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Article

Valorization of Mediterranean Species of Thyme for the Formulation of Bio-Herbicides

by
Rym Boukhalfa
1,
Claudia Ruta
1,*,
Saida Messgo-Moumene
2,
Generosa J. Calabrese
3,
Maria Pia Argentieri
4 and
Giuseppe De Mastro
1
1
Department of Soil, Plant and Food Sciences (DiSSPA), University of Bari Aldo Moro, 70126 Bari, Italy
2
Department of Biotechnologies, University Saad Dahleb, Blida 09101, Algeria
3
International Centre for Advanced Mediterranean Agronomic Studies-Mediterranean Agronomic Institute of Bari (CIHEAM-IAMB), 70010 Bari, Italy
4
Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, 70125 Bari, Italy
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(9), 2077; https://doi.org/10.3390/agronomy14092077
Submission received: 23 July 2024 / Revised: 4 September 2024 / Accepted: 9 September 2024 / Published: 11 September 2024
(This article belongs to the Special Issue Agro-Environmental Sustainable Exploitation of Halophyte, Medicinal and Aromatic Species from Marginal Areas)
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Abstract

:
This study focused on the evaluation of the phytotoxic activity of four essential oils (EOs) from the Mediterranean species of Thymus sp. pl., namely Thymus algeriensis Boiss. et Reut., T. ciliatus Desf. subspecies coloratus (Boiss. et Reut.) Batt., T. vulgaris L. ecotype Fasano and T. vulgaris cultivar L. Varico 3, to identify new biomolecules with herbicide potential. The chemical characterization of EOs was performed by GC-MS. The evaluation of the phytotoxicity of the EOs was conducted under in vitro conditions, and the inhibition of germination and seedling growth of Lolium perenne L. and Amaranthus retroflexus L. were assessed. Five concentrations (100, 250, 500, 750 and 1000 µL/100 mL) were considered. Phytochemical analysis revealed a great diversity of compounds. T. algeriensis and T. ciliatus EOs were characterized by the absence of carvacrol and a low content of thymol in T. ciliatus. On the contrary, T. vulgaris ecotype Fasano and T. vulgaris cultivar Varico 3 were characterized by an important content of p-cymene, thymol and carvacrol. All the EOs expressed a potent phytotoxic activity against the tested species. The total inhibition of seed germination and seedling growth were recorded for the highest concentrations of all the EOs. T. vulgaris ecotype Fasano expressed the most effective activity.

1. Introduction

Today, food security, undermined by a fast-growing human population, is the foremost challenge for governments, policymakers and producers. To increase yields and improve food quality, crop production worldwide is consequently still based on massive chemical inputs. In 2021, 3.53 million tons of pesticides were used, and herbicide consumption reached 1.73 million tons, representing almost half of the total pesticide use [1]. Indeed, weeds are the most damaging category for crop production. They compete for resources such as light, water and nutrients and act as a reservoir of plant pathogens, pests and disease vectors. Therefore, they are considered a major abiotic and biotic cause of yield losses and crop quality depreciation [2,3,4].
Although several control methods are available, including physical, mechanical and biological approaches, the most common is still the chemical approach, based on the use of herbicides [5]. The overuse of these herbicides has caused negative impacts on human health and the environment [6,7,8]. Moreover, synthetic chemicals affect both soil organic matter and microbial community composition by decreasing soil bacterial diversity and abundance, especially for rapidly growing species because they can be immobilized in the soil by adsorption or binding to colloids [9,10].
Furthermore, the continuous use of the same molecules, combined with transgenic crops tolerant to herbicides, has contributed to developing increasing herbicidal resistances in many weeds and farmland biodiversity depletion [6,7]. In 2024, 272 herbicide-resistant weed species had been reported in 100 different crops, affecting the efficacy of 168 different herbicides and 21 of the 31 known modes of action (MOAs) [11].
To overcome this issue, several studies have explored new sources of active molecules, which can be used in weed control. These studies have demonstrated that plants, through the release of volatile organic compounds, can alter the physiological processes of neighboring species and affect plant community composition [12]. In this context, plant-based products with herbicidal activity, such as essential oils (EOs), could be an alternative to chemical herbicides due to their allelopathic properties and easy degradation in the environment [8,13,14]. Among bio-herbicides, EOs could be promising candidates due to their high content of monoterpenes, sesquiterpenes and diterpenes, which have important phytotoxic effects, volatility and biodegradability [15].
Within the Lamiaceae family, several genera showed interesting potential in terms of molecules with biocidal action. Among these genera, Thymus is certainly one of the most important with more than 250 taxa, for which the effectiveness of their EOs and terpenes have been demonstrated to have biological activity, especially antimicrobial properties. Understanding the relationship between the structures of these compounds and their various biological activities is crucial. In an agro-ecological context, it is of great interest to study the potential bioherbicidal activity of certain Thymus species to analyze and valorize their biodiversity as an alternative to chemical herbicides with a view to sustainable agriculture. Some thyme species have already been considered for their herbicidal properties in previous research. However, few aspects have been studied, and none of the previous studies have performed large-scale screening [16,17]. Therefore, this genus deserves renewed consideration and further investigations.
Based on the important chemotype variability of Thymus species detected in different environments in Algeria and Italy, four species were identified as deserving further attention: two Algerian endemic species, T. algeriensis Boiss. et Reut., T. ciliatus Desf. subsp. coloratus (Boiss. et Reut.) Batt.; an Italian landrace, T. vulgaris L. ecotype Fasano; and a Swiss commercial hybrid, T. vulgaris L. cultivar Varico 3, cultivated in Italy. In this context, our study aims to perform a preliminary analysis of several thyme species to investigate potential allelopathic activity that could be exploited for sustainable management of weeds. The experiments were conducted under in vitro conditions using two common weed test species.

2. Materials and Methods

2.1. Plant Material and EOs Extraction and Characterization

Aerial part of plant samples of Thymus species, namely T. algeriensis Boiss. et Reut.; T. ciliatus Desf. subsp. coloratus (Boiss. et Reut.) Batt.; T. vulgaris L. cultivar Varico 3 and T. vulgaris L. ecotype Fasano, were collected at the full flowering stage during May and June 2022 (Table 1). T. algeriensis Boiss. et Reut. and T. ciliatus Desf. subsp. coloratus (Boiss. et Reut.) Batt were identified in the Hamma Botanical Garden (Algiers, Algeria) based on the Quezel and Santa (1963) classification. Botanical vouchers were deposited at the herbarium of the garden. T. vulgaris L. ecotype Fasano was identified in the Botanical Garden Museum University of Bari Aldo Moro (Italy) and maintained ex situ (field collection) at the experimental farm “Enrico Pantanelli” of the University of Bari A. Moro, located in Policoro (southern Italy, 40°10′20″ N, 16°39′04″). Furthermore, the T. vulgaris cultivar Varico 3 is a hybrid that was obtained in 2000 by crossing two accessions from the Agroscope Changins-Wädenswil Research Station (Agroscope ACW—Switzerland) breeding material.
Dried samples were submitted to hydrodistillation for three hours, using a Clevenger-type apparatus, according to the European Pharmacopeia 2022 (https://www.edqm.eu/en/european-pharmacopoeia-ph.-eur.-11th-edition#-, accessed on 24 May 2022). The essential oils (EOs) obtained were yielded and stored at 4 °C until their use.
Samples of the Thymus EOs were analyzed with a trace GC-FID Ultra Thermo Finnigan gas chromatograph equipped with an Agilent DB-5 (J&W Scientific, Milan, Italy) fused silica capillary column (30 m × 0.25 mm; 0.25 µm film thickness). Adopted analytical conditions were as follows: detector temperature was 300 °C; the column temperature was programmed from 60 °C (5 min isothermal) to 280 °C (30 min isothermal) at 4 °C/min. Hydrogen was the carrier gas (35 kP; 2.0 mL/min). Data were processed using a Chrom-Card 32-bit version 2.0 computing software. Analyses were run in the cold on-column mode.
GC-MS analyses were performed with a Hewlett Packard 6890 (MSD)-5973 (GC) GC-MS System interfaced with an HP Chemstation (Agilent, Scientific Instruments, Milan, Italy). The following analytical parameters were used: column oven program of 60 °C (5 min iso-thermal) to 240 °C (15 min isothermal) at 3 °C/min; injector, 280 °C. Helium was the carrier gas (flow rate, 1 mL·min−1). Chromatographic separation was performed with an HP-5 MS capillary column (30 m × 0.25 mm; 0.25 µm film thickness). MS operating conditions were as follows: ion source, 70 eV; ion source temperature, 200 °C; mass spectra acquisition, over 40–800 amu range at 1 scan·s−1. The ion source was operating in electron impact mode. Samples (1 µL) were injected using the splitless sampling technique.
The chemical composition of the analyzed EOs was achieved by a comparison of GC retention times of their constituents with authentic reference compounds in combinations with the Kovats index (KI) and by means of reference mass spectra from standard compounds and/or from library files [18,19].
KI values were calculated using an n-alkane series (C6–C32) under the same GC conditions as that for the samples. The relative amounts of individual components of the oil were expressed as percent peak area relative to the total peak area from the GC-FID analysis of the whole extracts without the use of correction factors. A linear proportion between the areas was used, assuming an equal response factor for all detected compounds.

2.2. Phytotoxicity of the EOs against Target Weeds

Two weed species, Amaranthus retroflexus L. and Lolium perenne L., were used to test in vitro the phytotoxic effect of the EOs on germination and early growth conditions. Seeds were purchased from Weberseeds Company and stored at 4 °C for use during the experiment.
Seeds were surface-sterilized with sodium hypochlorite (NaClO, 2%—v/v) for 2 min and rinsed three times with sterile distilled water [20]. The viability of the seeds was assessed by the germination test of 100 seeds in Petri dishes fitted with two layers of Whatman filter paper wetted with distilled water. Petri dishes were sealed immediately with parafilm and placed in a controlled growth chamber (24 ± 1 °C with adjusted light conditions of 16/8 h light/dark cycle).
Subsequently, the phytotoxicity of Thymus EOs against the weed test species was estimated in dose–response laboratory bioassays on seed germination and seedling early growth. Suspensions of T1, T2, T3 and T4 were prepared using 2% of Tween® 20 (Sigma-Aldrich, Milan, Italy) as an emulsifying agent, according to Abd-El Gawad et al. [21]. Briefly, five concentrations (100, 250, 500, 750 and 1000 µL/100 mL) of EOs were prepared by diluting the EOs in 2% of Tween 20, and then the volumes were adjusted to 100 mL with sterilized distilled water. A total of 2% of Tween® 20 solution was considered as negative control, while two pelargonic acid-based bio-herbicides (Finalsan Ultima 18.9% w/w pelargonic acid and Vithal 51.9% w/w pelargonic acid) were considered as positive controls. The concentrations of the positive controls were prepared as indicated on the labels of the products (1 L of Finalsan Ultima per 5 L of water and 2.2 L of Vithal per 5 L of water).

2.2.1. Germination Test

Ten seeds of A. retroflexus and L. perenne were placed in three Petri dishes for each EO and concentration, previously fitted with two layers of Whatman filter paper wetted with 3 mL of each treatment. Petri dishes were immediately sealed with parafilm to prevent loss of moisture and oil volatilization and placed in a controlled growth chamber at 24 ± 1 °C with adjusted light conditions of 16/8 h light/dark cycle and light: LEDs with a broad spectrum between 400 and 700 nm, a white/pink ratio of 2:1 and a light intensity of 50 µm s−1 m−2. Trials were conducted in a completely randomized design in triplicate. After 7 days, the number of germinated seeds was counted. Seeds showing radicles of more than 2 mm were considered germinated [22,23]. The results are expressed in a percentage of germinated seeds, determined as follows:
GP (%) = (n)/(N) × 100
where GP: Germination percent; n: Number of germinated seeds at final count; N: Total number of seeds [24].

2.2.2. Seedling Early Growth Test

Seeds of both target species were germinated in three Petri dishes (150 mm in diameter) for each EO and concentration. All the Petri dishes were fitted with two layers of filter paper wetted with distilled water and placed in a controlled growth chamber at the seeds’ optimal growth conditions for four days. Afterward, ten A. retroflexus and L. perenne seedlings were placed in 90 mm Petri dishes previously fitted with two layers of Whatman filter paper wetted with 3 mL of EO suspension at different concentrations. The Petri dishes were immediately sealed with parafilm to prevent loss of moisture and oil volatilization and placed in a controlled growth chamber at 24 ± 1 °C with adjusted light conditions of 16/8 h light/dark cycle and light: LEDs with a broad spectrum between 400 and 700 nm, a white/pink ratio of 2:1 and a light intensity of 50 µm s−1 m−2. Trials were conducted in a completely randomized design in triplicate. After 7 days, shoot and radical lengths were measured using a ruler and caliper.

2.3. Statistical Analysis

Data obtained were statistically analyzed using Minitab® version 19.2020.1 (Minitab software, State College, PA, USA). Analysis of the variance (one-way ANOVA) of the seed germination percentage and root and shoot growth was performed to assess the phytotoxicity of the treatments. Differences among means was run using Tukey’s test. Statistical significance was accepted when the probability of the result, assuming the null hypothesis, (p) was less than 0.05 (p < 0.05) [25].

3. Results and Discussion

3.1. Chemical Characterization of the EOs

The hydrodistillation of the aerial part of the samples resulted in different essential oil (EO) yields of the studied Thymus species, which ranged from 0.43% to 1.10% (v/w). The highest yield was observed in T. ciliatus subsp. coloratus (1.05%) and T. vulgaris cv Varico 3 (1.10%), while the lowest ones were found in T. algeriensis (0.43%) and T. vulgaris ecotype Fasano (0.60%) samples.
Gas chromatography analysis allowed for the identification of 43 components, representing 91.2–98.9% of the oils, with the remaining components present only in trace amounts (<0.1%). The percentage of the composition and indexes of the components are listed in Table 2. Based on the dominant compound of each EO, three different types were identified: α-pinene for T. algeriensis, linalool for T. ciliatus subsp. coloratus and p-cymene for T. vulgaris cv Varico 3 and T. vulgaris ecotype Fasano. The chemical analysis of the T. algeriensis EO revealed the dominance mainly of α-pinene (19.73%), α-terpenyl acetate (13.21%), borneol (11.31%), camphene (8.64%), isobornyl acetate (5.28%), β-pinene (4.37%), camphor (4.32%) and linalool (3.09%), while the T. ciliatus EO was characterized by an important content of linalool (93.06%), β-cedrene (1.98%) and limonene (1.36%). The chemical analysis of T. algeriensis (T1) and T. ciliatus subsp. coloratus (T2) EOs revealed the absence of carvacrol in both samples and a low content of thymol in T. ciliatus (0.55%).
Overall, these results are in agreement with other studies on Thymus species. Similar results for T. algeriensis were reported by [26,27,28]. They pointed out that the phytochemical profiles of T. algeriensis harvested from Chrea National Park at the same altitude and Medea (north-central Algeria) were characterized by the predominance of terpinyl acetate (18.0%), nerolidol (12.6%), geranyl acetate (16.4%), α-pinene (11.1–27.1%), borneol (9.0%), bornyl acetate (7.7%) and in some samples, the absence of thymol. In contrast, [26] observed that a sample collected in Chrea National Park at 800 m altitude was characterized by thymol as the predominant component. Great variability was also found in T. algeriensis populations from the central-northern, central-southern and eastern regions of the country [29,30,31,32]. These results confirm the important role that environmental conditions play in the synthesis of different essential oil compounds.
Concerning T. ciliatus subsp. coloratus, our results indicated that the chemical profile of the EOs of the species growing in Hammam Melouane differed significantly from what has been reported in the literature. Indeed, our findings identified linalool as the major compound (93.06%) for this species for the first time. Generally, the North Algerian T. ciliatus EO is characterized by the predominance of thymol, carvacrol, p-cymene and γ-terpinene [33,34,35].
T. vulgaris cv Varico 3 and T. vulgaris ecotype Fasano (T3 and T4) have similar chemical compositions, characterized by p-cymene (35.63% and 23.85%, respectively), thymol (20.35% and 21.77%, respectively), carvacrol (11.76% and 18.15%, respectively), α-cedrene (7.69% and 6.56%, respectively), caryophyllene oxide (5.57 and 2.09%, respectively), γ-terpinene (2.65% and 10.36%, respectively) and 1,8 cineol (2.35% and 3.30%, respectively) as major components.
T. vulgaris is the most studied Thymus species in terms of phytochemistry. Since 2000, more than one hundred articles have been published, but as far as we know and according to the literature, few studies have focused on the T. vulgaris cultivar Varico 3 and none on T. vulgaris ecotype Fasano. Regarding the T. vulgaris cultivar Varico 3, [36] obtained different results than ours. In their study on the chemical composition of the volatile oil and genetic fingerprint of ten T. vulgaris clones, they found that T. vulgaris Varico 3, cultivated in Switzerland, was rich in thymol (75.44%), with p-cymene as the second most abundant compound (8.14%). Previously, [37] revealed that thymol is the main compound (60.2%), followed by p-cymene (19.9%) and carvacrol (10.3%) for this species cultivated in the Czech Republic. Generally, the EOs of T. vulgaris can have different chemotypes depending on the main compounds. Our results showed that the main compounds of both EOs are p-cymene, thymol and carvacrol, suggesting that both belong to the p-cymene chemotype. These results are in accordance with those found by [38] in their cluster analysis on the compositions of 85 T. vulgaris EOs. In addition to the already-established seven chemotypes (thymol, carvacrol, linalool, geraniol, thujanol-4, terpineol and 1,8-cineole) [39], they recorded p-cymene/thymol as the second most common chemotype represented by 18 samples, even though the most dominant chemotype reported by the literature remains the “thymol chemotype”.
It is important to note that, apart from the main component that defines the chemotype of the species, some compounds are predictable, as they are metabolically related. For thymol and carvacrol chemotypes, p-cymene and γ-terpinene are expected to be present in considerable amounts, as they are their precursors [40]. Indeed, the metabolic pathway for the formation of thymol and carvacrol starts with the autoxidation of γ-terpinene to p-cymene, followed by hydroxylation to thymol [41].
The great variability and diversity of compounds observed in the chemical composition of Thymus species EOs could be attributed to several factors, such as response mechanisms to abiotic variations. In essence, environmental conditions can affect biosynthetic pathways [29,35,42,43].

3.2. Phytotoxicity of the EOs against Target Weeds

3.2.1. Phytotoxic Effect on Seed Germination

The phytotoxic effect of the different EOs extracted from aerial parts of thyme species on seed germination of L. perenne and A. retroflexus is summarized in Figure 1. All the treatments prevented seed germination of both species where their influence varied significantly depending on the EO and concentration. Seven days after treatment, T1 and T4 completely inhibited seed germination at 500, 750 and 1000 μL in a similar way to the positive controls. T1 and T4 also reduced seed germination at 100 μL; however, this reduction was not significant compared to the negative control. T3 completely suppressed seed germination at 750 and 1000 μL and reduced it considerably at 500 μL (83% less than negative control). In contrast, at 100 and 250 μL, the seed germination percentage was close to that of the negative control. T2 showed the least significant effect, suppressing seed germination only at 1000 μL. Germination percentages were almost similar to the negative control at 100, 250, and 500 μL. T4 exerted the most potent activity against the treated species (Figure 1a).
A similar pattern was observed for A. retroflexus. T4 completely inhibited seed germination at 250, 500, 750 and 1000 μL and reduced it by almost half at 100 μL. T3 showed strong effectiveness at 500, 750 and 1000 μL, extremely reduced seed germination at 250 μL and reduced it by half at 100 μL. T1 completely inhibited seed germination at 750 and 1000 μL and reduced it by almost 20% and 13% at 250 and 500 μL, respectively. T2 was the least effective treatment, inhibiting seed germination only at the highest concentration (1000 μL) and slowing it down at the other concentrations. As with L. perenne, it was noticeable that T4 exerted the strongest phytotoxic effect against A. retroflexus by totally inhibiting seed germination at four concentrations (Figure 1b).
Overall, all the EOs exhibited significant phytotoxic activity against the tested weed species. T4 was the most phytotoxic treatment, while T2 was the least effective.

3.2.2. Phytotoxic Effect on Seedlings’ Early Growth

The allelopathic effect of the EOs extracted from aerial parts of thyme species on L. perenne L. and A. retroflexus L. root and shoot growth is summarized in Figure 2. The results demonstrated a strong inhibitory effect of the EOs on the growth of the target plants. Root and shoot elongation was strongly inhibited in a dose-dependent manner. For L. perenne, T1, T2, T3 and T4 showed potent toxicity on root growth at all four concentrations (250, 500, 750 and 1000 μL), with T4 having a particularly strong effect, reducing root elongation by about 30 mm and 45 mm compared to the mean root elongation of the other treatments and the negative control, respectively, even at 100 μL (Figure 2a).
The same effect was recorded for A. retroflexus. All the EOs negatively affected root growth. They significantly reduced it at 750 and 1000 μL and decreased it by half at 250 and 500 μL. At 100 μL, no major effect was recorded. At the lower concentration, the effect was almost the same as that of the negative controls (Figure 2b).
For L. perenne, shoot growth responded to the application of EOs in a quite similar manner to root growth. T4 completely inhibited shoot growth at 750 and 1000 μL comparable to the positive controls and greatly reduced it at 500, 250 and 100 μL. In particular, the reduction in shoot growth was 25 mm less than the average of the other treatments at 250 μL and 30 mm for 100 μL. T1 also inhibited shoot elongation at 750 and 1000 μL and considerably reduced it at 500 and 250 μL, while at 100 μL, the effect was almost similar to that of the negative control. T3 prevented shoot growth only at 1000 μL but significantly slowed it down at 250, 500 and 750 μL. T2 was the least effective treatment compared to the other three. It did not prevent shoot growth; however, it significantly decreased it at 500, 750 and 1000 μL in comparison to the negative control with decreases of about 36, 39 and 47 mm, respectively (Figure 3).
It is noteworthy that all the EO-based treatments completely prevented the shoot growth of A. retroflexus, and T4 was the most effective in inhibiting or slowing down root and shoot growth of both target species in a dose-dependent manner.
The results obtained from the in vitro trials confirmed the phytotoxic effect of the studied Thymus EOs. All treatments inhibited seed germination and seedlings’ growth of the target weed species at higher concentrations, with T4 being effective even at lower concentrations. These results are highly consistent with those reported in the literature. Ref. [44] established that the T. vulgaris EO possessed potent activity against seven tested plants, negatively influencing their root growth and completely inhibiting their seed germination. Ref. [45] also revealed that the EO of T. algeriensis inhibited both shoot and root growth of Medicago sativa L. and of Triticum aestivum L. seedlings. To the best of our knowledge, no studies have been reported on the phytotoxic effect of T. vulgaris cultivar Varico 3, T. vulgaris ecotype Fasano and T. ciliatus subsp. coloratus. Nevertheless, the allelopathic activity of various Thymus sp. pl. EOs and their constituents has been reported to affect seed germination and plant growth of several weed species, thereby influencing plant community composition and dynamics [46]. Studies on EOs of T. pulegioides L., T. proximus Serg., T. vulgaris, T. kotschyanus Boiss. & Hohen., T. decussatus Benth., T. fontanesii Boiss. et Reut. T. eigii and T. daenensis Celak. revealed significant and strong phytotoxic potential against a wide range of weed species, such as Amaranthus retroflexus L., Poa anuua L., Sinapis arvensis L. and Avena fatua L. [16,17,43,47,48,49,50].
To explain this biological activity, research confirmed that this potential is due to the presence of terpenic compounds, most of which are volatile [51,52]. In fact, [21] established a structure–activity relationship between phytotoxicity and EO chemical composition based on chemometric analysis. Data analysis revealed that mono- and sesquiterpenes play major roles in the phytotoxicity of EOs, concluding that α- and β-pinene, 1,8 cineole, linalool and carvacrol are the most effective monoterpenes exhibiting an allelopathic effect on many plants. Additionally, caryophyllene, a sesquiterpene compound, and its metabolites, such as germacrene, spathulenol and hexahydrofarnesyl acetone, play an important role in the phytotoxic potential of EOs. This was also reported by [21], where monoterpenes like linalool and 1,8 cineole delayed weed germination time and reduced their development. This is in accordance with our results, where T. algeriensis, T. vulgaris cultivar Varico 3 and T. vulgaris ecotype Fasano, which are rich in α-pinene, p-cymene, thymol and carvacrol, respectively, exhibited the most significant activity.
In addition, it was assumed that the biological properties of EOs are generally induced mainly by their major components. However, recent research has demonstrated that compounds present in minor and trace amounts can also influence plant defense strategies and could act synergistically to enhance the biological potential of EOs [53]. T. ciliatus was the least effective treatment, even though it contained more than 90% of linalool. Furthermore, our EOs were generally rich in certain compounds, such as α-pinene, borneol, linalool, thymol, carvacrol and caryophyllene, but their chemical compositions were particularly different. T. algeriensis and T. ciliatus did not contain thymol and carvacrol, two compounds commonly well known for their strong allelopathic activity, or contained these only in trace amounts. This supports the view that there was a synergy between the major and minor compounds of the examined EOs that affected the germination and early growth of L. perenne and A. retroflexus.

4. Conclusions

The object of this study was to exploit plant sources to obtain active ingredients for use in crop protection, particularly for weed control. With this aim, a preliminary assessment of the phytotoxic potential of some Mediterranean Thymus sp. pl. EOs was performed, and the presence of bioactive compounds, potentially useful for weed management, was evaluated. The chemical analysis demonstrated the high variability of secondary metabolites in Thymus sp. pl essential oils (EOs). Forty-three chemical compounds were identified. The Algerian species were characterized by the absence of carvacrol and traces of thymol in T. ciliatus subsp. coloratus and the predominance of linalool, α and β-pinene, α-terpenyl acetate, borneol and camphene, while p-cymene, thymol, carvacrol and γ-terpinene were found as the main constituents of T. vulgaris species. Biologically, all the EOs demonstrated a significant phytotoxic effect on L. perenne and A. retroflexus. Thyme EOs strongly inhibited the seed germination and seedling growth of both weed species. T. algeriensis Boiss. et Reut., T. vulgaris cultivar Varico 3 and T. vulgaris ecotype Fasano completely inhibited the seed germination of both test species at 500, 750 and 1000 µL/100 mL, while T. ciliatus subsp. coloratus considerably reduced it at 500 and 750 µL/100 mL and completely suppressed it at 1000 µL/100 mL. The seedling growth of L. perenne and A. retroflexus was drastically slowed down under the effect of thyme EOs in a dose-dependent manner; at 1000 µL/100 mL, all the EOs almost inhibited the seedling growth. Overall, T. vulgaris ecotype Fasano exhibited the most potent phytotoxic activity, while T. ciliatus subsp. coloratus was the least effective.
This initial screening of species of genus Thymus endowed with biologically active compounds represents the first stage in a process of introducing into cultivation certain species that are currently under-utilized but have the potential to become industrial crops. The high cost of the natural product could thus be reduced in favor of a more efficient and less costly method of obtaining the raw material. All of this is perfectly in line with sustainability goals (Agenda 2030), including the replacement of synthetic compounds with plant-based compounds in agriculture, as in other contexts.
Further studies and applicative investigations on the effectiveness, selectivity, modes of action and application of thyme EOs under in vivo conditions, as well as their effects on crops and soil, are necessary to develop ongoing, tailored strategies for more sustainable weed control.

Author Contributions

Conceptualization, G.D.M., C.R. and S.M.-M.; data curation, R.B., S.M.-M., M.P.A., C.R. and G.D.M.; investigation, R.B., S.M.-M., M.P.A., C.R. and G.D.M.; project administration, G.D.M. and G.J.C.; supervision, R.B., S.M.-M., G.J.C., M.P.A., C.R. and G.D.M.; validation R.B., S.M.-M., M.P.A., C.R. and G.D.M.; writing—original draft preparation, R.B. and S.M.-M.; writing—review and editing, R.B., S.M.-M., G.J.C., M.P.A., C.R. and G.D.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was carried out within the Agritech National Research Center and received funding from the European Union Next-GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)—MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4—D.D. 1032 17/06/2022, CN00000022). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them.

Data Availability Statement

The original contributions presented in this study are included in the article, and further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Agronomy 14 02077 g001
Figure 1. Effect of the different treatments and doses on seed germination of L. perenne (a) and A. retrofelxus (b) after 7 days of exposure to a 100–1000 μL/100 mL range of concentrations of the four tested EOs. T1: T. algeriensis, T2: T. ciliatus, T3: T. vulgaris cultivar Varico 3, T4: T. vulgaris ecotype Fasano. Data are means of three replicates. At each dose, data followed by different letters are significantly different (p ≤ 0.05, Tukey’s test).
Figure 1. Effect of the different treatments and doses on seed germination of L. perenne (a) and A. retrofelxus (b) after 7 days of exposure to a 100–1000 μL/100 mL range of concentrations of the four tested EOs. T1: T. algeriensis, T2: T. ciliatus, T3: T. vulgaris cultivar Varico 3, T4: T. vulgaris ecotype Fasano. Data are means of three replicates. At each dose, data followed by different letters are significantly different (p ≤ 0.05, Tukey’s test).
Agronomy 14 02077 g001
Agronomy 14 02077 g002
Figure 2. Effect of the different treatments and doses on root elongation of L. perenne (a) and A. retrofelxus (b) seedlings after 7 days of exposure to a 100–1000 μL/100 mL range of concentrations of the four tested EOs and active compounds. T1: T. algeriensis, T2: T. ciliatus, T3: T. vulgaris cultivar Varico 3, T4: T. vulgaris ecotype Fasano. Data are means of three replicates. At each dose, data followed by different letters are significantly different (p ≤ 0.05, Tukey’s test).
Figure 2. Effect of the different treatments and doses on root elongation of L. perenne (a) and A. retrofelxus (b) seedlings after 7 days of exposure to a 100–1000 μL/100 mL range of concentrations of the four tested EOs and active compounds. T1: T. algeriensis, T2: T. ciliatus, T3: T. vulgaris cultivar Varico 3, T4: T. vulgaris ecotype Fasano. Data are means of three replicates. At each dose, data followed by different letters are significantly different (p ≤ 0.05, Tukey’s test).
Agronomy 14 02077 g002
Agronomy 14 02077 g003
Figure 3. Effect of the different treatments and doses on shoot elongation of L. perenne seedlings after 7 days of exposure to a 100–1000 μL/100 mL range of concentrations of the four tested EOs and active compounds. T1: T. algeriensis, T2: T. ciliatus, T3: T. vulgaris cultivar Varico 3, T4: T. vulgaris ecotype Fasano. Data are means of three replicates. At each dose, data followed by different letters are significantly different (p ≤ 0.05, Tukey’s test).
Figure 3. Effect of the different treatments and doses on shoot elongation of L. perenne seedlings after 7 days of exposure to a 100–1000 μL/100 mL range of concentrations of the four tested EOs and active compounds. T1: T. algeriensis, T2: T. ciliatus, T3: T. vulgaris cultivar Varico 3, T4: T. vulgaris ecotype Fasano. Data are means of three replicates. At each dose, data followed by different letters are significantly different (p ≤ 0.05, Tukey’s test).
Agronomy 14 02077 g003
Table 1. Data on the plant species used in the study.
Table 1. Data on the plant species used in the study.
SpeciesHabitatCodeCollecting AreaGeographical LocalizationAltitude
T. algeriensis Boiss. et Reut.Endemic wild speciesT1Chrea National Park, Blida (Algeria)N 36°27′08.5″
E 002°54′47.2″		1465 m
T. ciliatus Desf. subsp. coloratus (Boiss. et Reut.) Batt.Endemic wild speciesT2Hammam Melouane-Chrea National Park, Blida (Algeria)N 36°28′12″
E 003°00′36″		211 m
T. vulgaris L. cultivar Varico 3Cultivated hybridT3Policoro, Basilicata
(Italy)		40°10′28″ N
16°39′26″ E		15 m
T. vulgaris L. ecotype FasanoCultivated local speciesT4
Table 2. Chemical composition of the studied Thymus EOs.
Table 2. Chemical composition of the studied Thymus EOs.
N°CompoundKI 1KI 2T1T2T3T4
01santolina triene9089100.36------
02trycyclene9269240.47------
03α-thujene926924--0.200.480.66
04α-pinene93694019.730.211.031.15
05camphene9549508.640.290.880.93
06verbenene9679680.34--0.05--
07sabinene9759750.870.05----
08β-pinene9799804.370.140.21--
09myrcene9919922.770.360.521.35
10α-terpinene101710120.250.050.130.24
11p-cymene102210210.300.1335.6323.85
12limonene102910261.951.360.791.33
131,8 cineole103110282.990.163.533.30
14β(E)-ocymene105010471.170.07----
15γ-terpinene105910580.540.982.3510.36
16cis-sabinene hydrate107010670.450.10----
17terpinolene108810880.47--0.480.17
18linalool109611033.0993.062.572.54
191-octen-3γlacetate111011160.43------
20α-campholenal112511250.99------
21camphor114311424.320.381.661.20
22trans-verbenol114411452.61------
23pinocarvone116211580.56------
24borneol1165116611.31--1.471.22
25ρ-mentha-1,5 dien-8-ol117011741.72--1.761.65
26terpin-4-ol117711820.76------
27α-terpineol118911870.96------
28myrtenal119311900.90------
29verbenone120412150.73------
30isobonyl formate123312430.33------
31thymol methyl ester12351234----1.971.48
32linalool acetate125712623.96------
33isobornyl acetate128512895.28------
34thymol12901293--0.5520.3521.77
35carvacrol12981300----11.7618.15
36trans-carvyl acetate132813370.11------
37α-terpenyl acetate1350135313.21------
38α-copaene137613700.49------
39β-bourbonene138413760.22------
40β-cedrene141814040.511.987.696.56
41germacrene148014751.58------
42Δ-cadinene152415300.26------
43caryophyllene oxide15811573----5.572.09
Monoterpene hydrocarbons 42.233.7742.8540.04
Oxygen-containing monoterpenes 54.7194.1843.8951.31
Sesquiterpene hydrocarbons 3.061.987.696.56
Oxygen-containing sesquiterpenes --5.572.09
Others ----
KI: Kovats index; 1: the literature; 2: calculated.
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Boukhalfa, R.; Ruta, C.; Messgo-Moumene, S.; Calabrese, G.J.; Argentieri, M.P.; De Mastro, G. Valorization of Mediterranean Species of Thyme for the Formulation of Bio-Herbicides. Agronomy 2024, 14, 2077. https://doi.org/10.3390/agronomy14092077

AMA Style

Boukhalfa R, Ruta C, Messgo-Moumene S, Calabrese GJ, Argentieri MP, De Mastro G. Valorization of Mediterranean Species of Thyme for the Formulation of Bio-Herbicides. Agronomy. 2024; 14(9):2077. https://doi.org/10.3390/agronomy14092077

Chicago/Turabian Style

Boukhalfa, Rym, Claudia Ruta, Saida Messgo-Moumene, Generosa J. Calabrese, Maria Pia Argentieri, and Giuseppe De Mastro. 2024. "Valorization of Mediterranean Species of Thyme for the Formulation of Bio-Herbicides" Agronomy 14, no. 9: 2077. https://doi.org/10.3390/agronomy14092077

APA Style

Boukhalfa, R., Ruta, C., Messgo-Moumene, S., Calabrese, G. J., Argentieri, M. P., & De Mastro, G. (2024). Valorization of Mediterranean Species of Thyme for the Formulation of Bio-Herbicides. Agronomy, 14(9), 2077. https://doi.org/10.3390/agronomy14092077

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