Antifungal Activity of Essential Oils on Helminthosporium solani Causing Potato Silver Scurf under In Vitro and In Vivo Conditions

Abstract

Potato silver scurf, caused by the fungus Helminthosporium solani, is an important storage disease of potato (Solanum tuberosum L.), reducing the market value of tubers. Using in vitro and in vivo assays, the presented experiments aimed to determine the effect of selected essential oils (EOs: α-pinene, carvacrol, cinnamaldehyde, D-carvone, eucalyptol, L-linalool, L-menthol, L-menthone, (R)-(+)-limonene, and thymol) on H. solani growth. All EOs inhibited pathogen growth, but their antifungal activities differed significantly. Thymol, carvacrol, and cinnamaldehyde had the strongest inhibitory effects on mycelial growth under in vitro conditions. (R)-(+)-limonene displayed the weakest inhibition. The effectiveness of those EOs with the greatest antifungal activity was confirmed by in vivo experiments. EOs were applied through dressing and fumigation, with EOs bound to a biopolymer for dressing. Dressing and fumigation brought a highly statistically significant reduction in H. solani infection intensity and sporulation intensity on tubers. Although EOs had an insignificant effect on potato cooking quality, the taste of EO-dressed tubers was degraded by an off-odor and off-taste. EOs could provide an ecological alternative for reducing H. solani tuber infection during storage.

1. Introduction

Silver scurf, caused by the fungus Helminthosporium solani Durieu & Mont., (1849) [H. atrovirens syn. Spondylocladium atrovirens], is a disease of potato (Solanum tuberosum L.) that has become economically important in recent years [1]. Usually, it does not cause yield losses [2]. Predominantly, it induces cosmetic defects in tubers that can negatively affect potatoes’ marketability for consumption and seed uses. Considering the rising demand for washed potatoes, silver scurf reduces the market value of the tubers [3]. Light brown lesions increase the water permeability of the skin and thus reduce tuber weight during storage [4]. The disease can have an important impact on exports, especially of seed potatoes, as only a limited H. solani infection is allowed for certified seed potatoes [5]. French fries and chips produced from infected tubers often have unacceptable brown edges [6]. The growing incidence of silver scurf has been attributed to a lack of H. solani resistance in potato varieties [7] and ineffective control measures [1].

The fungus forms a dark mycelium, stromata, and conidiophores with conidia [8]. Hyphae are present in the phellem, phelloderm, and cortical tuber layers [9]. Lesions with abundant sporulation appear brown to burnt, whereas mature lesions are silvery [10]. Temperatures in the range of 15–32 °C and high humidity often result in conidial germination leading to tuber infection [9]. Mycelium penetrates the epidermis via lenticels and/or invades the periderm, causing a loss of pigmentation, which leads to the formation of silvery lesions [2,7,11]. Other than the tuber-bearing species S. tuberosum [11] and Solanum elaeagnifolium [12], no other plants are hosts of H. solani [13].

Progeny tubers can be infected from diseased seed tubers during the growing season [4], by soil inoculums [14], or during storage [7,15], when, under favorable conditions, the fungus sporulates and tubers becoming sooty [4]. Primary infection, occurring soon after tuber initiation, is often located at the stolon end of the tuber [15]. Most infections occur before harvest, although some infections can occur at harvest, or conidia may be transferred through irrigation [14]. The disease severity increases substantially during long-term potato storage, when the pathogen can spread by conidia [4]. Although H. solani is considered to be a tuber-borne pathogen, it can overwinter in soil as a saprophyte on plant debris [15,16]. Overwintering soil inoculum does not, however, play an important role in the epidemiology [13].

The name of this disease is derived from the characteristically metallic, silvery sheen of older lesions on the surface of tubers. Disease symptoms appear on tubers but on neither stems nor roots [17]. The silver discoloration of the periderm is caused by a loss of pigment, cell desiccation, and suberin deposition [18]. Tuber pigmentation is completely obscured in red-skinned potato varieties [15]. The first signs of silver scurf on tubers consist of light brown circular spots and/or lesions at the stolon end. These may also be dark olive in color [3,15]. Lesions on tubers in the soil remain small, but they enlarge during storage [15,16]. Individual lesions gradually merge [16]. Silver scurf and black dot, caused by Colletotrichum coccodes, are frequently confused. Both diseases cause spots on the periderm, and they often occur together [1].

The disease severity can be reduced by treating seed tubers with fungicides or by maintaining a good crop rotation [19]. The fungicide treatment of tubers against H. solani during storage has very little effect due to resistance development [20]. Intensive and inappropriate use of synthetic fungicides has led to this resistance development, as well as to negative effects on non-target organisms and an increase in soil contamination [21,22]. A global trend today is toward reducing the use of synthetic fungicides and that means there is a rising, strong tendency to search for safer and more ecological alternatives for managing plant pathogens [23]. Essential oils (EOs) extracted from aromatic plants [24,25] constitute an ecological alternative to synthetic fungicides. EOs are volatile, natural, complex compounds (complex mixtures of esters, aldehydes, ketones, terpenes, and other volatile compounds with low molecular weight) characterized by a strong odor and are formed by aromatic plants as secondary metabolites. They are usually obtained by steam or hydro-distillation [26,27,28]. In nature, essential oils play an important role in the protection of plants as antibacterials, antivirals, antifungals, insecticides, and also against herbivores by reducing their appetite for such plants [26]. EOs are isolated from different plant species, including the families Asteraceae, Lamiaceae, Cyperaceae, Zingerberaceae, Piperaceae, Apiaceae, Myrtaceae, Solanaceae, Apocynaceae, and Lauraceae [25,26,27,28]. They are derived from various plant parts, such as flowers, buds, seeds, leaves, branches, bark, wood, fruits, and roots [27]. Cinnamon oil EO can contain up to 90% cinnamaldehyde. Cinnamaldehyde is a viscous organic phenolic compound providing the characteristic flavor and aroma of cinnamon. It is found in the bark of the cinnamon tree and other species of the genus Cinnamomum [29]. Carvacrol and thymol are monoterpenoid phenols with significant antifungal activity, which were isolated, for example, from oregano (Origanum vulgare), marjoram (Origanum majorana), and thyme (Thymus sp.) [30]. Linalool is extracted from Coriandrum sativum, and menthol and menthone from Mentha piperita (=Mentha × piperita) [26]. EOs are readily biodegradable [31] and environment-friendly [32].

Using in vitro and in vivo assays, the aim of the experiments presented here was to determine the effects of selected EOs (α-pinene, carvacrol, cinnamaldehyde, D-carvone, eucalyptol, L-linalool, L-menthol, L-menthone, (R)-(+)-limonene, and thymol) on the growth of H. solani. This paper examines the antifungal properties of various EOs against H. solani in potatoes, which is important for the development of biopesticides.

2. Materials and Methods

2.1. Pathogen Culture

The fungus H. solani required for the experiments was isolated from naturally infected potato tubers expressing typical disease symptoms for silver scurf originating in the Czech Republic (Bohemian–Moravian Highlands). The pathogen was identified using a real-time polymerase chain reaction as described by Cullen et al. (2001). Isolate CCM F-511 originating from the Czech Collection of Microorganisms (Brno) served as a reference. H. solani was cultured at 25 ± 1 °C in Petri dishes (90 mm diameter) on Sabouraud Maltose Agar (SMA; HiMedia, Mumbai, India).

2.2. Essential Oils

Pure, natural EOs were selected for the experiments: α-pinene (98% purity; 147524-250ML), carvacrol (99%, W224511-100G-K), cinnamaldehyde (≥95%, W228613-100G-K), D-carvone (≥96%, W224928-100G-K), eucalyptol (≥99%, W246506-1KG-K), L-linalool (≥95%, natural), L-menthol (≥99%, W266523-100G-K), L-menthone (≥96%, W266701-1KG-K), (R)-(+)-limonene (97%, 183164-100ML), and thymol (≥98.5%, T0501-100G) (all Sigma-Aldrich, Germany).

2.3. In Vitro Antifungal Activity Assays

The antifungal activity of the evaluated EOs was determined as the inhibition of radial mycelial growth. The tested EOs were diluted with 96% ethanol (1:10); for perfect dispersion in the medium, several drops of (0.01% [v/v]) were added. Relevant volumes of diluted EO were added to sterilized SMA medium at a temperature of 40–45 °C to obtain final concentrations of 0–1600 ppm. When thoroughly mixed, SMA solutions were immediately poured onto 90 mm diameter Petri dishes (20 mL/dish). After cooling and the solidification of the medium, 6 mm agar discs with the fungal inoculum were cut from 3-week-old cultures using a sterile cork borer and placed into the center of the Petri dishes, which were then sealed with parafilm. Three replications were prepared for each concentration variant, and the dishes were incubated at 25 ± 1 °C for 10–12 days. Petri dishes without EOs were used as controls. Subsequently, the diameters of fungal cultures (mm) from individual experimental variants were measured, and individual mycelial growth characteristics were calculated. The results are expressed as the means of three independent replications for each pathogen–EO combination.

Mycelial growth inhibition (MGI) was calculated using the following formula as described by Albuquerque [28]:

MGI = [(DC − DO)/DC] × 100 [%],

where DC = radial mycelial growth of the control without EO (mm), and DO = radial mycelial growth with EO (mm).

Further, the nature of each EO’s effect on fungi (fungistatic and/or fungicidal) was determined. The minimum inhibition concentration (MIC) and minimum fungicidal concentration (MFC) of the evaluated EOs in fungi were established, as described by Plodpai [33]. MIC values were ascertained as the lowest EO concentration to completely prevent (inhibit) the apparent growth of fungi after incubation. To determine MFC, inhibited fungi on agar discs in Petri dishes treated with EO concentrations higher than the MIC were transferred to fresh medium (SMA) and incubated at 25 ± 1 °C for 14 days. The recovery of fungal growth was studied, thereby determining the concentrations of fungicidal effect. MFC was defined as the lowest concentration at which no fungal colony growth was recorded on fresh SMA medium after subculture. The half maximal inhibitory concentration (IC₅₀) values (concentrations creating a 50% inhibition effect) were graphically derived from dosage curves and from the response based on the measurement with various concentrations, as described by Chang [34].

2.4. In Vivo Antifungal Activity Assays

The effectiveness of EOs with the greatest antifungal activity as determined by the in vitro assays (i.e., carvacrol, thymol, and cinnamaldehyde) was verified directly on potato tubers using in vivo experiments, where the effect of EO vapors (fumigation) and that of tuber dressing with EO on H. solani growth were determined. The experiments to establish the fumigation effect on H. solani growth were performed in a desiccator (Super-Star-Desiccator, Sicco, Grünsfeld, Germany) with a usable volume of 42 L and a total volume of 45 L. Egg trays were uniformly distributed in the desiccators, and potato tubers (n = 7) of size 35–45 mm of the variety “Belana” (an early variety for fresh consumption, purchased in a supermarket) were placed into the trays. Potato tubers displayed mild symptoms of silver scurf (natural infection). Prior to EO application, photos of tubers were taken using an Olympus TG-6 camera (Olympus, Tokyo, Japan). Petri dishes with cellulose upon which had been applied 4.2 mL of pure essential oil (4.2 g [100 mL/m³] in the case of thymol) were placed into the desiccators. To obtain the required humidity, trays and filtration paper under the Petri dishes were moistened with distilled water. A control without EO application was included in the experiment. Three replications were made for each experimental variant. The experiment was assessed after 60 days of incubating the treated tubers at 10–15 °C and a relative humidity of 99%. For this purpose, photos were taken once again of the evaluated tubers. Tuber infection intensity (disease severity) was then assessed using ImageJ 1.53e freeware, (https://imagej.net//software/imagej/, 17 October 2023). The effectiveness of individual essential oils was determined as the difference in tuber infection intensity after and before EO application.

In another experiment, the effect of tuber dressing with EOs on H. solani growth was studied. Tubers (n = 7), again of size 35–45 mm and the variety “Belana”, expressing mild symptoms of silver scurf (natural infection), were immersed in EO at 2% concentration for 2 s. For greater effectiveness and stability, EOs were bound to a biopolymer as described in the patent by Matušinský [35]. The essence of that patent is that the biopolymer contains a stable form of EO as EO microdroplets enclosed in a coating formed by gelatin- and chitosan-containing biopolymer in the ratio 3:10. One hundred milliliters of the fungicidal product contains 0.5–1.5 mL EO, 9.5–8.5 mL rapeseed oil for EO dilution, 0.2 mL polyethylene glycol sorbitan monooleate, 13.3 mL biopolymer creating a microdroplet coating, and 76.5 mL 0.5% sodium tripolyphosphate solution. A control (variant without treatment) was included in the experiment. Tubers were placed on filter paper saturated with distilled water (150 mL) in closable plastic boxes (30 L). For each experimental variant, three replications were performed. The boxes were closed and placed into a warehouse at 10–15 °C and a relative humidity of99 %. Photos were taken of the tubers prior to establishing the trial. After 60 days of incubation, the extent of tuber infection intensity (disease severity) was determined in the individual experimental variants. For this purpose, photos were again taken of the tubers, and the extent of tuber infection was assessed using ImageJ 1.53e software. The effectiveness of individual EOs was determined as the difference in tuber infection intensity after and before application. In addition, the sporulation intensity of H. solani was studied on treated and non-treated tubers for both EO application variants. For this purpose, a zone of the skin with silver scurf symptoms was cut using a cork borer (7 mm diameter). The cut plugs (three plugs from each of seven tubers) were placed into closable tubes containing distilled water (5 mL), and the closed tubes were shaken at 100 rpm for 10 min on a Kavalier LT 3 shaker (Nedform, Valašské Meziříčí, Czech Republic) to release conidia. The intensity of sporulation (conidia/mL) of individual variants was determined using a Bürker chamber (Paul Marienfeld, Lauda-Königshofen, Germany).

2.5. Test of Table Quality after EO Application by Dressing and Fumigation

Finally, a test was made of potato table quality after EO application by dressing and fumigation. Prior to the test, tubers were transferred from storage and placed at room temperature. Tuber samples were cooked in steam within glass bowls. Prior to steaming, tubers were neither peeled, cut, nor salted. Five people conducted the assessment. Numbered tuber samples were served to tasters, who independently scored individual qualities according to Vidner [36]. Water was served for neutralization between individual samples. The test was conducted in daylight.

2.6. Statistical Assessment

The statistical assessment of the experimental results was performed using variance analysis (one-factorial ANOVA) and Tukey’s HSD test (p < 0.01; program STATISTICA 7; Statsoft, Tulsa, OK, USA).

3. Results

3.1. In Vitro Assays of the Antifungal Activity of EOs

The impact of EOs on the fungus H. solani was determined at different concentrations. The results of the experiments indicated all evaluated EOs have antifungal activity (

Table 1. Differences in the mean mycelial growth inhibition (MGI) of H. solani among various essential oil concentrations. Mean values within a column sharing the same superscript do not differ significantly from one another based on Tukey’s HSD test (p < 0.01). Different lowercase letters in table indicate statistically significant differences.

Essential Oil	Essential Oil Concentration (ppm)
	0	100	200	400	800	1600
(R)-(+)-limonene	0	3.1 d	7.1 e	14.2 c	31.4 b	51.1 b
α-pinene	0	7.4 cd	14.9 de	18.1 c	29.3 b	54.9 b
D-carvone	0	24.2 bc	39.5 c	100 a	100 a	100 a
carvacrol	0	100 a	100 a	100 a	100 a	100 a
eucalyptol	0	6.9 cd	31.8 cd	35.1 c	59.7 b	100 a
cinnamaldehyde	0	86.9 a	100 a	100 a	100 a	100 a
L-linalool	0	41.3 b	49.6 bc	92.6 ab	100 a	100 a
L-menthone	0	10.0 cd	14.3 de	50.9 bc	100 a	100 a
L-menthol	0	38.2 b	63.6 b	100 a	100 a	100 a
thymol	0	100 a	100 a	100 a	100 a	100 a

). A comparison of the data obtained at the concentration 100 ppm showed that all tested EOs were able to inhibit the mycelial growth of H. solani within a range from 3.1% to 100% (Table 1). Statistically highly significant differences were found in the antifungal activity among individual EOs. The highest level of mycelial growth inhibition (inhibition effect) against H. solani was obtained using thymol, carvacrol, and cinnamaldehyde, and there were no statistically significant differences among them. Therefore, these three EOs were selected for further in vivo experiments. The weakest inhibition effect was recorded for (R)-(+)-limonene.

The lowest IC₅₀ values were recorded for thymol and carvacrol, followed by cinnamaldehyde (

Table 2. Antifungal activity of essential oils on the mycelial growth of H. solani (mean values from three independent replications) for each pathogen–essential oil (EO) combination.

Essential Oil	IC50	MIC	MFC
(R)-(+)-limonene	1600	3200	3300
α-pinene	1600	3200	3400
D-carvone	175	350	350
carvacrol	40	80	100
eucalyptol	700	1400	1700
cinnamaldehyde	60	120	120
L-linalool	225	450	500
L-menthone	300	600	600
L-menthol	125	250	250
thymol	40	80	80

). The study of fungistatic and/or fungicidal activities revealed that EOs thymol, carvacrol, and cinnamaldehyde had fungicidal properties against H. solani at the concentrations 80, 100, and 120 ppm (Table 2). Thymol, carvacrol, and cinnamaldehyde had the lowest MICs among the tested EOs. These oils inhibited the growth of the pathogen in a dose-dependent manner. The strongest fungicidal capacity was determined in thymol (80 ppm), carvacrol (100 ppm), and cinnamaldehyde (120 ppm) (Table 2). The weakest fungicidal capacity was recorded for α-pinene (3400 ppm) and (R)-(+)-limonene (3300 ppm).

3.2. In Vivo Assays of the Antifungal Activity of EOs

EOs that were found to have a deleterious effect on the tested fungus were taken for further in vivo experiments. Tubers were dressed or fumigated, and differences between tuber infection intensity after and prior to EO application were determined. After fumigation, tubers were observed to have a statistically highly significant lower H. solani infection intensity compared to the non-treated controls for all tested EOs (Figure 1). Thymol had the strongest effect (99.82%) in reducing infection intensity. Cinnamaldehyde reduced infection intensity by 93.56% and carvacrol by 40.80%. After tuber dressing, a statistically highly significant reduction in H. solani infection intensity was determined also in all three tested EOs (Figure 1). The relative reduction in H. solani tuber infection ranged between 99.57% and 99.79%. Dressing had a stronger effect in reducing H. solani tuber infection, but in the case of carvacrol, this was without a statistically significant difference.

After tuber treatment with all tested EOs, a statistically highly significant reduction in H. solani sporulation intensity was found compared to the non-treated control for both application variants (Figure 2). Tuber dressing had a greater effect on H. solani sporulation intensity compared to fumigation. Reduction in sporulation intensity ranged between 97.44% and 100% and 86.69% and 97.73%, respectively. The application of thymol was most effective in both tuber treatment variants, with sporulation reduced by 100% and 97.73% compared to the non-treated control.

3.3. Test of Table Quality after EO Application by Dressing and Fumigation

At the end of the experiments, a test of potato cooking quality after EO application was performed together with tasting. EOs had no significant effect on tuber cooking quality (

Table 3. Evaluation of tuber cooking quality after EO treatment by dressing and fumigation.

Variant	Odor	Taste	Flesh Firmness and Cooking Behavior
carvacrol fumigation	6	21	14
carvacrol dressing	2 (essential oil odor)	17 (essential oil odor)	13
cinnamaldehyde fumigation	7	22	13
cinnamaldehyde dressing	2 (essential oil odor)	18 (essential oil odor)	13
thymol fumigation	7	22	13
thymol dressing	2 (essential oil odor)	18 (essential oil odor)	13
control fumigation	7	25	13
control dressing	7	27	13
Legend
Odor 5–8 pleasant, typical 1–4 satisfactory (occasionally off-odor) 0  unsatisfactory (off-odor)			Taste 31–40 excellent 21–30 very good 11–20 good 1–10   less good 0   unsatisfactory
Flesh firmness and cooking behavior 13–16 waxy, solid, fine texture, firm 9–12   slightly mealy, semi-solid, semi-fine, occasionally disintegrating slightly during cooking 5–8  mealy, semi-coarse texture, slight disintegration during cooking 1–4  strongly mealy, coarse texture, medium to strong disintegration during cooking 0   thin, very watery, soggy, strong disintegration during cooking

), but taste testing revealed that the quality was deteriorated due to the off-odor and taste of EOs; the taste of EO-dressed tubers was degraded by an off-odor and off-taste. The taste of EO-fumigation tubers was not degraded by an off-odor and off-taste.

4. Discussion

In this study, the antifungal activity of EOs α-pinene, carvacrol, cinnamaldehyde, D-carvone, eucalyptol, L-linalool, L-menthol, L-menthone, and (R)-(+)-limonene against H. solani causing potato silver scurf was determined using in vitro and vivo experiments. The control of H. solani is difficult because the fungus survives and spreads in the field and also in storage. Nevertheless, treating tubers with fungicides can reduce the occurrence of the disease. In our experiments, all evaluated EOs had an inhibitory effect on the pathogen’s growth under in vitro conditions. The antifungal activities of individual EOs differed significantly, however. The strongest antifungal activity was recorded for thymol, carvacrol, and cinnamaldehyde. Their effectiveness against H. solani was confirmed by in vivo experiments, wherein potato tubers were dressed and fumigated. EOs are volatile substances and that is why they were bound to a biopolymer for the dressing application. Incorporating EOs into the polymer matrix affects the oils’ physicochemical properties, in particular by improving their antimicrobial and antioxidant properties [37]. In our experiments, after the dressing and fumigation of tubers, a statistically highly significant reduction in H. solani tuber infection intensity was found.

Several authors have worked to verify the antifungal activity of EOs against H. solani, confirming various degrees of effectiveness using in vitro and in vivo experiments. Hartmans [38] demonstrated the promising antifungal activity of S-carvone derived from caraway (Carum carvi L.) against H. solani through in vitro and in situ experiments. The partial effectiveness of L-menthol contained in Mentha piperita in the pathogen’s inhibition was reported in a study by Coleman [32]. Al-Mughrabi [39] demonstrated the effectiveness of S-carvone, L-menthone, peppermint, and spearmint oils against H. solani under in vitro conditions. Bång [40] determined the effect of EOs on H. solani under in vitro and in vivo conditions, where the vapors of several EOs had a fungicidal effect provided that the concentrations were high enough and exposure times were long. Volatile substances from thyme (Thymus vulgaris) displayed an antifungal activity under in vitro conditions. An EO from common sage (Salvia officinalis) was effective for controlling disease development under in vivo conditions. Sharma [41] reported the inhibition effect of EOs extracted from Ageratum conyzoides, Lantana camara, Litsea cubeba, and Piper mullesua against H. solani under in vitro conditions, with EOs from Ageratum conyzoides and Piper mullesua being found most effective. Tripathi [42] demonstrated the antifungal activity of EOs from Litsea cubeba and Pipermullesua against H. solani through in vitro experiments. Frazier [43] determined a reduction in silver scurf severity and occurrence using frequent tuber applications of clove (Syzigium aromaticum) oil.

In our trials, after tuber treatment with all evaluated EOs (thymol, carvacrol, and cinnamaldehyde), we found for the dressing and fumigation application a significant reduction in H. solani sporulation intensity compared to the non-treated control. This is a very important finding for preventing the further spread of the pathogen during storage. The H. solani pathogen can sporulate and infect tubers in stored potatoes. Conidia from infected tubers could be distributed via a ventilation system to healthy tubers and may cause infection [7]. Tuber dressing had a greater effect on the infection intensity and H. solani sporulation intensity.

EOs had no significant effect on tuber cooking quality, but in tasting the cooked EO-dressed tubers, a deteriorated quality was detected as an off-odor and off-taste. The taste of EO-fumigation tubers was not degraded by an off-odor and off-taste.

Besides the characteristic strong aroma and flavor that can cause undesirable organoleptic changes in food, EOs are sensitive compounds that can easily degrade. Their low solubility in water is one of the biggest limitations in the use of essential oils; therefore, the concentration of these hydrophobic agents is low in the aqueous phase [44,45]. The encapsulation of essential oils may overcome such problems. Encapsulation is defined as an efficient method of preserving the quality of sensitive substances and improving the delivery systems of essential oils, enabling a controlled release of ingredients [46]. Encapsulation assists in reducing evaporation or slows the mass transfer of the volatile compounds to the external environment, modifying the physical characteristics of the material inside the particle core. This procedure also changes the physical properties of compounds, facilitating their application by converting a liquid into a solid phase, providing a controlled and targeted release of the active ingredients, improving the shelf life, and masking the flavors or odors of essential oils [47]. Encapsulation can be achieved by many techniques and is divided into the chemical method, physicomechanical method, and physicochemical method. The encapsulation process might involve more than one technique [48]. For the past decade, the use of biopolymer materials, instead of conventional and highly polluting materials, has increased substantially. Biopolymers can be obtained from various sources; the most used are those based on polysaccharides (sodium alginate, agar, chitosan, carrageenan, starch, and cellulose), lipids (waxes and fatty acids), and proteins (gelatin, collagen, and soy protein isolate) [49,50]. Another possibility is the encapsulation of EO in microporous structures as zeolites because these materials are cheap and non-toxic to biological environments [51].

EOs could offer an ecological alternative to synthetic fungicides for reducing potato tuber infection by H. solani during storage.