the influence of climate parameters (temperature and precipitation) [39]. Moreover, the activity thereof differs depending on the production area or the variety of gettou. According to Ramos et al. [40], the major constituents of A. zerumbet var. variegata EO are 1,8-cineole (39%), β-pinene (11%), and β-caryophyllene (10%). As Daito-gettou EO used in this study contained a lesser amount of p-cymene and was abundant in terpinen-4-ol, this result supports the concept that the variety of gettou inhabiting the Daito Islands is different from that present in Okinawa main island.
Terpinen-4-ol, which was abundant in Daito-gettou EO, exhibited antifungal effects (MIC = 0.075%) against A. brasiliensis ( Table 3). As the MIC of Daito-gettou EO, which contains approximately 17% of this component, was 0.40%, the activity of Daito-gettou EO could mainly be attributed to terpinen-4-ol. Moreover, Maior et al. [41] and Ninomiya et al. [42] have reported that terpinene-4-ol possess antifungal activity. These findings strengthen our claim of terpinene-4-ol being the main component responsible for the antifungal action of Daito-gettou EO. Additionally, regarding the activity of p-cymene, which is another component, Aznar et al. [43] reported that it completely inhibits the growth of Candida lusitaniae for at least 21 d at concentrations above 1 mmol/L at 25 • C. However, MIC of p-cymene against Rhizopus oryzae is >1024 µg/mL, indicating a poor antifungal effect [15]. Daferera et al. [44] postulated that the antifungal activity of EOs is primarily due to their major components; however, other phenomena, such as synergy and antagonism with minor components, are also possible. These findings suggest that the antifungal activity of Daito-gettou EO is most likely attributable to terpinen-4-ol and the combined effect of other components, such as 1,8-cineole and p-cymene. The activity of Shima-gettou 1 and 2 EOs, which are terpinen-4ol-free, was considered to be mediated by α-pinene and limonene. However, we believe that these components alone cannot explain the antifungal activity of Shima-gettou EOs. It is likely that unknown antifungal components or their synergy with other components are involved in the antifungal activity of Shima-gettou EOs.
Moreover, the mechanism underlying the antifungal action of Daito-gettou EO was investigated by measuring its effects on the mycelial growth of A. brasiliensis. Daito-gettou EO showed an inhibitory effect on mycelial growth at different MICs (Figures 2 and 3). Terpinen-4-ol also exerted a significant effect on mycelial growth. This result suggests that Daito-gettou EO inhibits mycelial growth, which could mainly be attributed to terpinen-4-ol.
Furthermore, we aimed to elucidate the mechanism underlying the antifungal action in detail. To achieve this, we used a microbial calorimeter to obtain growth thermograms of fungi growing in the presence of various antifungal agents. Growth thermograms showed different heat generation patterns depending on the action of antifungal agents. One of the authors of this study, Takahashi [45], had reported the differences in growth thermograms between bactericidal and bacteriostatic agents. According to this report, imidazolidinyl urea, which shows bactericidal action, caused a delay in the rise of the growth thermogram, but it did not alter the initial growth rate. Propylparaben, which exhibits bacteriostatic action, caused a change in only the initial growth rate. The bactericidal or bacteriostatic action of antimicrobial agents are not classified properly, and most drugs are considered to be bacteriostatic up to a certain concentration and bactericidal above that concentration. Therefore, several drugs are considered to exhibit both bacteriostatic and bactericidal activities. However, we believe that the information obtained from growth thermograms will provide key clues for investigating the mechanisms of action of antifungal drugs.
A mechanism of action of TBZ has been reported by Allen and Gottlieb [46]. Per their findings, TBZ inhibits the terminal electron transport system of mitochondria and exhibits highly selective toxicity against fungi. Additionally, Kano et al. [31], by measuring the growth thermograms of TBZ, showed that TBZ inhibits the mycelial growth of Aspergillus niger in a concentration-dependent manner. In contrast, AMPH-B, a polyene macrolide antibiotic that selectively binds to ergosterol in cell membranes, is known to disrupt cell membranes, leak intracellular substances, and kill cells [47]. Based on these reports, the theory to quantitatively characterize the antimicrobial action of drugs [45], and the pattern of the f (t) curves obtained in this study, it can be hypothesized that TBZ exerts a fungistatic effect at low concentrations and exhibits fungicidal activity at high concentrations, and that AMPH-B is a fungicidal agent. Furthermore, we analyzed the f (t) curve of Daito-gettou EO and found that there was little change in the slope of the curve between concentrations of 0% and 0.05%, and the slope increased with the increase in concentration (>0.1%). The antifungal action of Daito-gettou EO did not change the initial growth rate but caused only a delay. Therefore, these results suggest that the antifungal action of Daito-gettou EO is fungicidal. Additionally, the f (t) curve of Daito-gettou EO was notably similar to that of terpinen-4-ol as shown in Figure 5, thereby implying that the fungicidal action of Daito-gettou EO is mainly due to the action of terpinen-4-ol.
In this study, the antifungal activity of Daito-gettou EO was compared by focusing on terpinen-4-ol, which is one of the main components. Terpinen-4-ol is a hydrophobic terpene that can strongly interact with microbial membrane lipids and affect membrane permeability. Polec et al. [48] reported that the antifungal activity of terpinen-4-ol is directly related to its incorporation into cellular membranes and is affected by the lipid composition of various pathogenic membranes. Furthermore, Li et al. [49] mainly attributed the antimicrobial activity of tea tree oil to the presence of terpinen-4-ol, and tea tree oil penetrated the cell wall and cytoplasmic membrane of the tested bacterial and fungal strains. In addition, their findings suggest that tea tree oil also penetrates fungal organelle membranes and exerts its antimicrobial effects by compromising the cell membrane, resulting in cytoplasm loss and organelle damage, which ultimate lead to cell death. These reports support the results of this study regarding the antifungal activities of Daito-gettou EO and terpinen-4-ol. It is presumed that Daito-gettou EO and terpinen-4-ol interact with the cell membrane of A. brasiliensis, affect the fluidity and permeability of the cell membrane, and substantially inhibit the growth of mycelia. Furthermore, Daito-gettou EO and terpinen-4-ol might disrupt cell membranes and cause intracellular substance leakage, and thereby exert a fungicidal effect against A. brasiliensis. However, it is apparent that the content of terpinen-4-ol in Daito-gettou EO is lower than that in tea tree EO, as determined using GC/MS analysis, and that Daito-gettou EO contains many other trace components in addition to terpinen-4-ol. Therefore, further investigations are necessary to clarify the detailed mechanism of antifungal action of Daito-gettou EO.
Daito-gettou EO has been closely associated with the life of people inhabiting Okinawa and the Daito Islands by supporting a safe food environment. It is used extensively and is considered to be a proxy for guaranteed food safety. Daito-gettou EO is considered particularly useful as a natural preservative for food to maintain hygiene. However, it is assumed that the EO composition of gettou fluctuates with the climate factors (such as temperature and rainfall) [39], which can lead to a corresponding fluctuation in its antifungal activity. In addition, Daito-gettou EO may contain unknown antifungal components, similar to those of Shima-gettou EOs, and therefore, further studies (for example, studies on synergistic effects between EO components and the influence of EO extraction methods) are required to elucidate the mechanisms underlying this activity.

Conclusions
In this study, the mechanism underlying the antifungal action of Daito-gettou EO was investigated, and the following major results were obtained. (1) Daito-gettou EO showed an antifungal effect against A. brasiliensis (MIC = 0.4%). (2) The main chemical components of EO were identified as γ-terpinene, terpinen-4-ol, 1,8-cineole, 3-carene, and p-cymene, which differed from the three kinds of Shima-gettou EOs used in this study. (3) Terpinen-4-ol, which is present in Daito-gettou EO at 17.24%, showed a higher antifungal activity than the other components (MIC = 0.075%), and the activity of Daito-gettou EO against A. brasiliensis could be attributed to this component. (4) Daito-gettou EO inhibited mycelial growth. (5) The pattern of growth thermograms, which were calorimetric observations of fungal growth in the presence of Daito-gettou EO, was similar to that of the fungicide amphotericin B. These findings imply that the mode of action of Daito-gettou EO is fungicidal; however, to confirm this, further studies on the molecular mechanisms of action are needed.