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Article

The Use of Thyme (Thymus vulgaris) Essential Oil for Controlling Cercospora Leaf Spot (Cercospora beticola) on Sugar Beets (Beta vulgaris)

by
Agnieszka Kiniec
1,*,
Maciej Spychalski
2,
Wojciech Miziniak
1,
Magdalena Palacz
2 and
Rafal Kukawka
2,*
1
Regional Experimental Station, Institute of Plant Protection, National Research Institute, Ul. Pigwowa 16, 87-100 Torun, Poland
2
Poznan Science and Technology Park, Adam Mickiewicz University Foundation, Ul. Rubiez 46, 61-612 Poznan, Poland
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(11), 2017; https://doi.org/10.3390/agriculture14112017
Submission received: 14 August 2024 / Revised: 3 November 2024 / Accepted: 5 November 2024 / Published: 8 November 2024
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Versions Notes

Abstract

:
Decreasing efficacy of fungicides and the withdrawal of further hazardous active ingredients in pesticides from use have prompted the search for alternative methods of crop protection. Essential oils (EOs) are secondary metabolites of plants and have been proven to show antibacterial, antifungal, and pest-repellent properties. This study was undertaken to determine the activity of grapefruit, rosemary, pine, sage, and thyme EOs against the fungus Cercospora beticola, which is the most dangerous pathogen of sugar beet and the causal agent of Cercospora leaf spot. According to the determined Minimum Inhibitory Concentration (MIC), thyme EO was found the most effective against C. beticola. For most of the fungal isolates tested, the MIC of this EO was 0.313 mL/L. Thyme EO also inhibited the growth of multi-resistant isolates. Based on the results obtained, thyme EO was subjected to further testing in field conditions, where its efficiency in controlling C. beticola was also proven. The results indicate that the use of thyme EO may be a promising method for the protection of sugar beets, although it requires further optimization in the context of its inclusion in sustainable protection programs assuming a reduced number of synthetic fungicide treatments.

1. Introduction

Modern agriculture is geared towards obtaining high yields to supply food for the growing world population. Achievement of satisfactory yields requires efficient crop protection. According to Integrated Pest Management, chemical methods should only be used as a last resort [1]. In practice, it is generally not possible to protect crops without the use of plant protection products. The consumer requires that agricultural products of top quality are produced with the lowest possible pesticide usage. Such a trend makes it necessary to look for alternative methods of protecting crops against pests and diseases. Modern agriculture should be sustainable, with no adverse effects on the health and life of people and other living organisms, and not burdening the natural environment. Other challenges currently faced in crop protection are the withdrawal of some effective but environmentally hazardous active ingredients and declining effectiveness of hitherto commonly used compounds. Pathogens develop resistance to active substances that have been repeatedly used [2,3,4]. New active synthetic substances scarcely ever appear on the market because the registration process is very expensive and time-consuming. Therefore, in recent years, increasing attention has been paid to alternative methods of crop protection, such as those based on the use of essential oils (EOs) [5,6,7,8,9].
EOs are secondary metabolites of plants that serve to protect them against pests and diseases. They show antibacterial, antiviral, antifungal, and pest-repelling properties [10], which is the reason for using EOs in medicine, pharmacy, cosmetics, and veterinary sciences, as well as for preservatives, food additives, and insect repellants [11]. EOs are volatile, complex mixtures with strong aromas. They can be composed of about 20 to 60 ingredients, with two or three predominant ones, while the others occur in small, often trace amounts [12,13,14]. The composition of EOs is influenced by the growing conditions and part of the plant from which the oil is obtained, and the plant genotype [15,16].
The composition of EOs is complex; their biological activity may be a result of the synergy of numerous molecules [17]. However, a vast body of evidence indicates that the major constituents of particular EOs are responsible for a given activity, while the minor components are only responsible for its modulation.
In general, the antifungal activity of EOs depends on numerous factors, such as the MIC values, different quantitative and qualitative ratios of its components, and the target microorganism. There are also synergistic or antagonistic effects on microbial cells that can affect overall antifungal activity. Important features of EO components are their poor water solubility and hydrophobicity, which allow them to attach to the cell surface and penetrate its interior [10]. Thus, it has been suggested that the EOs antimicrobial mechanism of action is related to degradation of the cell wall, disruption of cytoplasmic membrane, or membrane proteins. This results in cell lysis, cytoplasmic leakage, and eventually the cell death. However, the exact mechanism of EOs remains poorly understood, as each EO used in a particular experimental setup should be investigated separately. Attempts have been made to use EOs in plant protection against pests [18,19], bacteria, and fungi [20,21].
In this study, the biological activity of five commercially available EOs obtained from the following plants: grapefruit (Citrus grandis), rosemary (Rosmarinus officinalis), pine (Pinus sylvestris), sage (Salvia officinalis), and thyme (Thymus vulgaris) were analyzed. As for the EO subjects of this study, their activity has been tested against the various pathogens indicated above, but to the best of our knowledge, this is the first report on thyme EO activity in controlling C. beticola in field cultivation of sugar beets (Beta vulgaris).
Sugar beet is one of the two main sources of sugar in the world. More than half of the world’s beet sugar is produced in the European Union. In 2020, Poland was the third among sugar beet producers, second only to France and Germany [22]. Sugar beets are exposed to various pathogens, the most severe of which is C. beticola, causing the Cercospora leaf spot disease (CLS). The growth of the fungus is favored by temperatures from 25 to 30 °C and air humidity above 90% [23]. The vital activity of the fungus decreases at temperatures below 15 °C, and at a temperature of 10 °C, sporulation and infection do not occur. The infection worsens during dry periods throughout the day and humid periods at night [24]. In the climatic conditions of Poland, the first symptoms of the disease are observed already in the second half of June. Under optimal weather conditions, the pathogen can lead to complete leaf dieback. There are two types of toxins produced by C. beticola, i.e., cercosporin and beticolins. These toxins participate in the infection process and are responsible for the formation of necrosis on the leaves. In plants, beticolins induce dramatic loss of solutes such as amino acids and β-cyanin from root tissues. Moreover, toxins of this type inhibit ATP-dependent H+ transport [25]. Cercosporin has an impact on many physiological functions of the cell, including ATPase activity and proton transport. Cercosporin dissolves cell membranes, which, in turn, gives the fungal hyphae access to nutrients [26]. This is due to the production of reactive oxygen species by the toxin [27]. When not properly protected, sugar beets can lose up to 40% of beet root yield, and up to 10% of sugar yield which must be used to rebuild leaves [28,29].
The main means of protection against C. beticola is chemical treatment. Other ways of providing protection for sugar beet against CLS involve the use of appropriate agricultural technologies (e.g., appropriate crop rotation, deep tillage, or elimination of secondary hosts) and the sowing of resistant varieties. During the growing season, the pathogen’s development cycle may be repeated several times, which requires several protective treatments [28]. However, frequent protective treatments increase the pathogen’s resistance to the active ingredients of fungicides, especially if the number of substances authorized for use is limited. In the European Union, fungicides are based on active ingredients from three different groups: triazoles (e.g., epoxiconazole, tebuconazole, and difenoconazole), morpholines (e.g., fenpropidin), and strobilurins (e.g., azoxystrobin). Besides the above-mentioned ones, copper salts and sulfur-based agents (e.g., copper hydroxide) can also be used to protect sugar beet against CLS. Most of the eastern European isolates of C. beticola show high resistance to azoxystrobin; very often they are resistant to triazoles, and to morpholine. C. beticola is a pathogen that attacks all beet species: sugar, fodder, red beet, and chard. The last two are used directly for human consumption as well as for farm animals (fodder). This fact illustrates the urgency of the search for replacements for synthetic fungicides for beet’s protection against C. beticola that can be used in organic farming.
The aims of the present study were (i) to test the in vitro activity of commercially available EOs from grapefruit, rosemary, pine, sage, and thyme against a number of C. beticola isolates which are resistant to varying amounts of fungicides commonly used in sugar beet cultivation, and (ii) to evaluate selected EOs in the field cultivation of sugar beets. Field assays were not only focused on assessing the activity of EOs in controlling this pathogen, but also on determining and describing qualitative and quantitative parameters of sugar beet yield.

2. Materials and Methods

2.1. Collection of C. beticola Isolates

C. beticola isolates obtained from sugar beet leaves from central Poland were used for the study. Sugar beet leaves were collected in October 2020. The leaves were air dried and stored in paper bags. Sections of the dried leaves with CLS symptoms were incubated for 48 h in a wet chamber (RH > 80%) at room temperature (20–22 °C). The CLS symptoms are small spots with a characteristic reddish-brown border (Figure 1). The colonies of C. beticola that grow on PDA (Potato Dextrose Agar; Oxoid Ltd., UK) are light gray to brown in color. The mycelium is velvety and low, growing a little above the medium. The conidial spores are long and needle-like. It has smooth walls and, depending on environmental conditions, 3 to 24 cross-walls, but usually 3 to 14 [26]. C. beticola forms unbranched, slightly geniculate conidiophores that are light brown at the base and become lighter toward the top [4]. The conidial spores of C. beticola were collected from the leaf fragments using a preparation needle and used to prepare an aqueous spore suspension for the inoculation of PDA (Potato Dextrose Agar; Oxoid Ltd., Hampshire, UK) in 90 mm Petri dishes. The dishes were incubated at room temperature (20–22 °C) for 7–10 days. Subsequently, the morphological features of the colonies were verified and conidial spores [30] were transferred to new agar plates. The isolates are stored at the Regional Experimental Station in Toruń. The collection is stored in a refrigerator at 4 °C on PDA plates protected with parafilm and renewed every 12 months.

2.2. Resistance of Fungal Isolates Against Synthetic Fungicides

The commercial fungicide preparations (Table 1) used in this study were: Safir 125 SC (ADAMA, Kraków, Poland), Horizon 250 EW (Bayer Crop-Science, Leverkusen, Germany), Dafne 250 EC (INNVIGO, Warszawa, Poland), Amistar 250 SC (Syngenta, Basel, Switzerland), and Andros 750 EC (ADAMA, Kraków, Poland). The fungicides were dissolved in sterile distilled water. The solutions were added to the chilled PDA to provide the same active ingredient concentration of 1 µg/mL in the medium. Mycelial plugs (1 mm2) taken from a two-week-old colonies’ edge were placed in PDA medium amended with the fungicides. The controls were the colonies growing on PDA without the addition of the fungicides. Each isolate was tested on three Petri dishes and the tests were repeated twice. On each of them, 4 fragments of mycelium were inoculated. The radial growth was measured after seven days. Twelve measurements were made for each isolate and averaged. The percentage of growth of the culture on the fungicide medium relative to the controls was determined. The isolates were considered resistant when they showed more than 50% growth compared to the control colony.

2.3. Tested Essential Oils and Analysis of Their Composition

Five commercially available natural essential oils were subjected to composition analysis and further study. According to the manufacturer’s (ETJA Corp., Elbląg, Poland) instructions, grapefruit EO was obtained from C. grandis; rosemary EO from R. officinalis; pine EO from P. sylvestris; sage EO from S. officinalis; and thyme EO from T. vulgaris. The investigated EOs did not contain additives or solvents and were declared to be natural by the manufacturer. In both tests, i.e., antifungal activity assay and the field experiment, the emulsifier provided by the Poznan Science and Technology Park, was used. The emulsifier consisted of a polycarboxylate with a molecular weight of approximately 30 kDa. This polycarboxylate belongs to a group of star polymers based on substances of natural origin such as glycerin, citric acid, or adipic acid and PEG, which together form a branched polymer. The used polycarboxylate was in the form of sodium salt of carboxylic acid. The polycarboxylate was developed by a group of chemists from the Poznan Science and Technology Park, and this particular one was selected based on the results of thorough analysis of its emulsifying properties. Moreover, no direct influence of the tested emulsifier on C. beticola was also proven in the antifungal activity assays of EOs (Section 2.4) or field experiments (Section 2.5). For both indicated tests, the emulsifier was used at a concentration of 1%.
GC-MS analyses of the EOs compositions were carried out on a Bruker 436-GC SCION SQ operating in the EI mode at 70 eV equipped with a BR-5 capillary column (5%-phenylo-95%-dimethylpolisiloxane; (0.25 mm × 30 m, df = 0.25 µm) column). The MS detector was operated in the scan mode in the 50–500 m/z range, with ion source and transfer line temperatures held at 300 °C. One μL of diluted essential oil was injected in the split mode at the split ratio (20:1), and the inlet temperature was held at 300 °C. Helium was used as a carrier gas at a constant flow (1 mL/min). The oven temperature was programmed as follows: 60 °C held for 4 min, then raised to 180 °C (12 °C/min), then to 240 °C (8 °C/min) and to 300 °C (25 °C/min) held for 4.1 min.
Chromatographic signals were identified in two ways: by comparing the obtained mass spectra with the NIST 11 library, and on the basis of experimentally determined Kováts retention indices (in the range of 800–2000) based on the C8–C20 hydrocarbon standard.

2.4. Antifungal Activity Assays of Essential Oils

In the antifungal activity tests of EOs against C. beticola, 21 isolates characterized by different levels of resistance to the active ingredients, expressed as resistance to a different number of the fungicides, were selected. The isolates were not numbered sequentially, as the original laboratory numbers were retained. No medium-resistant isolates were used (Table 1). The isolates were divided into those resistant to all fungicides, four, three, two or one of them. From among the isolates showing similar resistance characteristics, at least two representative ones were selected. The antifungal activity of EOs was determined by assessing the Minimum Inhibitory Concentration (MIC), which was defined as the lowest concentration that fully inhibited the growth of the fungal mycelia.
Sections with an area of 1 mm2 were taken from the edge of a two-week-old culture and transplanted into PDA plates containing EOs. The EOs studied were used in the following concentrations: 10 mL/L; 5 mL/L; 2.5 mL/L; 1.25 mL/L; 0.625 mL/L; 0.313 mL/L; and 0.079 mL/L. The controls were the colonies growing on PDA without EOs. Each isolate was tested in three replicates. After a seven-day incubation period at room temperature (20–22 °C), the growth of the colonies was checked. The MIC was assumed to be the EO concentration at which the growth of C. beticola was fully inhibited. The tests were repeated twice.

2.5. Field Experiments

2.5.1. Locations of the Experimental Fields and Their Characterization

The experiments were conducted in the years 2022 and 2023 in the location of Falęcin in the Kuyavian–Pomeranian Voivodeship in Poland. The parameters characterizing the experimental fields are provided in Table 2. An appropriate crop rotation was maintained on each field. The meteorological conditions during the experiments are illustrated in Figure 2.

2.5.2. Experimental Design

In the field experiment, the sugar beet variety Pacific (Maribo, Denmark), which is a normal-type variety, was used. This variety is characterized by resistance to rhizomania (caused by beet necrotic yellow vein virus—BNYVV), and enhanced resistance to powdery mildew (caused by Erysiphe betae), Cercospora leaf spot (caused by C. beticola), seedling rot, and beet root tip rot (caused by Aphanomyces cochlioides and Fusarium fungi, respectively). The seeds were sown with a 6-row drill at a row spacing of 0.45 m in the second half of April.
Each experiment was set up in four replicates, in a randomized block design. The five-row experimental plots were 22.5 m2 (10 m long and 2.25 m wide). The roots of sugar beets were harvested manually from an area of 10.08 m2 in early October.
Treatments were performed using a wheelbarrow sprayer applying 0.9 L of spray liquid per 22.5 m2 (equivalent to 400 L of spray liquid per hectare). Based on the results of in vitro tests, thyme EO was selected for testing in field experiments. The standard fungicide program (henceforth referred to as SFP) as well as the untreated control (henceforth referred to as UTC) were used as a reference in the field experiment. The selection of fungicides used was made with the idea of applying two different active substances in each treatment. The treatments with fungicides were performed according to the labels. The fungicides Makler 250 SE and Eminent 125 ME were used in a mixture. Thyme EO was applied in two different concentrations, i.e., 0.05% and 0.1%, in 4 treatments. The concentration of thyme EO for field tests was chosen in such a way that the MIC value obtained for this EO was averaged to 0.5 mL/l, which corresponds to a concentration of 0.05%, while the second thyme EO concentration was chosen as twice that of the first one. The detailed schedules of treatments are provided in Table 3 and Table 4.

2.5.3. Assessment of C. beticola Infections

The assessment of the disease incidence and severity was made on plants from two middle rows within each block separately, consistent with the EPPO Standard scale [31]. The cercospora leaf spot incidence and severity were assessed on the day of sugar beet harvest. For each repetition, assessment was made on 10 consecutive plants from each row and expressed as a percentage of the infected area of the leaves.

2.5.4. Assessment of Qualitative Parameters of the Yield

A sample of approximately 30 kg of beetroots was taken from each plot. The evaluation of the qualitative parameters of beets was performed using a Venema auto-analyzer IIIG (Venema Consulting, Groningen, The Netherlands). The washed sugar beetroots were ground and clarified with 0.3% Al2(SO4)3 solution. Potassium and Na contents were determined by flame photometry, and α-amino-N (AmN) was analyzed by the fluorometric ortho-phthaldialdehyde (OPA) method. The polarimetry was used to determine sucrose content (SC) in the sugar beetroots. White sugar yield (WSY) was calculated according to the Brunswick formula [32]. Sugar processing losses were calculated according to the formula of Buchholz et al. [33]:
CT = 0.12 × (K + Na)+ 0.24 × (N-α-amines) + 0.48 [%],
where CT is the sugar processing loss [in %]; K, Na, N-α-amines are the potassium, sodium, and α-amino nitrogen contents [mmol (100 g−1 fresh roots)].
Refined sugar contents were calculated according to the formula of Buchholz et al. [33]:
RSC = Pol − CT − 0.6 [%]
where RSC is the refined sugar content [%]; Pol is the biological sugar content in root–sugar polarization [in % fresh weight]; and CT is the sugar processing loss [%].
Technological sugar yield was calculated according to the formula of Trzebiński [34]:
YST = YR × RSC × 100 − 1 [t ha−1]
where YST is the technological sugar yield [%]; YR is the root yield [t ha−1]; and RSC is the refined sugar content [%].

2.5.5. Statistical Analysis

The data related to the parameters describing beetroot yield, sugar polarization, potassium content in pulp, sodium content in pulp, α-amino nitrogen content in pulp and technological sugar yield were analyzed from 4 blocks for each variant of treatment. All recorded and calculated data were evaluated by analysis of variance (ANOVA), and the mean differences were compared by post hoc test at a p < 0.05 level, according to Tukey’s HSD. Statistical analyses were performed using the OriginLab 2022 software for Windows (OriginLab Corp., Northampton, MA, USA).

3. Results

3.1. Resistance of Fungal Isolates Against Synthetic Fungicides

The results of the tests of the effects of synthetic fungicides on the fungal isolates are given in Table 5 and Table S1. For the present study, 173 C. beticola isolates were prepared and the effects of five of the most often applied commercial fungicides on CLS used in the European Union were tested. The resistances of each of the isolates to active substances of the selected fungicides, i.e., epoxiconazole, tebuconazole, difenoconazole, azoxystrobin, and fenpropidin, were evaluated (Table S1). For each tested isolate, the number of active substances to which the isolate showed resistance was established. For the tests of the antifungal activity of EOs against C. beticola, the 21 isolates listed in Table 5 were selected. Each of the isolates was characterized by different resistances to the active ingredients (Table 1).

3.2. Essential Oils Composition Analysis

The compositions of EOs from the following plants were studied: grapefruit; rosemary; pine; sage; and thyme. The main components of the EOs used in the tests were identified on the basis of GC-MS spectra and are listed in Table 6. The table shows the retention index (RI) and the percentage of the area under a peak corresponding to a given substance in relation to the total percentage of the area under the other peaks recorded for a given sample. The total number of chemical compounds identified in the studied essential oils was 40 for grapefruit EO (representing 85% of the total oil content), 35 for rosemary EO (representing 95%), 25 for pine EO (representing 95%), 32 for sage EO (representing 90%) and 15 for thyme EO (representing 98%). Table 6 presents all compounds whose percentage content was higher than 1.2%.
The GC-MS analyses showed that the main component of grapefruit EO was limonene (83.5%). In rosemary EO, three components were identified at similar levels: camphor (19.8%), α-pinene (18.4%), and 1,8-cineole (17.4%). Pine EO was dominated by α-pinene (21.9%). The main component of sage EO was camphor (20.7%), while in thyme EO it was thymol (57.0%).

3.3. Antifungal Activity Assay of Essential Oils

The results of activities of selected EOs against C. beticola are presented in Table 7, where the MIC of each studied EO is indicated. The thyme EO was found to be the most effective inhibitor of pathogen growth (Figure 3A). The MIC values for this EO ranged from 0.313 to 1.25 mL/L. Significantly lower MIC values for thyme EO were observed for all isolates, i.e., those resistant to different amounts of active substances.
Grapefruit EO was the weakest in inhibiting C. beticola growth (Table 7 and Figure 3B). Only the MIC of isolate 4 was determined as 10.0 mL/L. For the remaining isolates, their MICs were over 10.0 mL/L. Sage EO turned out to be slightly more effective than grapefruit EO. For six fungal isolates, their MIC values were of 10.0 mL/L, while for the remaining ones, higher MIC values were found. Rosemary and pine EOs showed similar efficacy against C. beticola. For rosemary EO, the MIC values ranged from 5.0 mL/L to over 10.0 mL/L, and eleven fungal isolates showed MIC values greater than 10.0 mL/L. For pine EO, the MIC values ranged from 2.5 mL/L to over 10.0 mL/L. Only two isolates showed MIC values greater than 10.0 mL/L.
Fungal isolate 77 was the most resistant in all assays as the MIC values determined for rosemary, pine, and sage EO were 10 mL/L, and higher than 10 mL/L for grapefruit EO. Isolate 77 was the least sensitive to thyme EO from among all the tested isolates, as this EO showed MIC of 1.25 mL/L. Moreover, isolate 77 was also multi-resistant to the tested synthetic fungicides. Isolates 102 and 141 were the most sensitive to the EOs used in our assays. For the two isolates, the same MIC values were found for the following EOs: grapefruit (above 10.0 mL/L), rosemary (5.0 mL/L), and thyme (0.313 mL/L). The lowest concentration of pine EO that inhibited the growth of isolate 102 was 2.5 mL/L. This isolate was also most sensitive to pine EO. The MIC value of pine EO towards isolate 141 was 5.0 mL/L. Sage EO showed fungicidal activity against isolate 102 at a concentration of 10.0 mL/L. However, for isolate 141, the effective inhibiting concentration of sage EO exceeded 10.0 mL/L.

3.4. Field Experiments

3.4.1. Experiments in 2022

The highest beetroot yield was obtained for the sugar beets treated according to SFP variant, while the lowest one was obtained for the plants of the untreated control (Table 8). Root yield of sugar beets treated according to the thyme 0.05% variant was higher compared to that of the plants treated according to UTC variant, but the difference is not significant. The treatment of sugar beets according to the thyme 0.1% variant resulted in obtaining higher root yield comparing to that of the plants of untreated control. Analogous/similar dependencies were observed for the parameter related to technological sugar yield. The other parameters describing sugar beet yield were not affected by tested variants of treatment.
The lowest percentage of leaf area covered with cercospora leaf spot was observed for the plants treated with the SFP variant, while the highest percentage of symptomatic leaf area was observed for the UTC plants. Treatment of plants with thyme EO resulted in a reduction in the degree of leaf symptoms when compared to that of the UTC plants. More efficient protection against cercospora leaf spot was provided by the application of a higher concentration of the tested EO, i.e., the variant of thyme EO at 0.1%.

3.4.2. Experiments in 2023

The results of the field experiments conducted in 2023 are in line with those obtained in the experiments conducted in 2022. The highest beetroot yield and the highest technological sugar yield were observed for the sugar beets treated with the SFP variant (Table 9). As for the variants of the treatment with thyme EOs, higher root yield and technological sugar yield were obtained for the plants treated with the thyme 0.1% variant than for those treated with the thyme 0.05% variant. The other parameters describing sugar beet yield were not affected by tested variants of treatment.
As for the efficiency of protection against cercospora leaf spot, the highest was obtained after treatment according to SPF variant, while the lowest was seen in the UTC plants. As in the previous year, the treatment of plants with thyme EO resulted in a reduction in the degree of leaf infection compared to the UTC plants. More efficient protection against cercospora leaf spot was provided by the EOs applied at a higher concentration, i.e., the thyme EO 0.1% variant.

4. Discussion

European Union legislation indicates the need to undertake actions aimed at reducing the use and dependence on pesticides. Therefore, it is of key significance to search for new methods providing efficient protection against pathogens. The use of EOs may be one of such alternatives. EOs are generally recognized as safe, easy to use, and not requiring any special precautions [35,36]. An additional advantage of EOs is related to their multidirectional mechanism of action in fungal cells. As EOs do not bind to a specific gene like some fungicides’ active substances (e.g., strobilurins or benzimidazoles), it is unlikely that fungi will acquire resistance to EOs [37]. This feature is of key importance for controlling C. beticola as, recently, almost the entire Polish population of it has become resistant to azoxystrobin and thiophanate-methyl [3]. The resistance of the fungus to triazoles increases with each passing year, which is alarming as most of the fungicides available in European Union for the control of CLS are based on triazoles. C. beticola is one of the pathogens that quickly acquires resistance to chemical compounds used for plant protection [38], which is the reason why repeated infections may occur during the growing season. Moreover, the pathogen produces a large number of conidial spores and exhibits a high genetic variability [2,39]. The tests of the EO activity against C. beticola were performed with the isolates showing varying degrees of resistance to the fungicides’ active ingredients most commonly used in Poland (Table 2). No relationship was found between the resistance to different number of active ingredients of the fungicides and the sensitivity to EOs. This result is of high importance, as it provides a strong basis for concluding that the activity of a given EO against C. beticola will not vary depending on the degree of isolate resistance.
Various components are supposed to play a role in defining the EOs’ properties, such as cell penetration and lipophilic or hydrophilic attraction and fixation on membranes and cell walls. When comparing various activities of the major EOs’ components with those of the EOs from which they were isolated, similar trends were observed [40]. Thus, a general conclusion is that for biological purposes, when studying the effects of a particular EO, it should be treated as a whole, as the concept of synergism appears to be more meaningful [10,41].
We have demonstrated that thyme EO inhibited the growth of C. beticola at significantly lower doses compared to other EOs tested for all isolates subjected to study. The results obtained by us are consistent with those reported elsewhere. Amini et al. [42] have demonstrated a strong fungicidal effect of thyme EO against Rhizoctonia solani, Fusarium graminearum, and Sclerotinia sclerotiorum. Soković et al. [13] have found that a concentration of 0.25 μL/mL of thyme EO inhibited the growth of Alternaria alternata, Fusarium tricinctum, and Aspergillus niger. Thyme EO also strongly inhibited the growth of F. graminearum, F. verticillioides, F. subglutfinans, F. oxysporum, Diaporthe helianthi, Diaporthe phaseolorum var. caulivora, Phomopsis longicolla, P. viticola, Helminthosporium sativum, and Colletotrichum coccodes. The main component of the thyme EO used in this study was thymol (57.0%) [42]. Similar results have been reported by Bozin et al. [12], Soković et al. [13], and Ebani et al. [14]. However, not all fungi are sensitive to thyme EO. Thanatephorus cucumeris is insensitive not only to thyme EO but also to clove (Eugenia caryophyllus, Myrtaceae) EO, rosemary EO, sage EO, pine EO, mint (Mentha piperita, Lamiaceae) EO, lavender (Lavandula angustifolia, Lamiaceae) EO, caraway (Carum carvi, Apiaceae) EO, and neroli (Citrus aurantium, Rutaceae) EO [5]. Ebani et al. [14] have shown that only two out of seven tested fungi were sensitive to thyme EO: Candida tropicalis and A. niger, while the MIC of thyme EO values against C. albicans and Aspergillus fumigatus were high. The observations concerning C. albicans are not in agreement with those of Bozin et al. [12] who concluded that C. albicans was sensitive to the thyme EO. In addition, they found the MIC of thyme EO was five times lower than that of the standard antifungal agent used to control C. albicans.
As for the probable antifungal mechanism of thyme EOs, it is suggested that this EO affects fatty acid metabolism, in particular ergosterol, in the fungal cell [43]. Ergosterol is a unique sterol found only in the cell membrane of fungi, important for their proper growth and functioning. The application of thyme EO leads, among other effects, to an increased concentration of reactive oxygen species and oxidative stress, which causes a decrease in the extracellular polymer matrix and capsular polysaccharide. It has been reported that the amount of ergosterol in the cell membranes of Candida and Cryptococcus treated with thymol decreases, which causes disruption of membrane integrity, membrane-associated enzyme disturbances, extensive damage, and, as a consequence, cell death [13,44].
Al-Shahrani and co-workers have studied the antifungal activity of thyme EO on clinical strains of Fusarium spp., Aspergillus spp., and Candida spp. [45]. According to their results, this EO exhibits strong antifungal properties against all fungal strains when applied in concentrations ranging from 0.5 to 10 mg/mL. It was concluded that it can be an effective alternative to currently used antifungal medications such as amphotericin B, which have a high toxic potential.
In our study, grapefruit EO turned out to be the least effective against C. beticola; its MIC could be determined for only one isolate. As for the others, the MIC was higher than 10.0 mL/L. Similar results have been obtained by Gwiazdowski et al. [46], who studied the activity of 20 different EOs against oilseed rape pathogens. The EOs with the weakest fungistatic effects included the EOs from grapefruit (Citrus grandis, Citrus paradisi, Rutaceae), and orange (Citrus aurantium var. bergamia, Citrus aurantium var. dulcis, Rutaceae). Sharma and Tripathi [23] have also investigated the effect of EOs obtained from plants of the genus Citrus on phytopathogenic fungi of mangoes, tomatoes, and apples. They indicated that Citrus sinensis EO showed fungicidal activity against Aspergillus niger, Alternaria alternata, Botrytis cinerea, and Penicillium expansum. However, Sharma and Tripathi [23] used several times higher concentrations of grapefruit EO when compared to those used in our study. At the lowest concentration of 25.0 mL/L, the grapefruit EO was ineffective against all tested pathogens. The main component of the grapefruit EO used in this study was limonene (43.5%). Limonene is a cyclic monoterpene, commonly found in citrus fruits [47,48]. Some researchers claim that limonene has an antifungal and antioxidant effect, as well as that it inhibits aflatoxin production [49]. However, the results of our study show that EOs whose main ingredient is limonene are ineffective in limiting the growth of C. beticola mycelium. Also, Kishore et al. [50] have shown that limonene has a weak antifungal effect.
Sage EO showed slightly greater effectiveness against C. beticola than grapefruit EO. For most of the tested isolates, the MIC values exceeded 10.0 mL/L. Similar results have been obtained by Cosić et al. [5] who studied the activity of sage EO against 12 pathogens, including fungi of the genus Fusarium. All fungal isolates grew well in the presence of sage EO. The poor fungicidal effect of EOs obtained from plants of the genus Salvia has also been described by Er et al. [21]. When examining the activity of nine essential oils against Plasmopara halstedii, the authors showed that the EO obtained from Salvia triloba was the weakest in inhibiting the growth of the pathogen. The main components of the sage EO used in this study were camphor (20.7%) and 1,8-cineole (11.8%). Camphor was also found to be the main active ingredient of sage EO, although the same authors also emphasize the importance of α-thujone [17,51]. Both these components belong to oxygenated monoterpenes and are used in medicine, as ingredients of ointments and inhalations. After oral administration, they show a toxic effect [52,53]. It is known that α-thujone shows antimicrobial activity [54]. However, Mighri et al. [55] have proved that Artemisia herba-alba EO has significantly better antimicrobial properties when α- and β-thujone are present in the EO in similar proportions rather than when one of these components predominates. Camphor has repellent, antimicrobial, and antiviral effects [37]. Some researchers, however, indicate that pure camphor does not have very strong bioactivity. Such an activity is demonstrated by camphor-rich essential oils, containing also other compounds, e.g., 1,8-cineole [56]. However, the sage EO used in our study showed weak activity against C. beticola, despite the dominant presence of camphor and 1,8-cineole in its composition.
Bozin et al. have found that the MIC of rosemary EO used against Candida albicans was lower than that of sage EO [12]. Our studies confirmed this observation, although it was also shown that rosemary EO also weakly inhibited the growth of C. beticola. The MIC values of this EO ranged from 5.0 mL/L to over 10.0 mL/L. Similar results have been reported by Cosić et al. [5]. They tested the activity of rosemary EO against 12 different pathogens, including fungi of the genus Fusarium. Most of them grew well in the presence of rosemary EO. Djeddi et al. [40] have reported no fungistatic effect of rosemary EO at a concentration of 20% against C. albicans. However, the concentration used by these researchers was sufficient to inhibit the growth of Staphylococcus aureus, S. epidermis, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli. Ebani et al. [14] have determined the MIC value of rosemary EO towards C. albicans as 9.14 µg/µL. On the other hand, the MIC values of rosemary EO towards Candida tropicalis, Aspergillus niger, and A.fumigatus were much lower and amounted to 0.29 µg/µL. The main components of rosemary EO used in this study are camphor (19.7%), α-pinene (18.4%), and 1,8-cineole (17.4%). Similar results have been reported by Bozin et al. [12] and Ebani et al. [14]. Cosić et al. [5] have shown that rosemary EO even stimulated the growth of Fusarium subglutfinans. Our tests did not show that rosemary EO has such an effect on the growth of C. beticola. There is a strong variation in the pathogens’ responses to rosemary EO. This result points to the need to test the action of individual EOs against each of the relevant pathogens that cause damage to agriculture.
Pine EO showed similar efficacy against C. beticola as rosemary EO, which is consistent with the results presented by Cosić et al. who found that pine EO was poorly effective against 11 tested pathogens, including fungi of the genus Fusarium [5]. On the other hand, Amri et al. have reported growth inhibition of fungi of the genus Fusarium and Rhizoctonia in the range of 50% to 60% compared to a control when 4 µL/mL of pine EOs was added [57]. Moreover, the application of pine EO inhibited the growth of fungi of the genus Alternaria by about 40%, compared to the control [57]. The main component of pine EO used in our study is α-pinene (21.85%), which is in full agreement with the results presented by Ustun et al. [58] and Allenspach et al. [59]. This compound is monoterpene hydrocarbon and has moderate antifungal activity. Medina Romero et al. [60] have reported inhibition of the growth of fungi belonging to Fusarium genus by around 35% comparing to a control when 4 µL/mL of pine EO was used. For comparison, carvacrol completely inhibited the growth of mycelium when used at a four-times-lower concentration. Additionally, Cosić et al. [5] have described the stimulating effect of pine EO on the growth of Diaporthe helianthi and Helminthosporium sativum mycelium. In our study, the growth-stimulating effect of pine EO on C. beticola was not observed. Similar results have been reported by Bozin et al. [12,38]. It has been shown by these authors that oregano (Origanum vulgare) EO effectively inhibited the growth of antibiotic-resistant bacteria and that sage EO and rosemary EO effectively inhibited the growth of antibiotic-resistant bacteria [12].
Based on the research from plate tests, thyme EO was selected as that of the highest efficiency in limiting the growth of C. beticola. Demonstrating thyme EO activity in a plate test was an initial stage of our investigation, which was further continued in field cultivation of sugar beets. Tested variants of treatment assumed that a total of 4 applications of thyme EO either at a concentration of 0.05% or 0.1% were performed. Of the two concentrations tested, the variant of treatment with 0.1% EO concentration resulted in lower disease infestation and higher technological sugar yield. The values of these parameters were significantly higher than those obtained from the untreated control plants, but they were significantly lower than the values of these parameters obtained for plants treated with standard fungicide protection. However, it is still to be investigated whether the use of a higher concentration of thyme EO will result in an improvement in disease inhibition and an increase in technological sugar yield. A study on the best working concentration of this EO is scheduled to be conducted. However, even at the best-performing EO concentration, it might not be possible to provide protection at a level similar to that provided by a standard fungicide program. However, any method allowing a reduction in the number of fungicide treatments necessary to ensure adequate protection and yields will be of high significance. This area of research is consistent with the EU “Farm to Fork Strategy” which sets out a requirement for reducing the use of pesticides by 50% by 2030.
Another research topic that would be worth considering is the investigation of potential synergistic effects between EOs and Systemic Acquired Resistance (SAR) inducers, which also serve as an alternative to fungicides [61]. Of note, the use of SAR inducers is also free from the risk of pathogens acquiring resistance to active substances [62]. The mode of action of SAR inducers is directed toward the stimulation of a plant’s natural defense mechanisms and not toward pathogens directly [63]. In our previous study, we proved that the combined used of SAR inducer and fungicide (in a reduced number of treatments) resulted in similar levels of protection against C. beticola and obtaining technological sugar yield comparing to standard fungicide treatment [64]. The combination of EOs, which act directly on pathogens, and SARs, which activate plant resistance mechanisms, can lead to a synergistic effect on plants, which in turn can result in obtaining results comparable to those obtained after treatment of plants according to standard fungicide programs. Moreover, the use of SAR inducers provided protection against different pathogens simultaneously [65]. Cercospora leaf spot is the most severe disease affecting sugar beets; however, other diseases of fungal, bacterial, or viral origin can also affect particular sugar beet plantations [66,67]. Therefore, further research on the combined use of EOs and SAR inducers is justified from the point of view of potential application in agricultural practice.
Further development of technology related to the use of the best-performing thyme EOs will require more detailed field testing. The high activity demonstrated in field trials has to be proven under variable conditions. It will also be of key importance to maintain the stability of the composition of the tested EO to ensure that the obtained results are reproducible and reliable. As for the experimental variants of treatment to be tested during field studies, it will also be necessary to investigate the possibility of combining essential oils with available plant protection agents. Such combined use may take the form of interchangeable treatments within the established protection program or combined use in one treatment with a full or decreased dose of a fungicide. In the current situation in plant protection, related to the lack of availability of active substances, each new possibility that provides effective protection and reduces the risk of pathogens acquiring resistance will be of high significance for farmers.
However, there are certain difficulties related to the use of EOs in crop protection that need to be overcome. The EOs are poorly water soluble, volatile, and easily degraded by heat, light, and moisture [68]. These issues can be solved by using EOs in the form of nanoemulsions, which would allow for improving the solubility and stability, while reducing the volatility, of EOs [64,69]. Another important issue is maintaining a constant composition of EOs. Their composition is affected by weather and the growth conditions of the plant, from which EOs are obtained [70]. Moreover, the extraction method itself may also affect the contents of individual EOs components. What is more, even within a given method, the differences in temperature, extraction time, substances or techniques used, can also influence the composition of EOs. This can affect their aroma, taste, and therapeutic properties. For instance, the content of limonene, which is the main component of grapefruit EO, varies depending on the extraction method used [71,72]. Another modification of extraction methods might be enzyme pre-treatment with mixture of cellulose and hemicellulose from leaves mixed with distilled water. Such a modification has been reported to improve the extraction efficiency of essential oils from T. capitatus and R. officinalis [73].

5. Conclusions

Our study has shown that thyme EO can effectively limit the growth of C. beticola both in plate tests and field cultivation of sugar beet. Moreover, this EO also limited the growth of resistant fungal isolates. In the face of declining effectiveness of synthetic fungicides and withdrawal of some active ingredients from use, the inclusion of EOs in plant protection programs can be a valuable solution to improve the quality and size of yields.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture14112017/s1: Table S1, characteristics of tested C. beticola isolates related to the number of active substances to which the isolate showed resistance.

Author Contributions

Conceptualization, A.K. and W.M.; methodology, A.K. and M.P.; validation, A.K. and W.M.; formal analysis, A.K., M.P., M.S. and R.K.; investigation, A.K.; resources, A.K., M.P. and R.K.; data curation, A.K.; writing—original draft preparation, A.K., W.M., R.K. and M.S.; writing—review and editing, A.K., W.M., M.S. and R.K.; visualization, A.K.; supervision, A.K.; project administration, A.K. and R.K.; funding acquisition, A.K. and R.K. All authors have read and agreed to the published version of the manuscript.

Funding

The “New plant resistance inducers and their application as innovative approach to plant protection against pathogens” project is carried out within the TEAMTECH (POIR.04/04.00-00-5BD9/17-00) program of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. IOR–PIB Program Wieloletni—Task 1.4. “Monitoring the resistance of pests to plant protection products and creating risk reduction programs, taking into account the safety of bees”.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors would like to thank Renata Długosz and Jacek Piszczek for their help in laboratory work.

Conflicts of Interest

The authors declare no conflicts of interest.

Sample Availability

Samples of the EOs are available from the authors.

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Agriculture 14 02017 g001
Figure 1. Sugar beet leaf with cercospora leaf spot disease (CLS) symptoms.
Figure 1. Sugar beet leaf with cercospora leaf spot disease (CLS) symptoms.
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Agriculture 14 02017 g002
Figure 2. Weather conditions (average temperature and precipitation) at experimental location Falęcin in years 2022 and 2023.
Figure 2. Weather conditions (average temperature and precipitation) at experimental location Falęcin in years 2022 and 2023.
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Agriculture 14 02017 g003aAgriculture 14 02017 g003b
Figure 3. Antifungal activity of EO against C. beticola isolate 30B; (A) thyme EO showed a MIC of 1.25 mL/L, (B) grapefruit EO showed a MIC higher than 10.0 mL/L.
Figure 3. Antifungal activity of EO against C. beticola isolate 30B; (A) thyme EO showed a MIC of 1.25 mL/L, (B) grapefruit EO showed a MIC higher than 10.0 mL/L.
Agriculture 14 02017 g003aAgriculture 14 02017 g003b
Table 1. Commercial fungicide preparations and their active ingredients used in the experiments.
Table 1. Commercial fungicide preparations and their active ingredients used in the experiments.
FungicideActive Ingredients
Safir 125 SC125 g/L epoxiconazole
Horizon 250 EW250 g/L tebuconazole
Dafne 250 EC250 g/L difenoconazole
Amistar 250 SC250 g/L azoxystrobin
Andros 750 EC750 g/L fenpropidin
Table 2. Description of experimental sites.
Table 2. Description of experimental sites.
Year of
Experiment		Name and Symbol
of Location		Geographical
Coordinates		Soil CharacteristicspH
2022Falęcin53°13′06″ N 18°36′33″ Esandy loam6.7
2023Falęcin53°13′06″ N 18°36′33″ Esandy loam6.6
Table 3. The detailed schedule of treatments performed in the experiment in 2022.
Table 3. The detailed schedule of treatments performed in the experiment in 2022.
Variant of TreatmentThe Numbering and Dates of Treatments
4 July 202212 July 202218 July 202227 July 202217 August 2022
IIIIIIIVV
UTC
SFP Makler 250 SEEminent 125 ME Spyrale 475 EC
Thyme EO 0.05%Thyme EO 0.05%Thyme EO 0.05% Thyme EO 0.05%Thyme EO 0.05%
Thyme EO 0.1%Thyme EO 0.1%Thyme EO 0.1% Thyme EO 0.1%Thyme EO 0.1%
UTC—untreated control; SFP—Standard Fungicide Program; Makler 250 SE (Innvigo, Warszawa, Poland) active substances: azoxystrobin 250 g/L; Eminent 125 ME (UPL, Warszawa, Poland) active substance: tetraconazole 125 g/L; Spyrale 475 EC (ADAMA, Poland) active substances: fenpropidin 375 g/L, difenoconazole 100 g/L.
Table 4. The detailed schedule of treatments performed in the experiment in 2023.
Table 4. The detailed schedule of treatments performed in the experiment in 2023.
Variant of TreatmentThe Number and Dates of Treatments
7 July 202314 July 202320 July 202328 July 202321 August 2023
IIIIIIIVV
UTC
SFP Makler 250 SEEminent 125 ME Spyrale 475 EC
Thyme EO 0.05%Thyme EO 0.05%Thyme EO 0.05%Thyme EO 0.05%Thyme EO 0.05%
Thyme EO 0.1%Thyme EO 0.1%Thyme EO 0.1% Thyme EO 0.1%Thyme EO 0.1%
UTC—untreated control; SFP—Standard Fungicide Program; Makler 250 SE (Innvigo, Poland) active substances: azoxystrobin 250 g/L; Eminent 125 ME (UPL, Poland) active substance: tetraconazole 125 g/L; Spyrale 475 EC (ADAMA, Poland) active substances: fenpropidin 375 g/L, difenoconazole 100 g/L.
Table 5. Characteristics of tested C. beticola isolates.
Table 5. Characteristics of tested C. beticola isolates.
No.
Isolate		EpoxiconazoleTebuconazoleDifenoconazoleAzoxystrobinFenpropidinThe Number of Substances the Isolate Is
Resistant To
4+++++5
35+++++5
102++++−4
112++++−4
141+++−+4
58++−++4
68++−++4
73++−+−3
77++−+−3
88−−−++2
159−−−++2
2−−++−2
124−−++−2
30−+−+−2
80−+−+−2
11++−−−2
45−−−+−1
130−−−+−1
15−−−−+1
10−−−−−0
33−−−−−0
The isolates were considered resistant when they showed more than 50% growth compared to the control colony. + resistant isolate; − sensitive isolate.
Table 6. Chemical composition of the essential oils from grapefruit, rosemary, pine, sage, and thyme.
Table 6. Chemical composition of the essential oils from grapefruit, rosemary, pine, sage, and thyme.
ComponentRI * Area (%)
GrapefruitRosemaryPineSageThyme
α-pinene9372.618.421.94.6
camphene955 5.43.86.4
β-pinene982 9.85.9
δ-3-carene1012 3.19.8
ρ-cymene10283.14.5 16.0
β-phellandrene1030 1.8
limonene103383.51.22.61.4
1,8-cineole1037 17.4 11.8
α-thujone (cis)1099 25.5
linalool11003.8 2.2
β-thujone (trans)1110 5.4
isoborneol1153 1.9
camphor1155 19.7 20.7
borneol1177 2.9 3.5
menthol1182 5.1
α-terpineol1188 4.58.0 3.2
γ-terpineol1196 7.3
bornyl acetate1288 7.9
thymol1293 57.0
carvacrol1302 2.3
β-caryophyllene1415 3.3
α-caryophyllene1489 4.7
caryophyllene oxide1583 12.5
* RI—Retention indices relative to C8–C20 n-alkanes column, the highest values for each oil have been bolded. Compounds whose percentage content was lower than 1.2% were not included in the table.
Table 7. MIC values of the tested EOs against C. beticola isolates [mL/L].
Table 7. MIC values of the tested EOs against C. beticola isolates [mL/L].
IsolateGrapefruit EORosemary EOPine EOSage EOThyme EO
MIC Value [mL/L]
410.05.010.010.00.313
35>10.0>10.010.0>10.00.313
102>10.05.02.510.00.313
112>10.05.010.0>10.00.313
141>10.05.05.0>10.00.313
58>10.0>10.010.0>10.00.625
68>10.0>10.0>10.0>10.00.313
73>10.05.010.0>10.00.313
77>10.05.010.010.01.25
88>10.0>10.0>10.0>10.00.313
159>10.0>10.010.0>10.00.313
2>10.0>10.05.0>10.00.313
124>10.05.010.010.00.625
30>10.0>10.010.0>10.00.625
80>10.0>10.010.010.00.313
11>10.0>10.010.0>10.00.313
45>10.010.010.0>10.00.625
130>10.010.010.0>10.00.313
15>10.05.010.010.00.625
10>10.0>10.010.0>10.00.313
33>10.0>10.010.0>10.00.313
Table 8. Summary of the results of field experiments conducted in 2022.
Table 8. Summary of the results of field experiments conducted in 2022.
Variant of TreatmentRoot
Yield
[t ha−1]		Sugar
Polarization
[%]		Potassium
Content
[mmol 1000 g−1
of Pulp]		Sodium
Content
[mmol 1000 g−1
of Pulp]		α-Amino
Nitrogen
Content
[mmol 1000 g−1
of pulp]		Technological Sugar
Yield
[t ha−1]		Infected Leaf Area [%]
UTC56.46 a16.03 a36.30 a9.48 a17.05 a7.90 a100 c
SFP66.62 c16.88 a37.75 a7.78 a20.45 a9.83 c70 a
Thyme EO 0.05%59.26 ab16.29 a35.43 a9.83 a20.53 a8.40 ab85 b
Thyme EO 0.1%61.85 b16.35 a35.83 a11.05 a22.00 a8.77 b80 ab
Symbols for the table: within columns, mean values marked with the same letter do not differ significantly at p = 0.05 according to Tukey’s HSD. UTC—plants not treated either with fungicides or with thyme EO; SFP—plants treated with fungicides twice; thyme EO 0.05%—plants treated with thyme EO 0.05% four times; thyme EO 0.1%—plants treated with thyme EO 0.1% four times. A detailed schedule of treatments is provided in Table 3.
Table 9. Summary of the results of field experiments conducted in 2023.
Table 9. Summary of the results of field experiments conducted in 2023.
Variant of TreatmentRoot
Yield
[t ha−1]		Sugar
Polarization
[%]		Potassium
Content
[mmol 1000 g−1
of Pulp]		Sodium
Content
[mmol 1000 g−1
of Pulp]		α-Amino
Nitrogen
Content
[mmol 1000 g−1
of Pulp]		Technological Sugar
Yield
[t ha−1]		Infected Leaf Area [%]
UTC56.00 a17.10 a39.25 a5.35 a17.73 a8.44 a100 c
SFP69.71 c17.58 a39.53 a5.55 a19.00 a10.81 c57.5 a
Thyme EO 0.05%60.14 ab17.19 a38.80 a4.48 a18.88 a9.10 ab72.5 b
Thyme EO 0.1%64.34 b17.10 a40.20 a6.00 a18.78 a9.67 b70 b
Symbols for the table: within columns, mean values marked with the same letter do not differ significantly at p = 0.05 according to Tukey’s HSD. UTC—plants not treated either with fungicides or with thyme EO; SFP—plants treated with fungicides twice; thyme EO 0.05%—plants treated with thyme EO 0,05% four times; thyme EO 0.1%—plants treated with thyme EO 0.1% four times. A detailed schedule of treatments is provided in Table 4.
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Kiniec, A.; Spychalski, M.; Miziniak, W.; Palacz, M.; Kukawka, R. The Use of Thyme (Thymus vulgaris) Essential Oil for Controlling Cercospora Leaf Spot (Cercospora beticola) on Sugar Beets (Beta vulgaris). Agriculture 2024, 14, 2017. https://doi.org/10.3390/agriculture14112017

AMA Style

Kiniec A, Spychalski M, Miziniak W, Palacz M, Kukawka R. The Use of Thyme (Thymus vulgaris) Essential Oil for Controlling Cercospora Leaf Spot (Cercospora beticola) on Sugar Beets (Beta vulgaris). Agriculture. 2024; 14(11):2017. https://doi.org/10.3390/agriculture14112017

Chicago/Turabian Style

Kiniec, Agnieszka, Maciej Spychalski, Wojciech Miziniak, Magdalena Palacz, and Rafal Kukawka. 2024. "The Use of Thyme (Thymus vulgaris) Essential Oil for Controlling Cercospora Leaf Spot (Cercospora beticola) on Sugar Beets (Beta vulgaris)" Agriculture 14, no. 11: 2017. https://doi.org/10.3390/agriculture14112017

APA Style

Kiniec, A., Spychalski, M., Miziniak, W., Palacz, M., & Kukawka, R. (2024). The Use of Thyme (Thymus vulgaris) Essential Oil for Controlling Cercospora Leaf Spot (Cercospora beticola) on Sugar Beets (Beta vulgaris). Agriculture, 14(11), 2017. https://doi.org/10.3390/agriculture14112017

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