Impaginato 169 Adv. Hort. Sci., 2024 38(2): 169­176 DOI: 10.36253/ahsc­15659 The effect of thymol and carvacrol rich­ plant essential oils on controlling postharvest decay molds in orange fruit L. Mokhtarnejad 1 (*) , M. Farzaneh 2 1 Plant Protection Research Department, West Azarbaijan Agricultural and Natural Resources Research Center, AREEO, Urmia, Iran. 2 Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Evin, Tehran, Iran. Key words: Antifungal effect, Satureja spp., spoilage, Thymus spp. Abstract: The antifungal activity of essential oils of Thymus daenensis, Thymus vulgaris, Satureja hortensis and Satureja khuzistanica as well as their major compounds were studied against mold decays of orange fruit. According to GC­ MS analysis, the major compounds of T. danensis essential oil were thymol (65.5%) and alpha­terpinene (11.9%) whereas T. vulgaris was rich in thymol (59%) and p­cymene (15.6%). Carvacrol (88.4%) in S. khuzistanica oil and car­ vacrol (51%), gamma­terpinene (20.8%) and p­cymene (13.7%) in S. hortensis oil were charecterized as major compounds. The oil of S. khuzistanica and its major compound carvacrol exhibited the strongest fungicide activity against Penicillium digitatum, Rhizopus stolonifer and Colletotrichum gloeosporioides at 300 µL/L. The results on orange fruits exhibited that the use of S. khuzistani‐ ca and S. hortensis EOs as spraying and dipping treatments could considerably reduce spoilages decays in the fruit. 1. Introduction Post­harvest diseases of fruits are mainly caused by fungal species such as Botrytis spp., Colletotrichum spp., Aspergillus spp., Alternaria spp., Rhizopus spp. and Penicillium spp. (Agrios, 2005). The fruit decay caused by post­harvest diseases is usually more than what is thought, because with the decrease in yield the price of damaged fruits (Wills and Golding, 2016). Citrus fruits, especially oranges, are among the fruits that are highly sensitive to fungal infections. The use of fungicides, such as benomyl, thiabendazole and imazalil, is the most common method of controlling post­harvest decays of citrus fruits. These fungicides have health and environmental problems such as cumulative and carcinogenic properties in living organisms and acute or chronic poisoning effects. In addition, resistance to these fungicides is increasing in the population of pathogens (Sharifi­Tehrani and Farzaneh, 2018). Anyway, the increase in global demand for providing sufficient and healthy food, based on health standards, along with the policies of the World Food and Agriculture (*) Corresponding author: l.mokhtarnejad@gmail.com Citation: MOKHTARNEJAD L., FARZANEH M., 2024 ­ The effect of thymol and carvacrol rich‐plant essential oils on controlling postharvest decay molds in orange fruit. ­ Adv. Hort. Sci., 38(2): 169­176. Copyright: © 2024 Mokhtarnejad L., Farzaneh M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 1 December 2022 Accepted for publication 2 February 2024 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-15659 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2024 38(2): 169­176 170 Organization (FAO) and the Environmental Protection Organization (EPO) has caused extensive research to be carried out. According to the Food and Drug Administration (FDA), the essential oils (EOs) of some medicinal plants are known as natural and healthy alternatives to chemical fungicides and are more acceptable to the public (Brun et al., 2003; Carvalho de Sousa et al., 2004; Nazzaro et al., 2017). EOs are volatile and natural complex compounds that are characterized by their sharp and strong smell and are formed as secondary metabolites in aromatic plants. Some EOs that have antiseptic properties (antibacterial, antiviral and antifungal properties) are used in food and pharmaceutical industries (Burt, 2004; Bolouri et al., 2022). In nature, EOs play an important role in protecting plants against bacteria, viruses, fungi and insects (Regnault­Roger et al., 2012; Zitzelsberger and Buchbauer, 2015). They may also attract a number of insects to disperse pollen and seeds (Bakkali et al., 2008). Medicinal plant EOs not only have no side effects (at the right concentra­ tion), but due to their antioxidant properties, may increase the quality and storage time of fruits (Arras and Usai, 2001; Anthony et al., 2003; Plotto et al., 2003; Plaza et al., 2004). Research has shown that aromatic plants belonging to the Lamiaceae and Asteraceae families are rich in antimicrobial and antioxidant compounds (Barroso and Ruberto, 1998; Farzaneh et al., 2006 a, b; Farzaneh et al., 2015). The antifungal property of EOs is also related to some of their compounds such as carvacrol, menthol, cymene, thymol, cinnamaldehyde, eugenol, pinene, and linalool, which are known as compounds with high antifungal effect (Cimanga et al., 2002). The purpose of this research is to investigate the potential of EOs of plants rich in thymol and car­ vacrol, such as Thymus danensis, Thymus vulgaris, Satureja hortensis, and Satureja khuzistanica in pre­ venting rot and decay of orange fruit caused by Colletotrichum gloeosporioides, Aspergillus niger, Rhizopus stolonifer and Penicillium digitatum. 2. Materials and Methods Inoculum preparation of pathogens Four fungi that cause post­harvest decay of orange fruit, including C. gloeosporioides, A. niger, R. stolonifer, and P. digitatum, were obtained from the mycology collection of the Department of Plant pro­ tection, Agriculture and Natural Resources Campus, University of Tehran. In order to prepare the pathogen inoculum, 5 mL of distilled sterile water containing 0.05% Tween 80 was added to the seven­ day old culture of each fungus on PDA medium and the surface of the colony was scraped to provide spores and mycelia suspension. The resulting suspen­ sions were passed through four­layer cheesecloth, and then the spore population was adjusted to a con­ centration of 1*105 spores per milliliter using a hemacytometer. Plant EOs and their major compounds The aerial parts of tow thyme species, T. danensis and, Thymus vulgaris, at the flowering stage were collected from Semirom region of Isfahan province, while the aerial parts of two savory species, S. hort‐ ensis and S. khuzistanica were collected from Pol­ Dokhtar and Majin regions of Lorestan province, respectively. The collected plant parts were delivered to the Medicinal Plants and Drugs Research Institute (MPDRI), Shahid Beheshti University (SBU) in Tehran. After confirming the identity, the plants were dried at room temperature and shade. Each sample was pow­ dered using a mill, and then their EOs was extracted by distillation with water in a Clevenger according to the method recommended in the British Pharmacopoeia (1988). The standard compounds of thymol, carvacrol, para­cymene and gamma­ter­ pinene were purchased from Sigma­Aldrich Co. Analysis and identification of EOs compounds The EO obtained from each plant was identified with gas chromatography (GC) and gas chromatogra­ phy coupled with mass spectrometry (GC­MS) meth­ ods. First, one microliter of EO extracted from each plant was injected into the TRACETM GC 2000 gas chromatograph (ThermoQuest Italia S.p.A., Rodano, Milan, Italy) with a flame ionization detector (FID) and fused silica capillary DB­1 column (60 m × 0.25 mmi.d.; film thickness= 0.25 μm). Injector and detec­ tor temperatures were 250˚C and 300˚C, respective­ ly. Helium was used as the carrier gas at a flow rate of 1.1 ml/min; oven temperature was programmed from 60˚C to 250˚C at the rate of 4˚C/min, and finally held isothermally for 10 min. GC­MS analysis was also performed by using a ThermoQuest Finnigan Trace GC/MS (ThermoQuest Italia S.p.A., Rodano, Milan, Italy), equipped with a DB­1 column (60 m × 0.25 mmi.d.; film thickness= 0.25 μm). Gas chromato­ graphic conditions and the thermal programming were as given for GC. Helium was used as carrier gas Mokhtarnejad and Farzaneh ‐ Carvacrol/thymol rich oil control mold decays in storage orange 171 with ionization voltage of 70 ev. Ion source and inter­ face temperatures were 200˚C and 250˚C, respective­ ly. Mass range was from m/z 43­456. Identification of individual compounds was done by comparison of their mass spectra with those of similar compounds from a database (Wiley/NBS library) or with authen­ tic compounds and confirmed by comparison of their retention indices with authentic compounds or with those of reported in the literature. The percentage of each compound was determined according to its rel­ ative area percentages obtained by FID, without using correction factors (Adams, 2007). In vitro antifungal assay The main ingredients of EOs, including thymol, carvacrol, paracymene and gamma terpinene, were obtained from the Phytochemistry Department of MPDRI. Antifungal effect of EOs and main com­ pounds was investigated against four post­harvest decay fungi of fruit by mixing EO with PDA solid cul­ ture medium (Farzaneh et al., 2006 b). In short, Petri dishes containing concentrations of 75, 150, 300, 600 and 1200 microliters of EO/standard major ­com­ pound per liter of culture medium were prepared and after placing a fungal disk (with a diameter of 5 mm) in center of Petri dishes, they were kept at a temperature of 25 °C in darkness. The growth of each fungus colony was measured daily until the surface of control Petri dishes was completely occupied by the fungus. The percentage of growth inhibition was cal­ culated. The minimum inhibitory concentration (MIC) of the EOs was calculated to prevent the growth of fungi. To investigate whether the EO shows fungicidal or fungistatic activity, the fungal disk of the treat­ ments without fungal growth, was re­cultured on the PDA culture medium, and the growth or not growth of the fungus on the PDA was investigated after one week to calculate the minimum fungicidal concentra­ tion (MFC). In addition, the EC50 value (effective con­ centration causing 50% inhibition of mycelial growth) was calculated from the data by probit analysis. Antifungal assay on orange fruit The healthy orange fruits (Thomson cultivar) free of any chemical and physiological treatment and same in size and ripeness index were provided from Citrus and Subtropical Fruits Research Center, Ramsar, Mazandaran province, Iran. After disinfect­ ing the fruits surface by 70% ethanol for one minute, a wound of 1 mm in diameter and 2 mm in depth (limited to the albedo part in the equatorial region of the fruit) was created on each fruit in sterile condi­ tion. Then the fruits were treated with the concentra­ tion of 1/1000 (1000 ppm) EO by two methods; dip­ ping and spraying. Then treated fruits were inoculat­ ed by spraying of a suspension of 1×105 spores per milliliter. Control treatments included dipping and spraying of fruits with Tween 80 solution (0.05%) and thiabendazole fungicide. In this experiment, each treatment contained of 4 replicates and each repli­ cate consisted of 8 experimental units (fruits). The surface of treated fruits was dried under air flow for 2 h and then they arranged on special fiber plates before transferring to a storage room of 25°C and darkness. After the storage period (10 days), the diameter of the decay area on orange fruit was mea­ sured using a caliper. The efficacy of the EOs was determined by the formula: IP =(C–T/C)*100), where IP is the inhibitory percentage of the of spoilage decay, and C and T are the spoilage decay area in control and treatment, respectively. Statistical analysis To analyze the data, three software were used. At first, the normality of the data was normalized by Mini­tab software Version 17.1 (Minitab Inc. state college, PA). Then SAS software Version 9.1.3 (SAS Institute Inc., Cary, NC) with GLM method was used for variance analysis. After analysis the variance, the mean of the data was compared using Duncan’s multi­range test at the 5% level. The EC50 values were calculated from the data subjected to probit analysis using IBM SPSS statistics Version 26 (IBM Corp. Chicago, IL). 3. Results The main compounds of EOs The main compounds in T. danensis EO included thymol (65.5%), alpha­terpinene (11.9%) and para­ cymene (7.5%) (Table 1). Thymol (59%), paracymene (15.6%) and gamma­terpinene (4.2%) were the main compounds identified in the EO of T. vulgaris (Table 1). The main compounds in the EO of the S. khuzis‐ tanica included carvacrol (88.4%), para­cymene (3%) and gamma­terpinene (4.5%). Carvacrol (51%), gamma­terpinene (20.8%) and para­cymene (13.7%) were the main compounds identified in the EO of the S. hortensis species (Table 2). Adv. Hort. Sci., 2024 38(2): 169­176 172 Antifungal effect of EOs The results of the antifungal effect of the EOs on the growth of fungi are shown in Table 3. In general, the more the concentration of EO increased, the more antifungal activity was seen. In addition, the intensity of the EOs inhibitory effects against C. gloeosporioides and R. stolonifer was more evident (Table 3). According to the results (Table 3), for controlling the A. niger growth, only the EO of S. khuzistanica showed the highest antifungal activity with the MIC 300 μL/L. To inhibit the growth of P. digitatum, all EOs showed significant antifungal activity with the Table 1 ­ The major constituents (%) of chemical composition of Thymus daenensis and T. vulgaris essential oils * Retention indices relative to C6­C24 n­alkanes on the DB­1 column. No. Compound Retention indices T. daenenis (%) T. vulgaris (%) 1 Alpha­thujene 925 0.8 1.8 2 Alpha­pinene 933 1.6 1.6 3 Beta­pinene 974 0.7 2.6 4 Myrcene 981 1.1 1.9 5 Alpha­phelandrene 999 0.2 1.7 6 Para­cymene 1014 7.5 15.6 7 Gamma­terpinene 1053 ­ 4.2 8 Alpha­terpinene 1080 11.9 ­ 9 Thymol 1266 65.5 59.0 10 Carvacrol 1282 0.1 3.1 11 Carvacryl acetate 1345 2.5 2.0 12 Beta­caryophyllene 1424 3.8 1.5 13 Beta­bisabolene 1501 1.3 0.9 Total ­ 97.0 95.9 Table 2 ­ The major constituents (%) of the chemical composi­ tion of Satureja khuzistanica and Satureja hortensis essential oils * Retention indices relative to C6­C24 n­alkanes on the DB­1 column. No. Compound Retention indices S. Khuzistanica S. Hortensis (%) 1 Alpha­thujene 925 ­ 2.5 2 Alpha­pinene 933 ­ 2.9 3 Beta­pinene 974 0.2 1.1 4 Myrcene 981 0.2 1.5 5 Para­cymene 1014 3.0 13.7 6 1.8­cineole 1023 0.7 1.0 7 Gamma­terpinene 1053 4.5 20.8 8 Carvacrol 1282 88.4 51.0 9 Carvacryl acetate 1345 0.1 1.3 Total ­ 97.1 96.7 Table 3 ­ The inhibitory activity (%) of four plant essential oils at different concentrations against spoilage fungi of citrus fruit by poi­ sonous PDA medium method Essential oil concentration (µl/l) Inhibitory activity (%) A. niger P. digitatum C. gloeosporioides R. stolonifer T. daenensis 75 22.16 34.20 11.13 24.96 150 57.35 56.33 34.20 96.05 300 90.11 85.71 100 100 600 100 100 100 100 1200 100 100 100 100 T. vulgaris 75 20.94 27.11 3.38 0.60 150 65.45 55.55 31.77 90.22 300 75.01 73.34 82.29 100 600 100 100 100 100 1200 100 100 100 100 S. hortensis 75 16.22 28.92 2.49 14.25 150 27.94 39.11 58.33 51.55 300 88.32 78.92 100 100 600 100 100 100 100 1200 100 100 100 100 S. khuzistanica 75 24.10 11.50 26.32 25.81 150 67.91 60.00 80.77 96.26 300 100 90.66 100 100 600 100 100 100 100 1200 100 100 100 100 Mokhtarnejad and Farzaneh ‐ Carvacrol/thymol rich oil control mold decays in storage orange 173 MIC 600 μL/L., whereas the EOs of three species, including S. hortensis, S. khuzistanica and T. danensis showed the great antifungal activity against C. gloeosporioides with the MIC 300 μL/L. To inhibit R. stolonifer, all four EOs with the MIC 300 μL/L showed the noticeable antifungal activity. The results (Table 4) obtained from the re­culture of fungal disks, in the treatments which no fungal growth was observed, showed that none of the EOs had fungicide properties on the A. niger. Two EOs of S. khuzistanica and S. hortensis at a concentration of 1200 μL/L showed fungicidal properties against P. digitatum, whereas T. daenensis and T. vulgaris EOs showed the MFC values more than 1200 μL/L. Essential oil of S. khuzistanica showed MFC against C. gloeosporioides at MFC 600 μL/L, while S. hortensis and T. danensis EOs exhibited MFC of 1200 μL/L of culture medium. Howevere, T. daenensis oil didn’t show MFC value at the maximum concentra­ tion. To control of R. stolonifer, the EOs of S. khuzis‐ tanica and S. hortensis showed MFC at the concen­ trations 300 μL/L and 600 μL/L, respectively, while both T. vulgaris and T. danensis EOs exhibited MFC at the concentration of 1200 μL/L (Table 4). Antifungal properties of the main components of EOs In general, by increasing the concentration of the EO/standard main­component its antifungal activity increased (Table 5). Among the main compounds, carvacrol exhibited the highest antifungal activity. The MIC of carvacrol against the growth of R. stolonifer was 150 μL/L. Carvacrol at a concentration of 300 μL/L prevented the growth of other fungi as well. Thymol was anoth­ er main compound in the EOs, especially thyme, which showed considerable antifungal activity. Thymol at the MIC of 300 μL/L completely prevents Table 4 ­ Minimum fungicidal concentration (µl/l) of four essen­ tial oil against citrus fruit spoilage fungi. The experi­ ments were carried out in vitro by Poisonous PDA Medium method Fungi S. khusiztanica S. hortensis T. danensis T. vulgaris A. niger >1200 >1200 >1200 >1200 P. digitatum 1200 1200 >1200 >1200 C. gloeosporioides 600 1200 1200 >1200 R. stolonifer 300 600 1200 1200 Table 5 ­ The inhibitory activity (%) of four major compounds of essential oils at different concentrations against spoilage fungi of citrus fruit by poisonous PDA medium method. The percentage of inhibition in each treatment corresponds to 4 repetitions (4 Petri dishes with a diameter of 8 cm) Compound Concentration (µl/l) Inhibitory activity (%) A. niger P. digitatum C. gloeosporioides R. stolonifer Thymol 75 10.20 6.70 14.65 14.50 150 55.48 55.75 66.30 58.86 300 95.34 100 100 100 600 100 100 100 100 1200 100 100 100 100 Carvacrol 75 22.71 29.63 31.88 36.40 150 64.95 65.50 88.84 100 300 100 100 100 100 600 100 100 100 100 1200 100 100 100 100 Para­cymene 75 0 0 0 0 150 30.87 28.44 36.50 33.96 300 61.54 55.89 69.74 62.94 600 93.38 94.61 100 100 1200 100 100 100 100 Gamma­terpinene 75 0 0 0 4.69 150 19.85 15.32 26.58 27.63 300 63.35 88.84 89.12 85.38 600 96.48 100 100 100 1200 100 100 100 100 174 Adv. Hort. Sci., 2024 38(2): 169­176 the growth of three fungi; C. gloeosporioides, R. stolonifera, and P. digitatum whereas the growth rate of A. niger was inhibited by 95.3%. Para­cymene had also showed antifungal activity that was able to inhibit the growth of all the fungi at the concentra­ tion of 1200 μL/L. Among the fungi, R. stolonifer and C. gloeosporioides were more sensitive to para­ cymene and their growth was completely inhibited at the concentration of 600 μL/L. It is also necessary to mention that this compound did not show any signifi­ cant antifungal effect against any of the fungi at the low concentrations (<150 μL/L). Gamma­terpinen is one of the main components of EOs, especially in the savory plants that at the MIC concentration of 600 μL/L caused a complete inhibition of the growth of all fungi except A. niger. In the other hand, the fungus A. niger was the most resistant fungus to this com­ pound, whose MIC was 1200 μL/L. Low concentra­ tions of this compound did not show the inhibitory effect on the growth of the fungi (Table 5). The results of the MFC indicated that gamma ter­ pinene at any of the concentrations did not cause the death of the fungi. In addition, carvacrol and thymol showed strongest fungicidal activity with MFC 600 μl/l against R. stolonifer. Carvacrol also exhibited strong fungicidal activity (MFC 600 μl/l) against C. gloeosporioides. However, A. niger had the highest resistance to the compounds, and its MFC value was often more than 1200 μl/l (Table 6). In addition, the antifungal potency of each EO and its main compound was determined according to EC50 value as well (Table 7). The lower the EC50 indicates the less the concentration of antifungal compound that is required to inhibit 50% of fungal growth. In general, the lowest EC50 values were achieved by S. khuzistanica EO that showed EC50 values of 95.14, 108.00, and 120.93 μL/L against R. stolonifer, C. gloeosporioides, and A. niger, respectively. In confir­ mation of it, carvacrol showed the lowest EC50 values of 80­124 μL/L against four citrus fruit spoilage fungi. Spoilage decay control on fruit In general, the application of EOs by dipping method showed the greatest effect in reducing spoilage and fruit rot, whereas the spraying method also had significant effect. In addition, S. khuzistanica essential oil was the most effective oil to reduce A. niger (95.4%) and P. digitatum (86.8%) decays area on the fruit in dipping method. The EO of S. khuzis‐ tanica had the greatest effect against R. stolonifer and C. gloeosporioides decays on the fruits by both methods of dipping and spraying of the fruit which could completely (100%) inhibit the both decays Table 6 ­ Minimum Fungicidal Concentration (µl/l) of major compounds of essential oils; thymol, carvacrol, para­ cymene and gamma­terpinene; against spoilage fungi of citrus fruit. The experiments were carried out in vitro by poisonous PDA medium method Fungi Thymol Carvacrol Para­ cymene Gamma­ terpinene A. niger >1200 >1200 >1200 >1200 P. digitatum 1200 1200 >1200 >1200 C. gloeosporioides 1200 600 >1200 1200 R. stolonifer 600 600 1200 >1200 Table 7 ­ The EC50 value (effective concentration causing 50% inhibition of mycelial growth) of each essential oil and its major com­ pounds against spoilage fungi of citrus fruit on PDA (µL/L) calculated by probit analysis Essential oil/compound A. niger P. digitatum C. gloeosporioides R. stolonifer T. danensis 154.40 (118.69­197.18) (z) 151.86 (97.52­219.75) 164.02 (151.83­178.53) 95.84 (88.81­103.094) T. vulgaris 172.02 (80.34­324.16) 179.65 (108.88­283.32) 210.16 (194.97­226.88) 119.23 (111.86­125.91) S. hortensis 191.92 (176.59­209.02) 186.28 (136.57­256.56) 141.95 (134.20­150.60) 143.68 (132.68­156.79) S. khusiztanica 120.93 (111.04­131.95) 160.21 (118.11­213.54) 108.00 (99.28­117.03) 95.14 (88.11­102.40) Thymol 156.31 (131.03­187.02) 143.14 (133.79­154.54) 128.59 (119.27­139.13) 135.97(125.77­147.96) Carvacrol 124.70 (114.56­136.16) 119.49 (109.03­131.12) 97.53 (89.30­105.78) 80.92 (78.78­83.06) Para­cymene 289.02 (191.82­459.22) 230.03 (171.91­329.54) 296.60 (213.73­432.76) 245.27 (183.04­365.97) Gamma­terpinene 284.61 (213.66­403.15) 220.44 (206.91­234.68) 207.22 (193.60­221.86) 207.85 (193.04­224.10) (z) Numbers in parentheses indicate 95% confidence limits determined by probit analysis. Mokhtarnejad and Farzaneh ‐ Carvacrol/thymol rich oil control mold decays in storage orange 175 (Table 8). In addition, S. hortensis could completely inhibit R. stolonifera decay. However, T. danensis and T. vulgaris couldn’t completely inhibit of the any fruit fungal decay and exhibited weak fungicide activity on the fruit. In addition, fungicide tiabendazole could completely control R. stolonifera decay. It seems that P. digitatum and A. niger are the most resistance fungi to these EOs on the orange fruit. 4. Discussion and Conclusions In our study, all four plants EOs (belong to Thymus and Satureja geniuses, Lamiaceae) exhibited considerable antifungal activity against postharvest spoilage fungi. It has been found that some medicinal plants of the Lamiaceae family have high antifungal properties (Bakkali et al., 2008; Adeyinka and Richard, 2015). Thymol was included the main part (more than 50%) of T. danensis and T. vulgaris EOs whereas S. khuzistanica and S. hortensis EOs were rich in carvacrol (more than 50%). In addition, their major compounds and specially thymol and carvacrol resulted in strong fungictatic and fungicide activities. However, the lowest MFC and EC50 values were obtained by S. khuzistanica oil and carvacrol. The antibacterial and antimicrobial properties of the main components of EOs such as cinnamaldehyde, eugenol, thymol and carvacrol have been identified in several studies (Bakkali et al., 2008; Adeyinka and Richard, 2015). The antimicrobial and antifungal activity of the EO may be due to the characteristics of terpenes/terpenoids compounds, which, due to their high lipophilic nature and low molecular weight, that enable them destroying cell membranes, and inhibiting spore germination (Bakkali et al., 2008; Nazzaro et al., 2017). However, the dominant composition of the EO may cause the antifungal activity of the EO alone or in synergic manner with other compounds (Plotto et al., 2003). Therefore, in our study, the antifungal property of these EOs can be contributed to their thymol or carvacrol content, although other EO constitutes may act synergistically and increase the antifungal activity of the main compound. Research has shown that aromatic plants belonging to the families Lamiaceae and Asteraceae are rich in antimicrobial and antioxidants compounds and increase the quality of the fruit and the length of its storage period as well (Tajkarimi et al., 2010; Hyldgaard et al., 2012; Gyawali and Ibrahim, 2014). In addition, EOs could control post­ harvest diseases due to their antifungal effects on the both vapor and non­vapor phases (Tripathi et al., 2008). In our study, the application of EOs by dipping method showed the more fungicide activity than the spraying method in terms of reducing spoilage and fruit rot. None of the four EOs and their dominant compounds at the maximum concentration studied in this research (1200 µl/l) could completely controlled A. niger in vitro and on fruit conditions, which indi­ cates the high tolerance of this fungus to EO com­ pounds. In addition, although both savory oils could completely kill P. digitatum by 1200 µl/l in vitro, they couldn’t completely inhibit the P. digitatum decay on fruit. On the other hand, the sensitivity of P. digita‐ tum to EO would be reduced on fruit. However, savory oils could completely inhibit R. stolonifer and C. gloeosporioides decays on fruit. Although, the significant in vitro antifungal activity of the EOs studied in this research depended on the content of carvacrol and thymol, the EOs of both Table 8 ­ The control of orange fruit fungal decays by four medicinal plants essential oils (1 per 1000) trough spraying and dipping meth­ ods, after 10 days’ incubation in the dark condition at 25°C. An= Aspergilus niger; Pd= Penicillium digitatum; Cc= Colletotrichum gloeosporioides; Rs= Rhizopus stolonifer. Means followed by the same letter within a column are not significantly different at P≤0.05. Treatment Disease incidence (%) Spraying method Dipping method A.n P.d C.c R.s A.n P.d C.c R.s T. daenensis 25.8 c* 38.8 b 9.4 ef 11.0 e 16.3 d 17.6 d 4.5 fg 7.2 f T. vulgaris 25.4 c 33.5 b 13.7 de 12.4 de 16.1 d 25.4 c 5.0 fg 6.6 f S. hortensis 9.6 ef 27.2 c 0.0 g 3.3 fg 7.2 f 16.3 d 0.0 g 0.0 g S. khuzistanica 7.2 f 22.0 c 0.0 g 0.0 g 4.5 fg 12.5 de 0.0 g 0.0 g Tiabendazole 7.6 f 21.5 c 3.8 fg 3.3 fg 3.5 fg 7.8 f 3.3 fg 0.0 g Infected Control 97.5 a 97.5 a 95.0 a 95.0 a 97.5 a 95.0 a 95.0 a 95.0 a Adv. Hort. Sci., 2024 38(2): 169­176 176 savory species (rich in carvacrol) were more effective than thyme species oils (rich in thymol) in terms of controlling fungal decays on fruit. Finally, plant EOs rich in carvacrol are introduced as promising candi­ dates for the commercial production of natural fungi­ cides to disinfection and management of post­har­ vest decay molds of citrus fruits. References ADAMS R.P., 2007 ­ Identification of essential oils compo‐ nents by Gas Chromatography/Quadrupole Mass Spectrometry. 4th Edition. ­ Allured Publishing, Carol Stream, IL, USA, pp. 804. ADEYINKA A., RICHARD F., 2015 ­ Application of phyto‐ chemical extracts and essential oils in food products. ‐ Int. J. Biotechnol. Food Sci., 3(3): 31­35. AGRIOS G.N., 2005 ­ Plant pathology. 5th Edition. ­ Academic Press, London, New York, pp. 922. ANTHONY S., ABEYVIKRAMA K., WILSON W.S., 2003 ­ The effect of spraying essential oils of Cymbopogon nardus, Cymbopogon flexuosus and Ocimum basilicum on postharvest diseases and storage life of Embul banana. ‐ J. Horti. Sci. Biotechnol., 78(6): 780­785. ARRAS G., USAI M., 2001 ­ Fungitoxic activity of 12 essen‐ tial oils against four postharvest citrus pathogens: chemical analysis of Thymus capitatus oil and its effect in subatmospheric pressure conditions. ‐ J. Food Protec., 64(7): 1025­1029. BAKKALI F., AVERBECK S., AVERBECK D., IDAOMAR DM., 2008 ­ Biological effects of essential oil. ­ Rev. Food. Chem. Toxicol., 46: 446­475. BARROSO G., RUBERTO N., 1998 ­ Antimicrobial and antioxidant properties of some commercial oils. ­ Flavour Frag J., 13: 235­244. BOLOURI P., SALAMI R., KOUHI S., KORDI M. ASGARI LAJAYER B., HADIAN J., ASTATKIE T., 2022 ­ Applications of essential oils and plant extracts in different indus‐ tries. ­ Molecules, 27: 8999. BRUN S., AUBRY C., LINA O., FILMON R., BERGES T., CHABASSE D., BOUCHARA JP., 2003 ­ Relationships between respiration and susceptibility to azole antifun‐ gals in Candida glabrata. ‐ Antimicrob. Agents Chemother., 47(3): 847­853. BURT S., 2004 ­ Essential oils: Their antibacterial properties and potential applications in foods ‐ A Review. ­Int. J. Food Microbiol., 94: 223­253. CARVALHO DE SOUSE A., SALES ALVIANO D., FITZGERALD BLANK A., BARRETO ALVES P., SALES ALVIANO C., ROCHA GATTAS C., 2004 ­ Melissa officinalis L. essential oil: antitumoral and antioxidant activities. ­ J. Pharm. Pharmacol., 56: 677­681. CIMANGA K., KAMBU K., TONA L., APERS S., DE BRUYNE T., HERMANS N., TOTTÉ J., PIETERS L., VLIETINCK A.J., 2002 ­ Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. ­ J. Ethnopharmacol., 79: 213­220. FARZANEH M., AHMADZADEH M., HADIAN J., SHARIOFI TEHRANI A., 2006 a ­ Chemical composition and anti‐ fungal activity of the essential oils of three species of Artemisia on some soil‐borne phytopathogens. ­ Agric. Appl. Biol. Sci., 71: 1327­1333. FARZANEH M., GHORBANI GHOUZHDI H., GHORBANI M., HADIAN J., 2006 b ­ Composition and antifungal activity of essential oil of Artemisia sieberi Bess. on soil‐born phytopathogens Pakistan. ­ J. Biol. Sci., 9(10): 1979­ 1982. FARZANEH M., KIANI H., SHARRIFI RAEISI M., HADIAN J., 2015 ­ Chemical composition and antifungal effects of three species of Satureja (S. hortensis, S. spicigera, and S. khuzistanica) essential oils on the main pathogens of strawberry fruit. ­ Postharvest Bio. Tech., 109: 145­151. GYAWALI R., IBRAHIM S.A., 2014 ­ Natural products as antimicrobial agents. ­ Food Control., 46: 412­429. HYLDGAARD M., MYGIND T., MEYER RL., 2012 ­ Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components. ­ Front. Microbiol., 3: 1­12. NAZZARO F., FRATIANNI F., COPPOLA R., DEFEO V., 2017 ­ Essential oils and antifungal activity. ­ Rev. Pharm., 10(86): 1­20. PLAZA P., TORRES R., USALL J., LAMARCA N., VINASA I., 2004 ­ Evaluation of the potential of commercial post‐ harvest application of essential oils to control citrus decay. ­ J. Hortic. Sci. Biotechnol., 79(6): 935­940. PLOTTO A., ROBERTS D., ROBERTS R.G., 2003 ‐ Evaluation of plant essential oils as natural postharvest disease control of tomato (Lycopersicon esculentum). ‐ Acta Horticulturae, 628: 737­745. REGNAULT­ROGER C., VINCENT C., AMASON J.T., 2012 ­ Essential oils in insect control: Low‐risk products in a High‐stakes world. ­ Annu. Rev. Entomol., 57(1): 405­ 424. SHARIFI­TEHRANI A., FARZANEH M., 2018 ­ Fungicides: History, mode of action, resistance and application in plant protection. ­ University of Tehran, Tehran, pp. 458 (In Persian). TAJKARIMI M., IBRAHIM S., CLIVER D., 2010 ‐ Antimicrobial herb and spice compounds in food. ­ Food Control., 21(9): 1199­1218. TRIPATHI P., DUBEY N.K., SHUKLA A.K., 2008 ­ Use of some essential oils as post‐harvest botanical fungicides in the management of grey mold of grapes caused by Botrytis cinerea. ­ World J. Microbiol. Biotechnol., 24: 39­46. WILLS R.B.H., GOLDING J.B., 2016 ‐ Postharvest: An Introduction to the physiology and handling of fruit and vegetables. 6th edition. ­ University of New South Wales Press, Sydney, pp. 293. ZIZTZELSBERGER C., BUCHBAUER G., 2015 ­ Essential oils as “A cry for help. ­ Rev. Nat. Prod. Commun., 10: 1127­ 1138.