Antioxidant and Antifungal Effects of Six Plant Essential Oils Against Penicillium digitatum and Penicillium italicum

1. Introduction

Fruit and vegetables postharvest diseases caused by fungal infections (Aspergillus, Penicillium, Fusarium, Alternaria, …) due to wounds or insect bites produce elevated loss of food during storage, distribution and sale [1,2,3,4,5,6,7]. Citrus major sources of postharvest diseases are green mold (Penicillium digitatum (Pers.) Sacc.) and blue mold (Penicillium italicum Wehmer), which cause economic losses of 15–30% and affect 50–90% of production, particularly in developing countries [8,9,10,11,12,13]. Traditionally, several methods based on synthetic chemical fungicides have been developed to reduce post-harvest losses; however, intensive use of these methods generates resistance, reducing their effectiveness [9,14,15]. Moreover, consumer trends demand products that are free of chemical residues and more environmentally friendly. Together with legislative restrictions on the use of phytosanitary products, this creates the need for new, more effective and environmentally friendly postharvest management. These alternatives include biocontrol strategies involving the use of antagonist yeast or bacteria, immersion in aqueous extracts of medicinal plants or citrus fruits, vaporization of essential oils from medicinal plants, wax coatings containing essential oils or plant extracts, new biopolymers and heat treatments, among others [6,11,16,17,18,19,20,21].

Studies of the antifungal capacity of medicinal plants extracts or essential oils have reported the ability of fight various fungal infections caused by Aspergillus spp., Candida spp., Cryptococcus spp., Epidermophyton spp., Fusarium spp., Microsporum spp. Penicillium spp., and Trichophyton spp. [22,23,24,25,26,27]. Several studies have shown that essential oils from species such thyme, oregano, clove, cinnamon or citrus have a high inhibitory capacity against the in vitro growth of fungal colonies from Penicillium species such as P. digitatum and P. italicum) [28,29,30,31,32,33,34,35,36]. The antifungal properties of these essential oils have contributed to the development of new research aimed at preventing post-harvest infections caused by P. digitatum and P. italicum [37,38,39,40,41,42,43,44,45].

The use of essential oils from medicinal plants whose chemical composition includes antifungal compounds (e.g., thymol, carvacrol, terpinen-4-ol, etc. [46]) makes it possible to search for local medicinal species commonly used in traditional medicine that are rich in these kinds of compounds, creating a new local employment opportunity that are more environmentally friendly. For that purpose, the main objective of this research is to evaluate the inhibitory and antioxidant activities of different aromatic plants, native to the SW from the Iberian Peninsula, against two mold species (P. digitatum and P. italicum), which cause postharvest disease in citrus fruits (e.g., oranges).

2. Materials and Methods

2.1. Plant Material, Essential Oil Extraction, and Chemical Characterization of Essential Oils

Aerial parts of six aromatic plants (L. pedunculata subsp. sampaioana, L. stoechas subsp. luisieri, Mentha × piperita, O. vulgare subsp. virens., Th. mastichina, and Th. zygis subsp. sylvestris) were collected from the experimental crops at Institute of Agrarian Research “La Orden-Valdesequera” (CICYTEX) (near of Guadajira, Spain). Representative samples were collected during the flowering stage, which took place between May and June 2024.

Fresh stems, leaves, and flowers from each specie were cut in small pieces and submitted to hydro-distillation in Clevenger-type apparatus for 2 h. The essential oils (EOs) were stored in amber vial at 4ºC.

The chemical analysis of the essential oils was carried out using a combination of two gas chromatography techniques (GC-FID + GC-MS), chemical compounds were identified by CG-MS and quantified by CG-FID. The analysis was performed on Agilent 8890 GC paired with the 5977B MSD (Mass Selective Detector). Polar column DB-WAX UI (60 m long, 0.25 mm diameter and 0.5 µm film thicknesses) was employed using Helium carrier gas at constant flow of 2 mL/min. Apolar column HP-5MS UI (60 m long, 0.25 mm diameter and 0.25 µm film thicknesses) was employed using Helium carrier gas at constant flow of 1 mL/min. The column temperature stared at 50ºC and increased to 240ºC (polar column) and 285ºC (apolar column).

2.2. Antioxidant Activity

The antioxidant activity of each essential oil samples was determined by ABTS and DPPH assay method. The absorbance was measured using a spectrophotometer (Beckman Coulter DU^® 730).

The standard line from each assay was designed using Trolox (6-hidroxy-2,5,7,8-tetramethylchroman-carboxylic acid) (Sigma-Aldrich 238813) between 1mM and 2mM concentration and measured the absorbance at 734nm (ABTS) and 517nm (DPPH).

All the essential oil samples were analysed in triplicate. The sample volume used was 3 milliliters (2,95 ml from DPPH/ABTS + 50 μl from essential oil sample). The results, from both analyses (ABTS and DPPH) were presented as millimoles (mM) of Trolox equivalents and grams of Trolox equivalents per gram of essential oil, with the main objective of developing a data matrix comparable between each other.

2.2.1. ABTS [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] Assay

ABTS assay is based on the ability of molecules to scavenge the free radical of ABTS in comparison with Trolox [47]. Absolute ethanol was used to prepare the working solution of ABTS (Sigma-Aldrich A1888) at a concentration of 7 mM, which was then adjusted to obtain a final absorbance of 0.7 ± 0.02 (at 734 nm). To determine antioxidant activity, the essential oil samples remained in the dark at ambient temperature for 30 minutes and, thereafter, the absorbance was measured at 734nm.

2.2.2. DPPH (2,2-diphenyl-1-picrylhydrazyl) Assay

The DPPH protocol to measure antioxidant activity was based on the description in reference [48]. Methanol (100%) was used as the solvent to prepare a working solution 75 µmol/L of DPPH (Sigma-Aldrich D9132), which was then adjusted to a final absorbance of 0.7 ± 0.02 (at 517 nm). For antioxidant activity determination, the samples remained in the dark at ambient temperature for 120 minutes, after which the absorbance was measured at 517 nm.

2.3. In Vitro Antifungal Activity Assay

2.3.1. Fungal Isolation

The fungal species used are P. digitatum and P. italicum, obtained from infected Citrus aurantium L. fruit. The isolation was realized in Petri dishes containing Sabouraud Dextrose agar (6%) and incubated for 7 days at 27ºC ± 1ºC, in complete darkness. The differential isolations were transferred to new Petri dishes containing Sabouraud Dextrose agar and re-sown each week until a pure fungal culture of each species was obtained. Finally, the standardization of the fungal colonies was achieve using a 0.85% saline solution suspension to obtain the 0.5 McFarland standard (1,5 · 108 CFU/ml) [49]. Morphological characterization (macro and microscopic) was performed using dichotomous keys as a reference [50,51,52].

2.3.2. Antifungal Activity

The disk diffusion method was used to evaluate the antifungal activity of each of the essential oils [53,54]. The fungal suspension was sown in Petri dishes (87,8 mm diameter) containing 25 ml of Sabouraud Dextrose Agar. A sterile swab was used to spread the mold suspension evenly across the surface of the dish to ensure a homogeneous development of the mold. Essential oils were inoculate using a 10 mm diameter filter disk soaked with 25μl of each essential oil sample and placed in the center of the Petri dish.

The study included a control group and a study group with 3 repetitions of each for each species of essential oil. Thus, 12 Petri dishes were used for each essential oil species (3 control dishes + 3 study dishes for each one of the analysed molds, P. digitatum and P. italicum). The Petri dishes were incubated for 5 days (96 hours) in an incubator chamber at 27ºC ± 1ºC in complete darkness and in the normal position (not inverted) to avoid affecting the mold growth. Finally, the Petri dishes were checked, photographed and measured every 24 hours. Measurements were taken by evaluating the inhibitory halo of growth around the filter disk using a caliper.

2.4. Statistical Analysis

Descriptive and inferential statistical analysis were performed using R v 4.3.3 software) [55] to determine the relationship between the inhibitory halo of growth results and the antioxidant activity obtained from the samples. The 48-hour data from inhibitory halo of growth were used to developed the statistical analysis (to ensure a correct understanding of the data and avoid mixing up inhibition and natural absence of growth).

3. Results

3.1. Essential Oil Composition

Table 1 shows the essential oils yield obtained for each of the aromatic plant, expressed in grams of essential oil per kilogram of fresh plant and as a percentage (w/w). The highest yields were obtained in Thymus mastichina (L.) L., Lavandula pedunculata subsp. sampaioana (Rozeira) Franco, and Thymus zygis subsp. sylvestris (Hoffmanns. & Link) Cout. with values of 2.43%, 1.28%, and 0.88% respectively. The species with the lowest yields were Mentha × piperita L., Lavandula stoechas subsp. luisieri (Rozeira) Rozeira, and Origanum vulgare subsp. virens (Hoffmans. & Link) Bonnier & Layens (0.62%, 0.42% and 0.41%, respectively).

The essential oils have a rich monoterpene-based chemical composition (Table 2). The majority of the detected compounds are: thymol (68.83% in Th. zygis subsp. sylvestris and 36.72% in O. vulgare subsp. virens), 1.8-cineole (66.06% and 17.71% in Th. mastichina and L. stoechas subsp. luisieri respectively), camphor and fenchone (35.51% and 34.20% respectively in L. pedunculata subsp. sampaioana), gamma-terpinene (30.69% in O. vulgare subsp. virens), menthone and L-menthol (29.12 and 27.56% respectively in M × piperita), and trans-alpha-necrodyl acetate (20.46% in L. stoechas subsp. luisieri).

3.2. Antioxidant Activity

Obtained results (Table 3) show that the essential oils of the L. stoechas subsp. luisieri, O. vulgare subsp. virens and Th. zygis subsp. sylvestris species have higher antioxidant activity. These species have in common a high percentage of the chemical’s thymol, gamma-terpinene and trans-alpha-necrodyl acetate in their essential oils.

3.3. In Vitro Antifungal Activity Assay

3.3.1. Fungal Isolation

The P. italicum species was observed to grow more quickly and be less susceptible to contamination in in vitro conditions than P. digitatum (Figure 1).

3.3.2. Antifungal Activity

The Petry dishes were photographed every 24 hours, and it could be observed that the filter disk infused with the essential oils is able to inhibit the fungal species development (inhibition halo) and delay maturation of both species (Figure 2).

Table 4 displays statistical parameters obtained from the inhibition halo measurements obtained after 48 hours of growing. Measurement results show a higher inhibitory capacity from essential oil over P. digitatum specie than P. italicum (inhibition halo mean: 29.69 mm in P. digitatum and 27.81 mm in P. italicum). Besides, the observed skew shows a positive trend. On the other hand, the kurtosis of the antioxidant activity shows a platykurtic curve, indicating fewer extreme values than a normal distribution. However, the inhibition halo differs in the kurtosis depending on the fungal species (slightly leptokurtic in P. digitatum and platykurtic in P. italicum).

Finally, the data distribution in relation to each essential oil species is presented in a boxplot for each fungal species (Figure 3). It is possible to observe that the Th. zygis subsp. sylvestris essential oil produced the most extensive inhibition halo for both fungal species, with a clear difference compared to the rest of the samples.

3.3.3. Statistical Analysis

The Shapiro-Wilk test applied to the data base obtained in the study indicate the absence of normal distribution (Table 5), for that reason the statistical analysis was based on non-parametric correlation test (Spearman’s correlation).

The results of the Spearman’s correlation test show a p-value of less than 0.05 (significance level), indicating a linear relationship between the pairs of variables studied at the ordinal level and showing that this relationship is not due to chance (Table 6.).

A significant statistical linear correlation was found between the different measurements of antioxidant activity (using the ABTS and DPPH methods) and the inhibition halo using the various essential oil samples on the two species of mesophilic mold (P. digitatum and P. italicum) (Figure 4).

4. Discussion

The obtained data shows the essential oils of Th. zygis subsp. sylvestris, O. vulgare subsp. virens and L. stoechas subsp. luisieri to have high antioxidant activity and elevated antifungal activity for both ABTS and DPPH. Conversely, the essential oils of Th. mastichina, M. × piperita and L. pedunculata subsp. sampaioana exhibited low antioxidant and anti-fungal activity against the two evaluated fungal species, P. italicum and P. digitatum.

High antifungal and antioxidant activity from Th. zygis has been widely recognized in several studies [56,57,58,59,60,61]. Th. zygis essential oil can have different chemotypes (thymol, carvacrol, carvacrol/thymol, linalool, geranyl acetate/geraniol, …) [58,60,61,62,63]. However, only the carvacrol, thymol and carvacrol/thymol chemotypes, have demonstrated elevated antifungal and antioxidant capacity [47,49,50,51,52,53]. The presence of thymol and carvacrol compounds in essential oil from other species of Thymus L. genus is well known [33,64,65,66]. Furthermore, research into the antifungal activity indicates that they have a higher inhibitory capacity for fungal growth than the pure compounds – thymol or carvacrol [57,64]. Regarding P. digitatum and P. italicum molds, Th. zygis subsp. sylvestris essential oil has an elevated inhibitory capacity against “in vitro” growth, as observed in other Penicillium species [33,36,56].

On the other hand, the other thyme species included in this study, Th. mastichina, has an essential oil rich in 1,8-cineole [59,67,68,69], with a poor antioxidant and antifungal capacity against the Penicillium species studied. However, other studies have shown it to have good antifungal properties against other fungal species, such as Sclerotinia spp., Fusarium spp., Alternaria spp. or Candida spp. [59,70,71,72]. This makes it possible to use it to fight fungal infections in crops or on the skin.

O. vulgare subsp. virens essential oil has a thymol/gamma-terpinene chemotype, which is unusual for this species [73,74]. This coincides with what was observed in research involving the carvacrol chemotype of O. vulgare, which exhibits high antioxidant and antifungal activity against P. digitatum and P. italicum [36,74,75,76,77,78].

The two Lavandula L. subspecies studied exhibit different antifungal capacities, with L. stoechas subsp. luisieri demonstrating greater activity than L. pedunculata subsp. sampaioana [79], and notably the inhibitory effect on P. digitatum growth is higher than on P. italicum. The antioxidant activity of L. stoechas subsp. luisieri essential oil is very high, mainly due to the presence of necrodiol derivatives [80]. On the other hand, L. pedunculata subsp. sampaioana has an essential oil rich in fenchone, camphor and 1,8-cineole, which are compounds with low antioxidant capacity [59,68].

M. × piperita essential oil exhibits the lowest of all the essential oils studied in the present research. However, other studies indicate good inhibitory capacity against several species of the Penicillium genus, including P. digitatum [81,82,83,84]. This divergence in results could be due to variation in the essential oil’s chemical composition, including different percentages of menthol, menthone, limonene, alpha-pinene, and betha-pinene, among others.

5. Conclusions

The Th. zygis subsp. sylvestris, O. vulgare subsp. virens and L. stoechas subsp. luisieri essential oils have a high antioxidant capacity and can effectively inhibit the “in vitro” growth of the molds that mainly cause postharvest damages in Citrus genus fruits. Furthermore, all the essential oils studied exhibited a higher inhibition response against green mold (P. digitatum) than blue mold (P. italicum).