Inhibitory Mechanism of Oregano Essential Oil Emulsion Against Colletotrichum gloeosporioides in Mangoes and Its Regulatory Effects on Postharvest Quality

TL;DR

Oregano essential oil effectively fights a mango fungus and improves fruit quality after harvest, offering a natural alternative to synthetic fungicides.

Contribution

Oregano essential oil's dual role as a fungicide and quality preserver in mangoes is demonstrated for the first time.

Findings

Oregano essential oil disrupted the plasma membrane of Colletotrichum gloeosporioides, inhibiting fungal growth.

Oregano essential oil preserved mango quality by reducing weight loss and maintaining firmness and color.

Oregano essential oil boosted antioxidant activity and reduced harmful chemical buildup in mangoes.

Abstract

In response to the growing need for sustainable and safe postharvest strategies, plant essential oils have emerged as promising natural alternatives to synthetic fungicides. Mango (Mangifera indica L.), as a vital tropical fruit, suffers significant postharvest losses due to anthracnose caused by Colletotrichum gloeosporioides. This study investigated the antifungal efficacy of oregano essential oil (OEO) against C. gloeosporioides and its regulatory effects on the postharvest quality of mango fruit. The potent antifungal activity of OEO is demonstrated by its low MIC (0.005%) and MFC (0.01%) against C. gloeosporioides. The antifungal mechanism was primarily attributed to the disruption of plasma membrane integrity of C. gloeosporioides, as indicated by increased propidium iodide uptake, elevated extracellular conductivity, and leakage of cellular proteins. The OEO treatment inhibited peel color transformation, reduced weight loss, maintained firmness, and slowed the increase in the soluble solids content to acidity ratio. Furthermore, OEO enhanced the fruit’s antioxidant capacity by sustaining higher superoxide dismutase activity and suppressing the activities of polyphenol oxidase and peroxidase, leading to a marked reduction in malondialdehyde accumulation. These findings comprehensively demonstrate the dual functionality of OEO as a direct fungicidal agent and a systemic physiological regulator that delays senescence and preserves mango quality. This study underscores the potential of OEO as a sustainable alternative for integrated postharvest management of mango anthracnose, offering insights for its practical application in the fruit industry.

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Full text

1. Introduction

Fruits offer a rich source of vitamins, dietary fiber, and bioactive compounds that contribute to overall human health [1]. However, postharvest losses due to microbial contamination significantly compromise fruit quality, leading to economic losses and potential health risks [2]. Among the various fungal pathogens affecting tropical fruits, Colletotrichum gloeosporioides stands out as a devastating agent of anthracnose in mango (Mangifera indica L.) [3]. Global mango production was reported at 58 megatons in 2019, while direct and indirect losses caused by anthracnose during pre- and postharvest stages reached up to 30% [4]. This pathogen also poses a serious threat to other tropical fruits; for instance, anthracnose caused by C. gloeosporioides accounts for 25–40% of postharvest losses of the 13 megatons of papaya produced globally [4]. This disease manifests as black lesions on the fruit surface, eventually leading to tissue necrosis and extensive rotting during storage and transportation [5]. Despite their efficacy in controlling postharvest pathogens, the use of synthetic fungicides is associated with negative environmental impact, potential human health risks, and the development of microbial resistance [6]. Consequently, great efforts have been increasingly shifted to the development and utilization of sustainable antimicrobial agents for postharvest disease control.

Plant-derived essential oils have emerged as promising candidates for natural preservation due to their broad-spectrum antimicrobial activity, low mammalian toxicity, and biodegradability [7,8]. These complex mixtures of volatile secondary metabolites are primarily composed of terpenoids, phenylpropanoids, and oxygenated hydrocarbons, which collectively contribute to their potent antimicrobial and antioxidant properties [9,10]. In addition, these bioactive components of plant essential oils reduced the risk of resistance development associated with single-target synthetic fungicides through a multi-target mechanism [11]. Notably, oregano essential oil (OEO), extracted from Origanum vulgare, has emerged as a promising candidate in postharvest management owing to its potent antifungal efficacy against diverse pathogens [12]. Kosakowska et al. (2024) reported that OEO effectively inhibited the growth of multiple phytopathogenic fungi, including Fusarium culmorum, Alternaria alternata, Botrytis cinerea, and C. cladosporioides, with MIC values ranging from 0.016 to 2 μL/mL [13]. Similarly, Hosseinzadeh et al. (2025) highlighted the broad antimicrobial spectrum of OEO against spoilage fungi such as Aspergillus, Fusarium, Botrytis, Alternaria, and Penicillium species [14]. Additionally, studies on tropical fruits have confirmed OEO’s efficacy against Colletotrichum species in mango [15] and avocado [16]. Horst et al. (2025) provided mechanistic insights, demonstrating that OEO reduces ergosterol content and increases membrane permeability in C. gloeosporioides, while also effectively controlling Elsinoë ampelina and Phytophthora infestans [17].

Numerous studies have demonstrated the efficacy of OEO against various pathogens, whose antimicrobial mechanisms involve compromising fungal cell membrane integrity, altering redox homeostasis, and interfering with cellular metabolism [18,19]. Kolypetri et al. reported that the extracted OEO presented strong antimicrobial and antibiofilm actions against foodborne Salmonella typhimurium and Listeria monocytogenes, and the sub-lethal exposure to OEO resulted in significant downregulation of virulence genes [20]. The OEO was reported to affect the activity of oxidoreductase by promoting the accumulation of reactive oxygen species (ROS), which led to membrane peroxidation and damage to the cell membrane [21]. Similar findings were demonstrated by Luo et al., who revealed that OEO inactivated Vibrio vulnificus by generating ROS, which caused lipid peroxidation of cell membranes, thereby reducing the permeability and integrity of cell membranes and ultimately leading to cellular dysfunction and death [22]. In addition, the OEO treatment was found to effectively inhibit C. gloeosporioides on the mango surface to the same extent as the traditional fungicide “Prochloraz”, suggesting its potential application in microbiological control and managing mango anthracnose [23].

Beyond its antifungal activity, OEO has also shown regulatory effects on postharvest fruit quality by modulating physiological processes such as respiration rate, enzymatic browning, and antioxidant enzyme activities [24]. Sanchez et al. elucidated that the OEO coating contributed to delayed ripening and reduced quality deterioration in mangoes by lowering the respiration rate and retaining firmness, soluble solid content, and pulp lightness [25]. OEO treatment on grape tapes effectively preserved postharvest quality by delaying senescence-related weight loss and softening, retaining nutritional acids, and inhibiting enzymatic browning [26]. Moreover, fresh-keeping paper embedded with OEO treatment has been found to effectively retard strawberry ripening and senescence by coordinately enhancing antioxidant capacity, inhibiting microbial decay, and preserving key physicochemical and sensory qualities such as weight loss, total soluble solids, and color [27]. These findings underscore the dual functionality of OEO as both an antimicrobial agent and a quality-preserving compound that modulates key postharvest physiological processes to delay fruit ripening and quality deterioration.

It is noted that the practical application of essential oils in postharvest management faces several inherent limitations, including high volatility leading to rapid loss of bioactivity, poor physicochemical stability, particularly under exposure to light, oxygen, and temperature fluctuations, and relatively low water solubility, which collectively compromise their efficacy in practical agricultural scenarios [28]. Furthermore, at effective concentrations, essential oils may impart undesirable organoleptic effects on treated fruits or exhibit phytotoxicity, necessitating careful dose optimization, while production costs also remain a consideration for commercial scalability compared to synthetic alternatives [29]. These challenges have driven the development of advanced formulation strategies such as encapsulation, emulsification, and incorporation into edible coating matrices to enhance stability, enable controlled release, and improve the overall applicability of essential oils in postharvest management [30,31].

Despite the demonstrated antifungal potential of OEO, a comprehensive understanding of its specific mechanism against C. gloeosporioides in mangoes and its concomitant regulatory effect on fruit quality remains limited. This study aims to elucidate the fundamental mechanisms through which OEO compromises fungal viability and to comprehensively evaluate its role in modulating mango ripening and senescence processes. By integrating microbiological, biochemical, and sensory analyses, this research seeks to provide a scientific basis for the practical application of OEO in sustainable postharvest management strategies.

2. Results

2.1. Determination of MIC and MFC of Oregano Essential Oil

The antifungal activity of OEO against C. gloeosporioides was evaluated by determining the MIC and MFC, as shown in Table 1. After 48 h of incubation under aerobic conditions, no spore growth was observed in the PDB medium supplemented with 0.005% OEO or at higher concentrations. However, a concentration of 0.002% OEO did not completely inhibit spore growth. The MIC of OEO against C. gloeosporioides was thus determined to be 0.005%. Further incubation for another 48 h at 28 °C revealed spore growth in the group supplemented with 0.005% OEO, but not in groups treated with 0.01% or higher concentrations, suggesting spore viability was compromised. Consequently, the MFC was established as 0.01%.

2.2. Hyphae Growth of C. gloeosporioides Against Oregano Essential Oil

The mycelium growth and expansion of C. gloeosporioides under OEO treatment are shown in Figure 1. The colonies gradually expanded as incubation time increased, while OEO treatment inhibited mycelium spread (Figure 1A). The colony diameter was 68 mm in the control group after 5 days of incubation, whereas a 24.71% reduction was observed in the group with supplementation of 0.01% OEO (Figure 1B). When supplemented with 0.02% of OEO, no growth of C. gloeosporioides mycelia was observed during the whole incubation period, indicating a non-culturable state or loss of spore viability under such conditions.

2.3. Plasma Membrane Integrity and Permeability of C. gloeosporioides Against Oregano Essential Oil

2.3.1. Plasma Membrane Integrity in Response to Oregano Essential Oil

PI staining was used to determine cell membrane integrity, and the stained spores of C. gloeosporioides under fluorescent modes are shown in Figure 2. With the extension of incubation time, the spore staining rate of C. gloeosporioides gradually increased (Figure 2A). When incubated for 6 h, the PI staining rate was 76.36% in the group supplemented with 2 MIC of OEO, which was 1.8 times higher than that for 3 h (Figure 2B). Additionally, the elevated OEO concentration resulted in enhanced PI staining rate of spores. After 6 h of incubation, the PI staining rate in the group with 2 MIC of OEO was 52.7% higher than that in the 1 MIC group. Notably, the spore PI staining rate always maintained a low level in the group without the addition of OEO (Figure 2B). These results indicated that the destructive effect of OEO on plasma membrane integrity of C. gloeosporioides spores is concentration- and time-dependent. Higher doses of OEO and longer duration time caused more severe damage to cell membrane integrity, thus leading to the loss of normal cellular functions.

2.3.2. Intracellular Components Leakage in Response to Oregano Essential Oil

The extracellular conductivity of C. gloeosporioides in all groups gradually increased, whereas OEO supplementation intensified this increase (Figure 3A). After 24 h of incubation, the extracellular conductivity of C. gloeosporioides in the control group was 558.6 µs/cm, while 701.8 µs/cm and 606.6 µs/cm were observed in the group with 1 MIC and 2 MIC of OEO, representing an increase of 25.64% and 8.59%, respectively. When incubated for 48 h, the extracellular conductivity in the group with 2 MIC of OEO was 1109.6 µs/cm, which was 14.32% higher than that in the control group (Figure 3A). Additionally, the extracellular conductivity showed a dose-dependent response to OEO treatment, with higher OEO concentrations resulting in enhanced extracellular conductivity. The extracellular protein content increased with higher OEO exposure concentrations (Figure 3B). After 24 h of incubation, the extracellular protein content of C. gloeosporioides in the group with 0.5 MIC and 2 MIC of OEO was 173.97 µg/mL and 220.55 µg/mL, respectively, representing an increase of 13.29-fold and 16.85-fold compared to that in the control group (Figure 3B). The increasing OEO concentration accelerated leakage of intracellular protein, which was perhaps due to the more serious damage to cell membrane permeability caused by higher exposure to OEO. These results indicated that OEO treatment disrupted the cell membrane integrity and permeability of C. gloeosporioides, leading to leakage of intracellular components and subsequent disorder of normal cellular function.

2.4. Mango Color Change Against Oregano Essential Oil

Table 2 shows the color change in mango against OEO during storage. The L* value of mangoes in the control group showed an initial increase followed by a decrease, peaking at 60.00 on day 6, and then gradually declining. In contrast, the L* value in the group with MIC and 2 MIC of OEO showed a continuous upward trend, with their peaks delayed until day 12 (60.77 and 57.30, respectively). This indicated that OEO treatment effectively delayed the attenuation of peel brightness and maintained the visual gloss of the fruit. The a* value of mangoes increased continuously with storage time, reflecting the process of peel color changing from green to red. On day 12, the a* value of mango in the control group was 16.53, while that in the group with 1MIC and 2 MIC of OEO was 1.21% and 33.76% lower, respectively, indicating that higher concentrations of OEO showed a stronger regulatory effect on mango color transformation. The b* value of mangoes in the control group showed an initial increase followed by a decrease, peaking at 54.03 on day 6 before declining. The b* value in the group with 1MIC and 2 MIC of OEO continued to increase until day 12, with the value of 58.43 and 54.70, respectively, representing an increase of 59.78% and 49.58% compared to the control group at 12th day. This demonstrated that OEO exposure was conducive to inhibiting peel yellowing and maintaining commercial appearance stability.

2.5. Mango Weight Loss and Firmness Against Oregano Essential Oil

The weight loss rate of mangoes was found to gradually increase over time during storage, and OEO treatment reduced the weight loss rate compared with the control group (Figure 4A). After 12 days of storage, the weight loss rate of mangoes in the control group was 11.97%, which was 5.74% and 10.42% higher than that in the group with MIC and 2 MIC of OEO, respectively (Figure 4A). These results indicated that OEO treatment helped inhibit moisture loss in mangoes, and higher concentrations of OEO showed more effectiveness in reducing moisture loss. The firmness of mangoes gradually decreased over time, and a decrease of 84.8% was observed in the control group during 12 days of storage (Figure 4B), indicating the continuous transpiration and respiratory metabolism during storage. Notably, the OEO treatment was beneficial for the delay of firmness decrease during storage. After 12 days of storage, the firmness of mangoes in the group with 1 MIC and 2 MIC of OEO was 4.02 N and 5.13 N, respectively, representing an increase of 29.26% and 64.95% compared to the control group (Figure 4B). This suggested that OEO treatment helped to maintain mango firmness during storage, thus facilitating the maintenance of quality attributes.

2.6. Soluble Solid Content and Acidity of Mangoes Against Oregano Essential Oil Treatment

The ripening of postharvest fruits is often accompanied by increases in soluble solid content (SSC) and decreases in acidity. The SSC of mangoes showed an overall upward trend during storage, while OEO treatment suppressed the increase (Figure 5A). On day 3 of storage, the SSC of mangoes in the control group was 17.6%, which was 5.52% and 14.73% higher than that in the group with MIC and 2 MIC of OEO, respectively (Figure 5A). After 12 days of storage, the SSC of mangoes in the group with 2 MIC of OEO was 19.24%, which was 4.47% lower than that in the control group, demonstrating the retardance of the mango ripening process (Figure 5A).

The acidity of mangoes in all groups exhibited a continuous downward trend during storage, and OEO intervention slowed the decrease in acidity (Figure 5B). After 12 days of storage, the acidity of mangoes in the group with 2 MIC of OEO was 0.76%, which was 52% higher than that in the control group (Figure 5B). Throughout the whole storage process, the SSC—acidity ratio of mangoes gradually increased, indicating the gradual ripening process, while OEO treatment slowed the ratio increase (Figure 5C). After 12 days of storage, the SSC—acidity ratio in the group with 2 MIC of OEO was 25.32, which was 37.14% lower than that in the control group (Figure 5C). These results suggested that OEO treatment retarded the postharvest ripening process characterized by the change in SSC and acidity, thereby contributing to the extension of storage time.

2.7. Enzyme Activity of Mangoes Against Oregano Essential Oil Treatment

POD enzyme is involved in biochemical reactions related to enzymatic browning. The POD activity of mango in all groups showed an upward trend with storage time, and OEO treatment inhibited the increase in POD activity compared with the control group (Figure 6A). After 12 days of storage, the POD activity of the control group was 190.44 U/g, while a decrease of 12.48% was observed in the group with 2 MIC of OEO (166.67 U/g) (Figure 6A). These results suggested that OEO played vital roles in the regulation of POD-mediated enzymatic browning reactions, helping to relieve quality deterioration of mango during storage.

The SOD activity of mangoes in all groups was found to exhibit an initial increase followed by a decrease during storage, and OEO intervention helped maintain SOD activity at higher levels (Figure 6B). The SOD activity of mango in the control group reached its highest value (337.18 U/g) on day 9 of storage, while an increase of 4.11% and 1.84% was observed in the group with 2 MIC and 1 MIC of OEO, respectively. After 12 days of storage, the SOD activity of mangoes in the group with 2 MIC of OEO was 288.80 U/g, representing an increase of 3.27% compared with the control group (Figure 6B). Despite of the fluctuation of mango SOD activity during storage, OEO treatment was conducive to keeping SOD activity at higher levels, thereby enhancing the antioxidant capacity of mangoes during storage.

The PPO activity of mangoes in response to OEO treatment was shown in Figure 6C. Although PPO activity showed an overall increasing trend during storage, OEO treatment suppressed this increase. Following 12 days of storage, mangoes treated with MIC and 2 MIC of OEO showed reductions in PPO activity of 13.25% and 22.02%, respectively, compared to that in the control group (51.64 U/g). These findings indicated the curbing of PPO activity by the OEO treatment, thereby alleviating PPO-mediated enzymatic quality deterioration and contributing to prolonged storage quality of mangoes.

2.8. Malondialdehyde Content of Mangoes Against Oregano Essential Oil Treatment

The malondialdehyde (MDA) content of mangoes during storage under OEO treatment is shown in Figure 7. The MDA content in all groups exhibited an upward trend with storage time, indicating an increased membrane lipid oxidation during storage. However, OEO treatment was beneficial for the reduction in MDA content. After 12 days of storage, the MDA content of mangoes in the control group was 28.61 nmol/g, while a decrease of 11.11% and 24.78% was observed in the group with 1MIC and 2 MIC of OEO. These findings demonstrated the role of OEO treatment in reducing the level of membrane lipid peroxidation and thereby relieving oxidative damage and quality deterioration of mangoes.

2.9. Correlation Analysis

Correlation analysis of mango quality parameters under oregano essential oil treatment is shown in Figure 8. The L* value and b* value showed a positive correlation, indicating a synchronization of peel brightness increase with yellow tones deepening. The negative correlation between weight loss rate and firmness suggested that moisture loss during storage was an important driving factor for fruit softening, and OEO treatment reduced the weight loss rate, thereby delaying the decrease in firmness. In terms of physiological metabolism and oxidative stress, MDA content showed a positive correlation with POD and PPO activity, indicating that the degree of membrane lipid peroxidation was closely related to the increase in oxidative stress-related enzyme activity. SOD activity showed a negative correlation with MDA content in the early stage of storage, indicating that higher SOD activity helped scavenge reactive oxygen species and reduce membrane lipid peroxidation. However, SOD activity decreased in all groups with the extension of storage time while MDA accumulation accelerated, indicating a gradual imbalance in the antioxidant system. Additionally, the SSC-acidity ratio showed a positive correlation with most browning and oxidation indices, further confirming the intrinsic connection between substance transformation during fruit ripening and quality deterioration. In summary, correlation analysis revealed that postharvest quality changes in mangoes are a process involving multiple interrelated indices and coordinated regulation. The OEO treatment delayed fruit ripening and maintained storage quality through multiple physiological pathways, including reducing water loss, inhibiting oxidative enzyme activity, slowing color transformation, and regulating substance metabolism.

3. Discussion

The increasing demand for sustainable postharvest strategies has intensified research into plant essential oils as promising alternatives to synthetic fungicides [32]. The potent antifungal activity of OEO is demonstrated in this study by its low MIC (0.005%) and MFC (0.01%) against C. gloeosporioides. This efficacy aligns with findings from broader screenings of phenolic-rich essential oils; for instance, a comparative evaluation of 15 essential oils, including clove, thyme, and cinnamon, reported that concentrations between 0.005% and 0.04% were required for complete inhibition of Aspergillus flavus on agar [33]. Many recent studies have elucidated the specific antifungal mechanisms of various essential oils against C. gloeosporioides. Tan et al. [34] demonstrated that black pepper essential oil inhibited C. gloeosporioides by suppressing key enzymes in the tricarboxylic acid cycle and EMP pathway, thereby reducing respiration rate and fungal proliferation. Radice et al. [35] comprehensively reviewed essential oils inhibiting C. gloeosporioides and summarized that their antifungal mechanisms include disruption of membrane integrity, interference with ergosterol biosynthesis, inhibition of spore germination, and suppression of respiratory metabolism. Collectively, these studies provided a mechanistic framework for understanding how essential oils exert their antifungal effects against C. gloeosporioides. However, while such studies have established the in vitro antimicrobial potential of various essential oils, research focusing on their regulatory effects on fruit physiology beyond pathogen inhibition remains limited. Therefore, this study comprehensively investigated the dual role of OEO, demonstrating its direct fungicidal action against C. gloeosporioides and its systemic regulation on physiological activities to delay ripening and senescence in mango fruit.

Several studies have elucidated the antifungal mechanisms of OEO against C. gloeosporioides. Gundewadi et al. [36] evaluated the antifungal activities of OEO against C. gloeosporioides and demonstrated that the major constituents—thymol and carvacrol—are responsible for membrane disruption and metabolic inhibition. Horst et al. [17] demonstrated that OEO inhibited C. gloeosporioides by reducing ergosterol content and increasing membrane permeability, which corroborated the membrane disruption mechanism observed in our study. Sánchez-Tamayo et al. [23] investigated pectin-based OEO coatings and demonstrated that the antifungal mechanism against C. gloeosporioides involves the lipophilic phenolic compounds integrating into the fungal membrane, disrupting its architecture and increasing permeability, leading to cellular leakage and death. Yilmaz et al. [37] further elucidated that OEO exerts its fungistatic effects against C. gloeosporioides through carvacrol-mediated membrane disruption and ergosterol biosynthesis interference, limiting fungal growth and conidial germination. These findings comprehensively revealed that the antifungal efficacy of OEO is fundamentally rooted in its capacity to compromise cellular membrane integrity [38]. Our data further supported this mechanism by demonstrating a time- and concentration-dependent increase in propidium iodide uptake, extracellular conductivity, and protein leakage, which provided direct cytological evidence for the disruption of plasma membrane integrity. Zhao et al. [39] also revealed that cinnamon essential oil treatment damaged the cell membrane of Rhizopus stolonifera, resulting in a high increase in extracellular conductivity and protein content. The lipophilic phenolic compound of plant essential oil, with its hydrophobic character, allows for integration into the fungal plasma membrane, disrupting the lipid bilayer architecture and increasing permeability [8,40]. This initial damage triggers a cascade of dysfunction, leading to the fatal leakage of ions and vital macromolecules, ultimately collapsing electrochemical gradients and halting metabolism. Our findings align with existing observations across fungal species, reinforcing that membrane disruption is attributed to the disorder of substance exchange and the subsequent death of C. gloeosporioides.

The role of plant essential oil extends beyond pathogen inhibition to encompass a broad-spectrum modulation of postharvest fruit quality, effectively decelerating the ripening syndrome [41]. In this study, the OEO treatment delayed peel color transformation of mangoes by inhibiting the decline in brightness and the shift towards red tones, which is intrinsically linked to the observed suppression of PPO and POD activities, key enzymes responsible for enzymatic browning. In addition, the OEO application was conducive to the retention of water and firmness of mangoes, which might not be merely a secondary consequence of disease suppression but indicated a direct physiological intervention by influencing transpirational water loss and the activity of cell wall-degrading enzymes. This is consistent with the findings of Bruno et al. [42], who reported that the bergamot essential oil treatment reduced the weight loss and maintained the firmness of the flesh of strawberries. Furthermore, OEO treatment was found to retarded the metabolic shifts in mango ripening as evidenced by the suppressed increase in SSC and the slower decline in acidity. The resultant delay in the SSC—acidity ratio underscores OEO’s capacity to postpone the normal ripening process, thereby extending the shelf life of mangoes.

Postharvest ripening and senescence of fruits are often accompanied by the accumulation of reactive oxygen species (ROS), thereby triggering oxidative stress and accelerating quality deterioration [43]. SOD played key regulatory roles in scavenging ROS by catalyzing the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen [44]. In this study, the mangoes treated with 2 MIC of OEO showed a 4.11% increase in SOD activity compared with that in the control group after 9 days of storage. These results are consistent with those of Yang et al. [45], who demonstrated that the SOD activity of apples peaked at day 8 after Phoebe bournei wood essential oil treatment, with a 2.24-fold higher than that in the control group. These enhancements in SOD activity contributed to the improved ability to maintain redox balance and relieve oxidative damage. Excessive ROS accumulation disrupted redox homeostasis in fruit and caused membrane lipid peroxidation, while MDA can be used as an indicator product to assess the extent of lipid peroxidation within the membrane [46]. Our findings revealed that the MDA content represented a decrease of 11.11% and 24.78% in the group with 1MIC and 2 MIC of OEO compared with the control group after 12 days of storage, indicating a decreased level of ROS accumulation and an alleviation in membrane lipid peroxidation, thereby ultimately contributing to retardance of mango quality deterioration. The correlation analysis also demonstrated that SOD activity showed a negative correlation with MDA content in the early stage of storage, indicating that higher SOD activity helped scavenge ROS and reduce membrane lipid peroxidation.

PPO and POD are involved in enzymatic browning by catalyzing the oxidation of phenolic compounds to quinones, which then polymerize to form brown pigments, resulting in quality deterioration of mango fruit [47]. Our findings demonstrated that the POD and PPO activity in the group with 2 MIC of OEO exhibited a decrease of 12.48% and 22.02% compared with the control group after 12 days of storage, respectively, attenuating the enzymatic browning process and the risk of quality deterioration. Similar results were observed by Wen et al. [48], who found that the color browning of fresh-cut apples was prevented via the inhibition of POD and PPO activity, which reduced the oxidation of total phenolics and flavonoids. This observed suppression of PPO and POD activity can be linked to the inhibitory effect of specific bioactive compounds in OEO. The inhibition of PPO and POD by specific essential oil components represents a key mechanism for alleviating enzymatic browning and maintaining fruit quality [49].

This study comprehensively elucidated the dual role of OEO in direct fungicidal action against C. gloeosporioides and systemic regulation of physiological activities to delay ripening and senescence in mango fruit. While plant essential oils like OEO present a promising sustainable alternative in postharvest management, translating laboratory efficacy into reliable commercial practice remains a significant challenge. Since the plant essential oil was composed of a variety of volatile compounds with different flavors [50], sensory evaluations of essential oils to ensure consumer acceptance of any residual aromatic notes were required in future research. Embedding plant essential oil within engineered edible coatings or microencapsulation matrices could significantly enhance its persistence and controlled release on the fruit surface [51]. Further research should pivot towards innovating more advanced delivery systems of plant essential oils, overcoming inherent limitations of volatility and stability. Furthermore, developing integrated, multi-hurdle strategies that synergistically combine essential oils with physical treatments or biological controls could reduce effective doses and establish more resilient and sustainable postharvest management strategies for fruit.

4. Materials and Methods

4.1. Materials and Chemicals

The mangoes (Mangifera indica L. cv. ‘Tainong No.1’) used in this experiment was sourced from a commercial orchard located in Sanya, Hainan Province, China (18°34′ N, 109°43′ E). The unripe green mangoes were harvested and transported to the laboratory under dark conditions. Oregano essential oil (OEO, ≥98%, carvacrol (85.0 ± 1.2%), thymol (1.1 ± 0.1%)), chitosan, and propidium iodide were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Potato dextrose agar (PDA) medium was obtained from Beijing Luqiao Technology Co., Ltd. (Beijing, China). A protein concentration assay kit and lactophenol cotton blue staining solution were acquired from Solarbio Science & Technology Co., Ltd. (Beijing, China). Nylon cell filters (40 μm) were procured from Beijing Labgic Technology Co., Ltd. (Beijing, China). Superoxide dismutase, peroxidase, polyphenol oxidase activity assay kits, and a malondialdehyde determination kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

4.2. Preparation of Spore Suspension

The preparation of spore suspension followed the method described by Liu et al. [24] with slight modifications. C. gloeosporioides was inoculated onto PDA medium and purified through two consecutive generations of culture. Fresh plates cultured for 3~5 days were selected, and 2 mL of sterile water was added to gently scrape the spores and mycelia. After thorough mixing, the spore suspension was obtained by filtering through a cell filter to remove impurities. The spore suspension concentration was calculated by using a hemocytometer and stored at 4 °C for subsequent use.

4.3. Evaluation of Antifungal Activity

4.3.1. Determination of MIC and MFC

The determination of Minimum Inhibitory Concentration (MIC) and the Minimum Fungicidal Concentration (MFC) was conducted by using the broth microdilution method according to our previous study [52]. Briefly, 100 µL of C. gloeosporioides spore suspension (1 × 10^3^ CFU/mL) was evenly spread on PDA plates supplemented with different concentrations of OEO (0, 0.002%, 0.005%, 0.01%, 0.02%), followed by incubation at 28 °C for 48 h to observe fungal growth. A negative control consisting of PDB medium inoculated with spore suspension (without OEO) was included to verify normal fungal growth. The minimum concentration that completely inhibited fungal growth after 48 h of incubation was defined as the MIC. The plates showing no growth were further incubated for another 48 h, and the lowest concentration that still showed no growth after 96 h of incubation was defined as the MFC.

4.3.2. Growth and Expansion of Mycelium

For observation of hyphae expansion, 2 µL of C. gloeosporioides spore suspension (1 × 10^6^ CFU/mL) was inoculated onto PDA medium containing different concentrations of OEO (0.5 MIC, 1 MIC, 2 MIC). Plates without essential oil served as blank controls. Each treatment was replicated three times. The plates were incubated at 28 °C, and mycelium growth was recorded every day. The colony diameters were measured by using the cross method, and the results were expressed as mean values.

4.4. Integrity and Permeability of Plasma Membrane

4.4.1. Plasma Membrane Integrity

The membrane integrity was assessed by using the propidium iodide (PI) staining method based on our previous study [52]. 300 µL of spore suspension (1 × 10^6^ CFU/mL) was centrifuged, and the pellets were resuspended with 500 µL of PBS solution containing different concentrations of OEO (0, MIC, 2 MIC). The control group consisted of pellets resuspended in PBS without OEO. All samples were incubated at 28 °C and collected at an interval of 3 h. For each sample, 100 µL were centrifuged, and the pellets were suspended with 20 µL of PI staining solution (1000×). The mixtures were incubated at 37 °C in the dark for 30 min, centrifuged to discard the staining solution, and washed twice with PBS solutions. The stained spores were then examined under a fluorescence microscope.

4.4.2. Measurement of Extracellular Conductivity

The extracellular conductivity was determined by using a conductivity meter DDS-307A (INESA, Shanghai, China). 100 µL of spore suspension was inoculated into PDB medium and incubated at 28 °C for 7 days. The mycelia were harvested by centrifugation. 2 g of mycelia was weighed, and added with 5 mL of OEO solutions at different concentrations (0, 0.5 MIC, 1 MIC, 2 MIC). The mixtures were incubated at 28 °C with shaking. The extracellular conductivity was measured at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, and 48 h. Each treatment was replicated three times, with each replicate measured three times.

4.4.3. Leakage of Intracellular Protein

The protein concentration was determined by using the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). 50 µL of spore suspension was inoculated into 35 mL of PDB medium and incubated at 28 °C for 7 days to obtain wet mycelia. 0.2 g of mycelia was weighed and mixed with 200 µL of OEO solutions at different concentrations (0, 0.5 MIC, 1 MIC, 2 MIC). The mixture was incubated at 28 °C for 24 h, followed by centrifugation to collect the supernatant. After sufficient staining with Coomassie Brilliant Blue G250 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) solution, the absorbance was measured at a 595 nm wavelength.

4.5. Preparation and Application of Oregano Essential Oil Emulsion

The preparation of OEO emulsion followed the method described by Gutiérrez-Pacheco [53] with appropriate modifications. 3 g of chitosan was dissolved in 3 mL glacial acetic acid with magnetic stirring until completely dissolved, resulting in a final chitosan concentration of 1%. Different concentrations of OEO solution were added to the above solution to achieve final concentrations of 0.01% and 0.02%. The mixture was stirred and sonicated for 10 min. Mango fruits without fungal inoculation were immersed in the prepared OEO emulsion for 30 s, then removed and air-dried at room temperature. The present study was designed from an application-oriented perspective, focusing on the overall efficacy of the complete OEO-loaded chitosan formulation. Therefore, a chitosan-only (without OEO) control group was not included. Future work will aim to delineate the individual contributions of the chitosan matrix and OEO to the observed preservation effects.

4.6. Determination of Mango Quality Attributes

4.6.1. Color

During storage, the color of the mango was measured every three days. For color measurement, a colorimeter (CR-10, Konica Minolta, Tokyo, Japan) calibrated with a white plate was used. Three random points at the equatorial position of the mango fruit were selected for measuring L* (lightness), a* (green-red value), and b* (yellow-blue value). Each measurement was repeated three times, and results were expressed as mean ± standard deviation.

4.6.2. Firmness, Weight Loss Rate, Soluble Solid Content, and Acidity

Fruit firmness was measured by using a TMS-PRO texture analyzer (FTC Company, Sterling, VA, USA). The probe diameter was 0.5 cm, with a force sensor range of 250 N, trigger force of 0.08 N, speed of 60 mm/min, and penetration distance of 8 mm. The probe was vertically positioned on the equatorial surface of the fruit for puncture testing, and the maximum puncture force (N) was recorded. Each measurement was repeated three times.

The weight loss rate of mango fruit was determined by using the weighing method, calculated as follows: weight loss rate (%) = (initial fruit weight − fruit weight after treatment)/initial fruit weight × 100. Each measurement was repeated three times.

Clear juice was obtained from mango pulp for soluble solid content and acidity determination. Soluble solid content was measured by using a handheld digital refractometer (Pocket PAL-1, ATAGO, Tokyo, Japan). After zero calibration with distilled water, 800 µL of mango juice was used for measurement. Acidity was determined by using an acid meter (GMK-708, G-WON, Seoul, Republic of Korea). For measurement, 0.5 mL of mango juice was diluted to 50 mL with distilled water. After zero calibration with distilled water, 600 µL of the diluted solution was added to the prism slot of the acid meter for measurement. Each parameter was measured three times, and the average value was used, expressed as a percentage (%).

4.6.3. Enzyme Activity

Enzyme activity of mangoes was measured every three days during storage, with three fruits selected for each treatment. A certain weight of mango tissue was mechanically homogenized in an ice-water bath, followed by low-temperature centrifugation for 10 min. The supernatant was collected for subsequent determination.

Peroxidase (POD): 200 µL of supernatant was mixed with 3.6 mL of reaction buffer containing 0.1% guaiacol, 0.05% hydrogen peroxide, and 100 mM potassium phosphate buffer (pH 7.4). The mixture was incubated at 37 °C for 30 min, and absorbance was measured at 420 nm.

Superoxide dismutase (SOD): 50 µL of supernatant was added to a buffer containing 0.1 mM xanthine, 75 µM nitroblue tetrazolium, 0.1 mM ethylenediaminetetraacetic acid, and xanthine oxidase (0.01 U/mL). The mixture was incubated at 37 °C for 40 min, and absorbance was measured at 550 nm. One unit of SOD enzyme activity was defined as the amount of enzyme required to inhibit 50% of the nitroblue tetrazolium photoreduction reaction.

Polyphenol oxidase (PPO): 100 µL of supernatant was mixed with 1 mL of reaction buffer containing 100 mM potassium phosphate buffer (pH 7.4) and 50 mM catechol. The mixture was incubated at 37 °C for 10 min, then cooled under running water. After centrifugation, the supernatant was used to measure absorbance at 420 nm. One unit of PPO activity was defined as the amount of enzyme required to catalyze 1 nmol of catechol per minute.

4.6.4. Malondialdehyde (MDA) Content

MDA content was determined by using the thiobarbituric acid method with a kit. The measurements were carried out every 3 days with three fruits selected for each treatment during the whole storage. 1.0 g of mango tissue was weighed, ground with 5.0 mL of trichloroacetic acid, and centrifuged at 4 °C for 20 min. 2.0 mL of the supernatant was mixed with 2.0 mL of 0.67% TBA solution, heated in a boiling water bath for 20 min, and centrifuged. The absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm. The group without supplementation of mango tissue was set as the control group.

4.7. Data Analysis

All experiments were conducted with three replicates, and data were expressed as mean ± SD. Significant differences among treatments were determined by analysis of variance (ANOVA) followed by Duncan’s multiple range test using Stata 18, with statistical significance set at p < 0.05. OriginPro 2021 (OriginLab Corporation, Northampton, MA, USA) was used for graphical representation, and correlation analysis was conducted by using the Correlation Plot plugin.

5. Conclusions

This study demonstrates that OEO is an effective and multi-functional agent for the postharvest management of mango anthracnose caused by C. gloeosporioides. The OEO exhibited potent in vitro antifungal activity, with an MIC of 0.005% and an MFC of 0.01%. The disruption of fungal plasma membrane integrity was identified as the primary antifungal mode of action, as evidenced by increased propidium iodide uptake, elevated extracellular conductivity, and leakage of cellular proteins, leading to the collapse of cellular homeostasis and death of C. gloeosporioides. Beyond direct antifungal effects, OEO treatment delayed the ripening and senescence of mango fruit by retarding peel color transformation, reducing weight loss, maintaining firmness, and slowing metabolic shifts in SSC and acidity. Furthermore, the physiological regulation of mangoes was also mediated through the suppression of key enzymatic activities (PPO and POD) involved in the browning process and the enhancement of the fruit’s antioxidant defense system, as indicated by increased SOD activity and reduced MDA accumulation. Collectively, these findings provide compelling evidence for the dual role of OEO as both a natural fungicide and a physiology-modulating preservative, offering a sustainable alternative to synthetic fungicides for maintaining the quality attributes of mangoes. Future research should focus on elucidating the molecular mechanisms underlying OEO’s regulation of fruit physiology, evaluating the sensory impact of treatment on consumer acceptance, and developing advanced delivery systems or integrated strategies to facilitate its practical commercial application.