Application of Oregano Essential Oil by Spraying and Nebulization to Post-Harvest Tahiti Lime for Green Mold Control.

Abstract

This study investigated in vitro antifungal action of oregano essential oil (EO) and different application methods to Tahiti lime to control green mold and preserve the fruits. The chemical composition of oregano EO was analyzed by gas chromatography coupled to mass spectrometry (GC-MS), showing carvacrol (70%) and thymol (17%) as its major constituents. The antifungal activity against Penicillium digitatum was evaluated by the contact method in PDA culture medium with oregano EO at different concentrations, in addition to the volatile phase. In the in vivo tests, inoculated fruits were treated with oregano EO by spraying and nebulization in drums (200 L) for 24 hours, stored at 24 °C. Curative and protective effects of oregano EO on fruits were also studied by spraying and nebulization, with refrigeration at 9 °C for 13 days, plus six days at 23 °C. The results demonstrated the oregano EO fungicidal action by volatile compounds and by the contact method against P. digitatum. Spraying of oregano EO showed a tendency to inhibit green mold on fruits at 24 °C and reduced the incidence by 18% (protective) under refrigeration (F=ns). Nebulization (3000 mg L⁻¹ EO) reduced the disease incidence by 11% and 31%, in a curative manner at 24 °C and protective manner at 9 °C (F=*), respectively. Both types of application did not cause significant changes in fruit quality. Therefore, oregano EO is a promising solution to inhibit green mold on post-harvest Tahiti lime. Further studies are needed to improve application efficiency and economic viability.

Keywords:
Citrus latifolia; Origanum vulgare; Penicillium digitatum; alternative control; quality.

HIGHLIGHTS

Oregano essential oil has fungicidal action against Penicillium digitatum.

Spraying and nebulization of oregano oil has the potential to inhibit green mold.

Oregano essential oil does not affect the quality of Tahiti lime.

INTRODUCTION

In Brazil, citrus fruits are among the most important fruits in terms of production and export. The Tahiti lime [Citrus latifolia (Yu. Tanaka) Tanaka] is a highly appreciated fruit in the country and abroad. In 2023, production reached 1.6 million tons (t), in an area of 62.000 hectares (ha), with the state of São Paulo as the leader in production (70%). Exports reached 166.600 t, equivalent to 10% of the total production, with the third largest volume among the most exported fruits [¹].

However, post-harvest diseases can cause significant fruit losses. Green mold (Penicillium digitatum (Pers.) Sacc.) is considered the most common and important disease in post-harvest citrus fruits in all production regions. Other phytopathogens have also been reported, such as P. italicum, Phomopsis citri, Colletotrichum gloeosporioides, Geotrichum citri aurantii [²-⁴].

Post-harvest diseases in citrus fruits can be controlled with fungicides: imazalil, azoxystrobin/fludioxonil, pirimetanil and thiabendazole [⁵]. However, the selection of resistant strains of pathogens due to the continuous use of fungicides and global trend toward organic fruits and vegetables free from chemical residues, with an emphasis on sustainability and food safety, have encouraged the use of alternative processes and products, including essential oils with antifungal potential. Many studies have assessed the antimicrobial action of essential oils and their major constituents for medicinal, industrial, and agricultural purposes. Some aromatic plants stand out, such as cinnamon, cloves, thyme, oregano, mint, lemongrass, and citrus plants, as well as their constituents: eugenol, cinnamaldehyde, carvacrol, thymol, menthol, citral, limonene, and others [⁴,⁶-⁹].

For disease control in post-harvest fruits, many studies have reported positive results with essential oils applied by immersion, spraying, sprinkling, nebulization, vaporization, and nanoemulsions, directly or combined with coating and plastic packaging [⁶, ⁹-¹¹]. However, some limitations must be investigated, such as curative and preventive action, species of phytopathogen, application method, type of climacteric and non-climacteric fruit, compatibility with other post-harvest processes (thermotherapy, cooling, coating), as well as the standardization of the essential oil-based product [⁷, ¹¹].

Oregano EO (Origanum vulgare L.) and its main constituents have been classified as Generally Recognized as Safe (GRAS) for human use by the FDA, as well as traditional preparations and uses that do not have relevant toxicological properties. Oregano EO stands out as it is rich in thymol and carvacrol and has several health-promoting properties. These compounds have significant antioxidant, anti-inflammatory, antiangiogenic, anticancer, and antimicrobial activities [¹², ¹³]. Promising results have been reported for the control of Penicillium spp. in citrus fruits, including lemons [³, ⁶, ¹⁴-¹⁶]; however, further studies on the application methods are required.

This study aimed to evaluate the antifungal action of oregano EO in vitro on P. digitatum, as well as its application in different ways on post-harvest Tahiti lime, for green mold control and fruit preservation.

MATERIAL AND METHODS

Phytopathogen and essential oil

The target pathogen was an isolate of Penicillium digitatum (SISGEN A03215C), obtained from the mycotheque of the Centro de Citricultura Sylvio Moreira (IAC). Oregano essential oil was purchased commercially, batch LZ0603 from LASZLO®.

The analysis of the chemical composition of the oregano EO was performed on a Thermo Scientific gas chromatograph (model TRACE 1300 Series GC) equipped with a flame ionization detector (FID), mass spectrometer (model ISQ 7000) and Triplus RSH automatic injector. The injector was maintained at 220 ºC, with a carrier gas flow (helium, 99.9999% purity) in a split ratio of 1:20. The essential oil samples were diluted in ethyl acetate (chromatographic grade, 0.1 mg mL^-1) and 1 µL of solution was injected. The separation of the substances was performed in a Rtx-5 MS capillary column (30 m x 0.25 mm, 0.25 μm, Thermo Scientific), with a carrier gas flow rate of 1.0 mL min^-1, in the following temperature program: 60°C - 240°C, 3°C min^-1. The mass spectrometer (MS) operated in full scan mode, by electronic ionization (70 eV) and acquisition range of 40 to 450 m/z. The transfer line and ionization source were operated at 230°C and 250°C, respectively. Chromeleon software (Thermo Scientific-Waltham, MA, USA) was used for data acquisition and processing. The substances were identified through a comparative analysis of the mass spectra with the National Institute of Standards and Technology (NIST 14) and Flavour & Fragrance Natural & Synthetic Compounds (FFNSC3) libraries, and the Linear Retention Index (LRI) of the substances with the literature [¹⁷]. The linear retention indexes were obtained from the injection of a series of n-alkanes (C₉-C₂₄, Sigma-Aldrich, 99%) under the same chromatographic conditions as the samples, using the equation of Van den Dool and Kratz [¹⁸].

Antifungal activity of oregano essential oil assessed in vitro

In an in vitro contact assay, a stock solution of oregano EO was prepared with the addition of Tween⁸⁰ (0.2% v/v), in sterile distilled water, from which aliquots were taken for the composition of the treatments, incorporated into the melting Potato-Dextrose-Agar (PDA) culture medium (45 °C) and poured into Petri dishes (90x15 mm). The resulting concentrations were 0.0; 125; 250; 500; 1000; and 2000 mg L^-1. After 24 hours, a disc (4 mm) from the edge of the fungus colony (P. digitatum) was placed in the center of each Petri dish. The fungus was cultivated for seven days at 25 °C and a 12 hours photoperiod. Eight replicates were performed for each treatment.

To evaluate the antifungal effect of the volatile compounds of the oregano EO, Petri dishes with two compartments (90x15 mm) were used, depositing 10 and 20 µL aliquots of the pure oil on a sterile filter paper disc (20 mm) and placed in one of the compartments. The control consisted of 10 µL of sterile distilled water. The concentration of the essential oil in its volatile phase was calculated as a function of the free air volume of the Petri dish, resulting in about 180 and 360 µL L^-1 air for 10 and 20 µL, respectively [¹⁹]. A disc (4 mm) from the edge of the pathogen colony was placed in the other compartment of the plate containing PDA culture medium, totaling three treatments with six replicates. All plates were sealed with Parafilm® and stored in a BOD incubator at 25 °C.

The mycelial growth of the pathogen was assessed daily using a digital caliper, taking two orthogonal measurements of the colony diameter until some treatment reached the edge of the plate. With data obtained, the percentage of mycelial growth inhibition (MGI) was calculated:

(1) $$\mathrm{MGI}(\mathrm{\%})=[(\mathrm{dc}-\mathrm{dt})/(\mathrm{dc})\times 100]$$

In which: dc = colony diameter of the control; and dt = colony diameter of the treatment.

The mycelial growth velocity index (MGVI) was calculated using the equation below:

(2) $$\text{MGVI}\left(mmda{y}^{-1}\right)=\sum (D-Da)N^{-1}$$

In which: D = colony diameter on the evaluation day; Da = colony diameter on the day before the evaluation; and N = number of incubation days.

After the end of the incubation period, the fungus inocula that did not develop were transferred to other Petri dishes containing only PDA, to assess the fungistatic or fungicidal action of the EO. Both tests were repeated twice.

Oregano essential oil in the control of green mold on Tahiti lime

The P. digitatum isolate was grown in PDA culture medium in a BOD incubator at 25°C, with light-darkness alternance (12 hours) for seven days. A conidial suspension was obtained with the addition of Tween²⁰ (0.05 mL) and adjusted to 1x10⁵ cfu mL^-1, as determined by counting in a hemocytometer. The fruits were inoculated by making a micro injury (2 mm deep) with the help of a needle, at a point in the equatorial region, on which 10 µL of the conidial suspension was deposited with a micropipette.

Application by spraying

Tahiti limes from an organic production system in Mogi Mirim, in the state of São Paulo, were taken to the laboratory and checked for absence of defects. After that, they were subjected to surface disinfection using neutral detergent, rinsing, immersion in a sodium hypochlorite solution (200 µL L^-1 of active chlorine) for 2 minutes, rinsing, and drying under a fan. The fruits were inoculated, placed in plastic boxes and incubated at 24 °C in a humid chamber for about 18 hours. Later, the oregano EO was sprayed on the fruits using a manual sprayer until completely covered, applying approximately 2.0 mL per fruit, in the following concentrations: 0.0 (water), 125, 250, 500, and 1000 mg L^-1, with Tween⁸⁰ surfactant (0.05% v/v). After drying with a fan, the fruits were stored in a chamber at 24°±1 °C/75-85% RH for up to five days. The experimental design was completely randomized, with five replicates per treatment and eight fruits in each plot. The experiment was repeated twice.

Application by nebulization

‘Tahiti’ limes, after surface disinfection, were inoculated and incubated. The fruits were then stored in drums (200 L) containing a mini-fan and hermetically sealed. Oregano EO was then applied by nebulization using an OMRON NE-C701 inhaler/nebulizer device through a hole in the drum lid. Two trials were performed: E1) control (water); 1000 and 2000 mg L^-1 EO; E2) control (water); 2000 and 3000 mg L^-1 EO. Tween⁸⁰ was added at 0.05, 0.10, and 0.15% (v/v) for 1000, 2000, and 3000 mg L^-1 EO, respectively, and 0.05% for control. The volume applied by nebulization was 10 mL for 60 minutes, equivalent to 0.05, 0.10, and 0.16 mg L^-1 EO in the headspace of the drum, where they remained for 24 hours. Then, the drums were opened and the fruits were stored in a chamber at 24°±1 °C/75-85% RH for up to five days. The experimental design was completely randomized, with six replicates per treatment and eight fruits in each plot.

The incidence and severity of green mold on the fruits were assessed daily using a digital caliper (mm). The incidence was measured by the number of fruits with symptoms of the disease and the inhibition rate (%) was calculated. The measurements of the lesion area of green mold were used to calculate the Area Under the Disease Progress Curve (AUDPC):

(3) $$\mathrm{AUDPC}=\sum \left[(Y_{i+1}+Y_i)/2\right][T_{i+1}-T_i]$$

In which: Y_i+1 = lesion diameter at time T_i+1, and Y_i = lesion diameter at time T_i [²⁰].

Spraying and nebulization of essential oil, curative and protective mode, on fruits stored under refrigeration

Tahiti limes, after surface disinfection, were divided into two batches: L1) with inoculation and L2) without inoculation for analysis of quality attributes. For curative effect, the fruits were first inoculated and after 24 hours they received the treatments; for the protective effect, the fruits were subjected to the treatments and after 24 hours they were inoculated. The treatments of all fruits were: spraying - control (water) and 1000 mg L^-1 EO; nebulization - control (water) and 3000 m L^-1 oregano EO. L1 had a 2x2x2 factorial design, with factor A (application methods), factor B (doses), and factor C (inoculation time), creating eight treatments, with four replicates and eight fruits in each plot. All fruits were stored under refrigeration at 9°±1 °C/80-90% RH for 13 days, with transfer to 23°±1 °C/75-85% RH for another six days. The incidence and severity of green mold were assessed every two days.

Evaluation of quality attributes

The L2 (batch without inoculation) consisted of four treatments, with 20 fruits each, intended for physical and chemical analysis on the first day, after 13 days of refrigeration and after another six days under ambient conditions. The following analyses were performed:

• • Weight loss (%) with a Marte BL3200H semi-analytical balance, using four replicates and five fruits in each plot.

• • Peel color with a Minolta BC-10 colorimeter, L*a*b* system: L* luminosity (0=black; 100=white); a* parameter (-a* = green / +a* = red); b* parameter (-b* = blue / +b* = yellow). Two readings were taken in the equatorial region per fruit, with 10 replicates per treatment. The results were presented in Color Index (CI), calculated by the equation:

  (4)$$\mathrm{CI}=1000\times {\mathrm{a}}^{\ast }/({\mathrm{L}}^{\ast }\times {\mathrm{b}}^{\ast })$$

• This index ranges from -20 to +20; and the lower the CI, the greener the color of the fruit peel [²¹].

• • Soluble solids content (SS - °Brix) with ATAGO PR-101α digital benchtop refractometer - 0-45, using five replicates per treatment and samples consisting of the juice of two fruits.

• • Titratable acidity (TA - % citric acid) - titration with 1.0 N sodium hydroxide (NaOH) solution until pH 8.1 is reached using a TECNAL TEC-2 pH meter, with five replicates per treatment and samples consisting of the juice of two fruits.

• • Ascorbic acid (AA - mg 100 g^-1) - determined by the volumetric method, based on the reduction of the indicator 2,6-dichlorophenol indophenol sodium (DCFI), with five replicates per treatment and samples consisting of the juice of two fruits, in duplicate [²²].

Statistical analysis

In the in vitro contact test, the mean values of the treatments were analyzed by polynomial regression as a function of the dose. For the volatile compounds, analysis of variance was applied, followed by comparison of the mean values by the Tukey’s test (p<0.05). The incidence of green mold on the fruits was evaluated by means of the Kruskal-Wallis nonparametric test. Also, the mean values of AUDPC (severity) and quality attributes of the treatments were subjected to analysis of variance and compared by the Tukey’s test (p<0.05) and, when appropriate, analyzed by polynomial regression. Statistical analyses were performed using ESTAT 2.0 and MINITAB 14.

RESULTS AND DISCUSSION

Chemical composition of oregano essential oil

According to the analysis of the chemical composition of oregano EO, its major constituents are carvacrol (70%) and thymol (17%), (Table 1). It highlighted the differences in the chemical composition of this essential oil, in comparison to other studies [¹¹], such as 63.3% thymol and 7.8% carvacrol [¹⁴]; 30.5% carvacrol and 27.5% thymol [³]. Six subspecies of O. vulgare have been reported in different locations in the world, in addition to nine chemotypes, with volatile constituents in different proportions, representing a large source of monocyclic monoterpene (thymol, carvacrol, y-terpinene, p-cymene) [¹²,¹³].

Table 1
Chemical composition of oregano essential oil determined by GC-MS.

Substance	% Relative	LRI exp.	LRI lit.
α-pinene	0.42	936	932
camphene	0.32	955	946
β-pinene	0.23	985	974
myrcene	0.13	992	988
α-terpinene	0.07	1022	1014
limonene	0.22	1034	1024
1,8-cineol	1.26	1039	1026
linalool	0.15	1107	1095
camphor	0.26	1161	1141
borneol	0.07	1185	1165
terpinen-4-ol	0.42	1192	1174
α-terpineol	0.09	1208	1186
thymol methyl ether	0.04	1237	1232
bornyl acetate	0.06	1293	1284
thymol	17.32	1303	1289
carvacrol	70.27	1312	1298
E-caryophyllene	0.24	1426	1417
α-humulene	0.08	1463	1452
TOTAL	91,65

• ^*
  LRI exp.: experimental linear retention index; LRI lit.: linear retention index from literature [¹⁷].

These variations emphasize the importance of a detailed chemical analysis of each batch of essential oil, since external factors such as origin, climate, plant development stage, and extraction methods play crucial roles. Also, these differences in chemical composition can directly influence antimicrobial effects, which reinforces the need to understand these variations in order to standardize the use of essential oil in different applications, whether for agricultural or medicinal purposes [¹¹-¹³, ²³].

Thermochemical stability and proper storage conditions are very important for preserving the quality and efficacy of essential oils. Oregano EO was thermally degraded until reaching a maximum temperature of 211.6 °C. The stability of oregano EO tended to decrease with temperature (40 °C - 80 °C / 60 min) compared to cinnamon EO stored under closed conditions, with an approximate difference of at least thirteen degrees. However, the results confirmed the thermal stability of oregano EO and its resistance to degradation during heating and storage [²⁴].

Antifungal activity of oregano essential oil against P. digitatum assessed in vitro

The Figure 1A shows the antifungal activity by the contact method of oregano EO against P. digitatum, with fungistatic action at 500 mg L^-1 and fungicidal action above 1000 mg L^-1. The MGVI decreased with increasing oil concentration, and a third-degree regression was the model with the best fit to data. The Figure 1B shows the significant fungicidal effect of oregano EO volatiles on the pathogen (F test **). Preliminary, in vitro tests were carried out with exposure of the P. digitatum isolate to the fungicide imazalil (1000 µL L⁻¹), resulting in 100% MGI (data not shown), similar to the results obtained with oregano OE.

Figure 1
Mycelial growth velocity index (MGVI) and mycelial growth inhibition (MGI %) of Penicillium digitatum, cultivated in potato dextrose agar (PDA) medium with oregano essential oil by the contact method (A) and by volatiles (B), 180 and 360 µL L^-1 headspace for 10 and 20 µL, respectively, at 25 °C. Tests repeated twice (F test**; Tukey’s test, p<0.05).

The efficacy of carvacrol and thymol by contact and volatiles in the control of post-harvest pathogens, especially against Penicillium spp., has been widely explored due to their antifungal properties [⁶, ²⁵]. Studies indicate that the combination of thymol and carvacrol has more efficiently inhibited mycelial growth than the use of carvacrol alone [¹⁵]. The minimum inhibitory concentration (MIC) of these compounds varies according to the target pathogen. Both compounds, at the concentration of 125 mg L⁻¹, inhibited the mycelial growth of P. digitatum, while oregano EO required a higher concentration (500 mg L⁻¹) to achieve the same effect [³], in agreement with the MIC found in this study. This result shows that the antifungal action of essential oils is determined not only by the presence of their main compounds, but by the interaction of various components [¹⁶]. The volatile phase of essential oils, including oregano EO, has high antifungal action against Penicillium spp. [⁶, ¹⁶, ²⁵].

The most important phytochemicals of oregano EO are volatile and non-volatile phenolic compounds (phenolic acids and flavonoids) and the antimicrobial mechanisms include enzyme inhibition, efflux pump inhibition, ATP depletion, inhibition of biofilm formation, and cytoplasmic membrane damage [⁷, ¹⁰, ¹²]. Essential oils can slow down the process of lipid peroxidation and free radical elimination, and these antioxidant properties and the ability of structural modification that cause variation in fungal morphology lead to the inhibition of conidial germination. In addition, sesquiterpenes, monoterpenes, alcohols, esters, aldehydes, and phenols are reported as the main components found in EOs, causing strong inhibition of fungal mycelial growth [⁸]. These aspects reinforce the importance of essential oils as a sustainable and efficient alternative for post-harvest pathogen control.

Oregano essential oil for the control of green mold on Tahiti lime

Spraying

It is observed in Table 2 that, as the concentration of oregano EO increased, the incidence of green mold decreased in the fruits, with an inhibition rate of 3.8% at 1000 mg L⁻¹. Regarding disease severity (AUDPC), although it showed a tendency for inhibition above 500 mg L⁻¹, it did not differ statistically from the control after five days of storage under ambient conditions (Figure 2).

Table 2
Incidence of green mold on Tahiti limes inoculated with Penicillium digitatum and sprayed with oregano essential oil after five days of storage at 24°±1 °C/75-85% RH.

Treatments (mg L-1)	Incidence	Inhibition rate
	Mean ± standard deviation	%
0.0	39.0 ± 1.0Z 38.5 ± 0.5 38.5 ± 0.5 38.0 ± 0.0 37.5 ± 0.5	-
125		1.3
250		1.3
500		2.6
1000		3.8

• ^Z
  Mean values of two trials with 40 fruits per treatment.

Figure 2
Area Under the Disease Progress Curve (AUDPC) of green mold severity on Tahiti lime with Penicillium digitatum after inoculated and spraying with oregano EO, stored for five days at 24°± 1 °C/75-85% RH. Mean values of five replicates with eight fruits per plot. Trial replicated twice. Treatment means did not differ significantly from each other (F test = ns; Tukey’s test, p<0.05, data transformed into $\sqrt{x+0.5}$).

The analysis of this and other studies reveals a promising outlook for the use of essential oils in post-harvest fruit preservation and control of fungal diseases, showing different types of application using the contact method and the efficacy of the compounds.

Lemons inoculated with P. digitatum immersed in wax with carvacrol or thymol for 10 minutes had a significant reduction in the incidence of green mold, suggesting that pure EO compounds had a stronger antimicrobial action than the unchanged EO [¹⁵]. The addition of oregano EO nanoemulsion in an alginate-based coating helped extend the shelf life of tomatoes, with a significant reduction in mold and yeast for up to 14 days under ambient conditions (24 °C) [⁹]. The application of wax with oregano EO delayed the ripening of mangoes and effectively controlled the natural incidence of anthracnose for 18 days at 14 °C [²⁶]. The inclusion of these compounds in the wax coating prolonged the effect, reinforcing the importance of the formulation and application method to maximize efficacy.

Oranges inoculated and immersed in thymol for 05 minutes showed inhibition of P. digitatum and G. candidum when stored at 22 °C, with a positive factor of absence of phytotoxicity [³]. Spraying thyme EO reduced the severity of gray mold (Botrytis cinerea) on inoculated and refrigerated (1 °C) bunches of Italia grapes [²⁷]. These data highlight the relevance of storage conditions for the stability of active compounds.

Nebulization

Nebulization of Tahiti limes with oregano EO showed higher efficiency at 3000 mg L^-1 (0.16 mg L^-1 EO headspace), with an inhibition rate of 11%; as well as a tendency to delay disease development (AUDPC). Although promising, the mean values did not differ significantly from the control (Table 3), so the method can still be improved. Positive results of the application of EOs by nebulization and vaporization are reported for several post-harvest fruits [⁷]. Vaporization of essential oils from Thymus vulgaris and Origanum heracleoticum effectively reduced gray mold (B. cinerea) in strawberries while maintained fruit quality [¹⁰]. Likewise, fumigation with oregano EO at 60 µL L^-1 reduced rot caused by B. cinerea and Alternaria alternata in post-harvest blueberries [²⁸]. These studies highlight that optimized vaporization can act as a solution in post-harvest management.

Table 3
Incidence, Area Under the Disease Progress Curve (AUDPC - severity), and inhibition rate (%) of green mold on Tahiti lime with Penicillium digitatum after inoculated and treated with oregano essential oil by nebulization, stored at 24°±1 °C/75-85% RH for four and five days for trials I and II, respectively.

Oregano EOZ (mg L-1)		Trial I			Trial II
		IncidenceY	Inhibition rate		IncidenceY	Inhibition rate
0.0		42	-		37	-
1000		43	-2.4%		-	-
2000		45	-7.1%		37	0.0%
3000		-	-		33	10.9%
	AUPDCX			Inhibition rate	AUPDCX	Inhibition rate
0.0	76.2			-	111.3	-
1000	72.6			4.7%
2000	71.4			6.3%	111.6	-0.3%
3000	-			-	105.6	5.1%

• ^Z
  Nebulization (10 mL) of EO solution equivalent to 0.05; 0.10, and 0.16 mg L^-1 in the headspace of the drum (200 L), stored for 24 hours in a chamber.
• ^Y
  Each treatment had 48 inoculated fruits. Data did not differ significantly by the Kruskal-Wallis nonparametric test (p<0.05).
• ^X
  Mean values of six replicates with eight fruits per plot. AUDPC mean values of each trial did not differ significantly when compared to each other (F test = ns, Tukey’s test p<0.05, data transformed into $\sqrt{x+0.5}$).

Spraying and nebulization of essential oil on fruits stored under refrigeration - curative and protective mode.

This study provides a comparison of two types of EO application to post-harvest fruits, with curative and protective effects. According to Table 4 and Figure 3, both types of oregano EO application showed a similar effect of reduced incidence of green mold in Tahiti limes inoculated before (curative) and after (protective) treatments and refrigeration, with a significant protective effect of 18.5% and 30.8% for spraying and nebulization, respectively. Regarding mold severity (AUDPC), it was higher for the fruits inoculated before the treatments (curative) when compared to those inoculated later (protective) (F test**, Table 5). It is justified by the incubation time (24 hours), plus the treatment time (24 hours) before storage under refrigeration. Also, at low temperatures, a tendency to inhibit mold severity was observed in treatments with EO, in both types of application, but they did not differ from the controls. Rapid refrigeration of the fruits associated with the application of oregano EO was a more adequate solution to delay the lag phase of pathogen development.

Table 4
Incidence and inhibition rate of green mold in Tahiti limes with Penicillium digitatum after inoculated and treated with oregano EO (S = spraying control; SO = oregano EO by spraying - 1000 mg L^-1; N = nebulization control; NO = oregano EO by nebulization - 3000 mg L^-1). After 13 days under refrigeration at 9°± 1 °C/80-90% RH, plus 6 days at 23°±1 °C/75-85% RH.

Oregano EO	Curative		Protective
	IncidenceZ	Inhibition rate	IncidenceZ	Inhibition rate
S	27 a	-	27 a	-
SO	28 a	-3.7 %	22 ab	18.5 %
N	29 a	-	26 a	-
NOY	30 a	-3.4 %	18 b	30.8 %

• ^Z
  Curative treatment - 24 hours after inoculation; protective treatment - 24 hours before inoculation. Each treatment had 32 fruits. Data followed by the same letter in the column do not differ significantly by the Kruskal-Wallis nonparametric test (p<0.05).
• ^Y
  NO = equivalent to 0.16 mg L^-1 in the headspace for 24 hours.

Table 5
Analysis of variance (F) of the AUDPC mean values for the factorial design (2x2x2) to evaluate oregano essential oil with two types of application (A), two doses (B), and curative and protective effect (C) on green mold on Tahiti limes after refrigerated storage.

Factors	F
	9 °C - 13 days	23 °C - 6 days
Factor A (2 - application)	ns	ns
Factor B (2 - dose)	ns	ns
Factor C (2 - inoculation)	**	**
Factor AxB	ns	ns
Factor AxC	ns	ns
Factor BxC	ns	ns
Factor AxBxC	ns	ns

• Significance of F test for treatment effects: ns = not significant.
• ^*
  (p<0.05) and
• ^**
  (p<0.01).

Figure 3
AUDPC of green mold on Tahiti lime with Penicillium digitatum after inoculated and treated with oregano EO (S = spraying control; SO = oregano EO by spraying - 1000 mg L^-1; N = nebulization control; NO = oregano EO by nebulization - 3000 mg L^-1). After 13 days under refrigeration at 9°±1 °C/80-90% RH, with transfer to 23°±1 °C/75-85% RH for another 6 days. The bars represent the mean value of four replicates with eight fruits per plot. The mean values of the treatments did not differ significantly from each other for the curative and protective effects (Tukey’s test. p <0.05).

The results of this study indicate that the oregano EO nebulization system is more effective than spraying for Tahiti limes. Spraying EO covers the fruit and acts more through contact, healing wounds during a short storage period. Nebulization of EO stored in a chamber for 24 hours allows greater exposure of the fruits to volatile compounds, with protective action and possible induction of disease resistance mechanisms. Both methods, despite their specific properties, can be complementary to maximize efficacy.

Induction of disease resistance promoted by essential oils has been reported by several authors [⁸]. Cinnamon EO effectively controlled P. italicum in citrus fruits by activating the gene expression of defense-related enzymes [²⁹]. It was found that a nanoemulsion of cinnamaldehyde, carvacrol, and eugenol induced resistance in Newhall oranges against P. digitatum by promoting phenylpropanoid metabolism [³⁰]. Likewise, fumigation of blueberries with oregano EO induced the activity of enzymes related to pathogenesis (phenylalanine ammonia lyase - PAL; polyphenol oxidase - PPO; peroxidase - POD; chitinase - CHI; B-1.3-glucanase - GLU), with reduced rot during storage [²⁸].

In vitro studies with EOs often demonstrate fungicidal activity against pathogens, since they are directly exposed to volatile and contact compounds. However, when EOs are applied to post-harvest fruits, the results do not always show the same efficacy observed under in vitro conditions. This limitation is associated with the complex interaction between host, pathogen, environment and antimicrobial agent [²⁷]. Notably, oregano EO and its main component, carvacrol, were effective against fungal isolates resistant to the fungicide imazalil [³].

Suppression of quiescent infections and/or wounds, while preserving fruit quality, can be enhanced by integrating several preand post-harvest control methods [³¹]. It is important to optimize concentrations, formulations, and application technologies, such as spraying, nanoemulsion, and nebulization, in order to ensure more consistent and commercially viable results. These analyses reinforce the innovative role of EOs in fruit conservation, highlighting their multifunctionality and the potential for integrated use for post-harvest management.

Effect of oregano essential oil on the quality of Tahiti lime

According to the results of the quality attributes of Tahiti limes (Table 6), oregano EO application by spraying or nebulization caused no significant changes in post-harvest fruits regarding signs of phytotoxicity or noticeable odor, as observed in other studies [³, ²⁷]. The color index and luminosity suggest the natural degradation of chlorophyll and expression of carotenoids during storage, an expected characteristic of fruit senescence [³², ³³]. During the period under refrigeration, fruit weight loss was around 6%, and after another six days at 23 °C, it was between 2-3%, which indicates water loss and metabolic changes. Soluble solids content (SS) and titratable acidity (TA) of the fruits remained practically constant. The ascorbic acid (AA) content increased slightly over time, which may be attributed to weight loss, also observed in other studies with lemon [¹⁶, ³², ³³].

Table 6
Peel color index (CI) and luminosity (L^*), weight loss (WL), soluble solids (SS), titratable acidity (TA), and ascorbic acid (AA) of Tahiti limes treated with oregano EO (S = spraying control; SO = EO applied by spraying - 1000 mg L^-1; N = nebulization control; NO = EO applied by nebulization - 3000 mg L^-1), at the beginning of the experiment, after 13 days under refrigeration at 9°±1 °C/80-90% RH and another 6 days at 23°±1 °C/75-85% RH.

Attribute	Days	Treatments				CV% FW
		S	SO	N	NO
CIZ	beginning	-7.71±1.12	-	-	-	-
	13	-6.50±1.26	-6.31±1.16	-6.67±0.73	-7.14±1.98	20.37ns
	13+6	-5.45±1.25	-5.98±1.13	-5.82±1.60	-6.02±1.36	23.11ns
L*Z	beginning	50.6±2.5	-	-	-	-
	13	53.0±3.2	53.8±4.0	53.0±2.2	52.3±4.5	6.75ns
	13+6	56.5±3.9	54.8±3.6	55.4±4.3	55.3±3.9	7.07ns
WL (%)Y	13	5.6±0.6	5.6±0.6	5.6±0.6	5.9± 0.9	5.63ns
	13+6	3.6±2.0	2.1±0.4	2.3±0.4	2.7±0.8	15.96ns
SS (°Brix)X	beginning	7.06±0.23	-	-	-	-
	13	7.64±0.32	7.66±0.36	7.46±0.23	7.26±0.30	4.06ns
	13+6	7.58±0.16	7.74±0.40	7.72±0.30	7.76±0.24	3.79ns
TAX	beginning	6.22±0.37	-	-	-	-
(% citric acid)	13	6.47±0.16	6.06±0.26	6.31±0.42	6.49±0.06	2.00ns
	13+6	6.36±0.27	6.34±0.19	6.36±0.46	6.36±0.22	2.23ns
AAX	beginning	19.31±1.32	-	-	-	-
(mg 100g-1)	13	20.90±0.71	20.90±0.71	21.53±0.87	21.53±0.87	3.72ns
	13+6	25.33±1.12	25.65±0.71	24.38±1.42	25.33±0.0	3.85ns

• ^Z
  Mean±standard deviation of 10 replicates.
• ^Y
  Mean±standard deviation of 4 replicates with 5 fruits per plot.
• ^X
  Mean±standard deviation of 5 replicates of a sample of 2 fruits.
• ^W
  F test = ns (not significant). mean values analyzed by Tukey’s test (p<0.05).

Seedless limes (C. latifolia) packaged in perforated packages and stored at 10°C maintained their quality and bioactive compounds for 21 days, with changes similar to those observed in this study: increased luminosity (L*), reduced green color (IC); around 4-5% mass loss; 8°Brix soluble solids; 6.5% titratable acidity; 30 mg mL^-1 ascorbic acid for 14 days, but reduced subsequently [³³].

The application of thymol and carvacrol with wax to lemons reduced the respiratory rate and ethylene production, while maintaining the soluble solids content and titratable acidity during storage [¹⁵]. Similarly, it was found that exposing strawberries to EOs vapors prevented weight loss and soluble solids degradation [¹⁰]. However, concentrations above 1% of oregano EO in mangoes increased the respiratory rate, causing peel damage, which indicates adjustments are required in the formulation for different fruits [²⁶].

Citrus fruits are classified as non-climacteric, maintaining a relatively low and stable respiratory rate after harvest. Fruit cells can protect themselves from oxidative stress by producing low molecular weight antioxidant molecules, such as ascorbic acid and phenolic compounds [¹⁶]. Nanoemulsion of essential oils in oranges maintained the firmness of the fruits and increased flavonoids, lignin, and phenolic compounds content, in addition to proteins related to pathogenesis [³⁰].

Although oregano EO is classified as GRAS, adverse reactions to specific compounds have been reported. The cytotoxicity analysis showed that oregano EO was potentially toxic, with cell survival rates below 20% when in contact with 25 μg mL^-1 concentrations of the oil. Toxicological studies are thus necessary and may vary due to several factors, including dose and frequency of exposure [³⁴].

The results of this work indicate that oregano EO did not compromise the physical and chemical characteristics of Tahiti limes, reinforcing the viability of using this essential oil in post-harvest fruits, considering that maintaining quality is essential for commercialization.

CONCLUSION

Based on the conditions in which this study was conducted, we concluded that oregano EO has in vitro fungicidal activity against P. digitatum both by the contact method (≥1000 mg L⁻¹) and through volatile compounds. The application of oregano EO by spraying showed a tendency to inhibit green mold in fruits stored at 24 °C while under refrigeration, it showed a protective effect, with an inhibition rate of 18% (F=ns). Nebulization of oregano EO (3000 mg L⁻¹) showed the best results, achieving mold inhibition rates of 11% at 24 °C (curative effect) and 31% at 9 °C (protective effect), with statistically significant differences in relation to the control. Although a trend towards reduced disease severity (AUDPC) was observed, the differences were not significant (Tukey, p<0.05). Also, both types of oregano EO application did not cause significant changes in the physical and chemical attributes of Tahiti limes. Therefore, oregano EO is a promising solution for the inhibition of green mold in post-harvest limes. However, further studies are required to improve application efficiency and evaluate the economic viability of this solution.

• Funding:
  This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) number 2022/00454-0. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
• Institutional Review Board Statement:
  “Not applicable”.
• Informed Consent Statement:
  “Not applicable.”

Acknowledgments:

We would like to thank FT/UNICAMP and Instituto Agronômico (IAC) for the availability of laboratories. Andrade Sun Farms Agrocomercial Ltda. for providing the fruits. Daniela Matsumoto Soares for contribution to chromatography analysis. FAPESP (grant # 2018/25812-1 and 2017/50338-9) for supporting the financing of the chromatograph (GC-MS) used in the analysis of essential oils. The authors thank Espaço da Escrita - Pró-Reitoria de Pesquisa - UNICAMP - for the language services provided.

Use of Generative Artificial Intelligence

The authors declare that large language models and other generative artificial intelligence (AI) or AI-assisted technologies cannot be credited as authors and have not been listed as authors of this paper.

The authors declare that generative artificial intelligence (AI) or AI-assisted tools were used under full human supervision. The tool(s) and version(s) used, and their purpose, are described here: COPILOT. No confidential or sensitive data were uploaded to such tool(s), and all AI-assisted content was checked, corrected and approved by the authors, who take full responsibility for the integrity and originality of the manuscript.

Data Availability Statement:

Research data are available in the repository (https://redu.unicamp.br/dataset.xhtml?persistentId=doi:10.25824/redu/SEKZ9T).

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• Editor-in-Chief:
  Bill Jorge Costa

• Associate Editor:
  Ana Cláudia Barana

Publication Dates

• Publication in this collection
  22 June 2026
• Date of issue
  2026

History

• Received
  15 May 2025
• Accepted
  02 Feb 2026