Allelopathic Effect of Salvia pratensis L. on Germination and Growth of Crops

Abstract

Salvia pratensis L. is a valuable medicinal plant rich in bioactive compounds, yet its allelopathic potential remains underexplored. This study evaluated allelopathic effects and total phenolic (TPC) and flavonoid (TFC) contents of water extracts from the dry aboveground biomass of S. pratensis. To assess their selectivity and potential application in sustainable weed management, extracts at five different concentrations were tested on the germination and early growth of lettuce, radish, tomato, and carrot. The results demonstrated that the phytotoxic effects of S. pratensis extracts were both concentration- and species-dependent. Higher extract concentrations significantly inhibited germination and seedling growth, while lower concentrations of extracts stimulated shoot elongation by up to 30% compared to the control. Phytochemical analysis revealed that S. pratensis extracts contain notable TPC and TFC contents, with their concentrations increasing consistently with the extract concentration. Correlation analysis showed that higher TPC and TFC contents were strongly negatively correlated with germination and seedling growth parameters. Radish exhibited the highest sensitivity to the extracts, while lettuce was the most tolerant. Further research under field conditions is needed to assess the efficacy, selectivity, and practical potential of S. pratensis extracts in sustainable crop production systems.

1. Introduction

Allelopathy represents a biological phenomenon during which plants release chemical substances—allelochemicals—into the environment that can directly or indirectly affect the germination, growth, and development of other plant species, with these effects being either positive or negative [1,2]. Allelochemicals are synthesized in all parts of the plant, including roots, stems, leaves, flowers, and seeds, and are released into the environment through root exudates, leaching, volatilization, or the decomposition of plant residues [1,3]. Generally, allelochemicals are secondary metabolites not essential for primary plant metabolism, consisting of a variety of compounds such as phenolic acids, flavonoids, tannins, alkaloids, coumarins, and others [4,5,6]. Phenolic compounds represent one of the most significant classes of allelochemicals, capable of influencing seed germination and plant growth through their effects on enzyme activity, hormone synthesis, photosynthesis, respiration, nutrient cycling, and soil microbial dynamics [6].

In natural ecosystems, allelopathy plays a key role in plant species succession, biodiversity, and the distribution of plant species within communities, and can also facilitate the spread of invasive species into new habitats [7,8]. In agroecosystems, allelopathy emerges as a sustainable and eco-friendly alternative to the use of synthetic herbicides, whose widespread application leads to environmental pollution, development of herbicide-resistant weed populations, herbicides residues in soil, water, and agricultural products, as well as harmful effects on human health [2,9]. Plant species characterized by significant allelopathic potential can be utilized for weed management or to enhance crop growth—for example, as biostimulants. They can be incorporated into agricultural systems through crop rotation, cover cropping, intercropping, mulching, residue incorporation, green manure, the selection of allelopathic genotypes, or the application of plant-based extracts [2,10]. Recent studies have emphasized the value of aromatic and medicinal plants—particularly wild species—due to their rich content of secondary metabolites and essential oils with various ecological functions, making them the subject of increasing interest in natural herbicide research [11,12,13,14,15].

The family Lamiaceae includes a diverse group of aromatic plants, with more than 200 genera and 7000 species, and it is one of the most economically significant plant families. Notable genera include Salvia, Lavandula, Ocimum, Mentha, Thymus, Origanum, Rosmarinus, Hyssopus, and Satureja [16,17]. Beyond its economic value, many species of this family—particularly those from the Salvia genus—have been extensively researched for their allelopathic effect [11,14,17,18]. Common sage (Salvia officinalis L.) allelopathy has been particularly well documented, with studies reporting phytotoxicity of its residues and extracts [14,19,20,21], essential oils [12,18], and even aromatic waters (hydrosols) [22]. Sadeqifard et al. [14] screened 123 plant species for allelopathic potential and found Salvia ceratophylla L. to be the most effective, completely inhibiting both root and shoot growth (100%) of lettuce (Lactuca sativa L.), with several other Salvia species—Mediterranean sage (Salvia aethiopis L.), Salvia leriifolia Benth., Salvia macrosiphon Boiss., woodland sage (Salvia nemorosa L.), and clary sage (Salvia sclarea L.)—also showing strong inhibitory effects. Expanding upon allelopathic research in Salvia genus, this study targets meadow sage (Salvia pratensis L.).

Salvia pratensis is a perennial herbaceous plant, widely distributed across Europe and southwestern Asia. It thrives in warm, sunny locations, including semi-dry meadows, grasslands and pastures, as well as edges of woodland and ruderal habitats. The plant produces blue or dark-purple flowers, which bloom from May to August, and emits a strong, pleasant scent due to the presence of essential oils [23,24]. Salvia pratensis is valued for its medicinal, culinary, and cosmetic uses. Both its flowers and leaves are used in infusions to alleviate digestive and respiratory ailments, and as a spice to enhance the flavor of soups, meat or fish dishes, and to infuse vinegar. In addition, it is appreciated both as a honey plant and as an ornamental species [23,25]. The leaves and aboveground parts of S. pratensis contain essential oils ranging from 0.008% to 0.1%, with the main components being caryophyllene, thymol, and p-cymene [26,27]. The presence of varying content of phenolics and flavonoids was also reported, with rosmarinic acid, caffeic acid, salvianolic acid, chlorogenic acid, rutin, and luteolin as the major components [13,28,29].

Allelopathic and herbicidal potential of S. pratensis has been explored in a limited number of studies. Itani et al. [30] found that S. pratensis leaves caused reductions in lettuce root length up to 50%, while according to Gruľová et al. [29], the phytotoxicity of S. pratensis extracts depended on several factors. Previously, we recorded a considerable inhibitory effect of S. pratensis extracts, even at lower concentrations, on weed species velvetleaf (Abutilon theophrasti Medik.) and common corn-cockle (Agrostemma githago L.) [31]. This prompted us to further investigate the allelopathic potential of this species on crops, to assess whether its effects are harmful or potentially beneficial. Understanding the impact of S. pratensis extracts on crops is crucial for evaluating their selectivity and practical suitability in sustainable weed management strategies. Therefore, this study aimed to evaluate the allelopathic effects of various concentrations of S. pratensis water extracts on the germination and growth of four vegetable crops, as well as to determine their total phenolic and flavonoid contents. A better understanding of crop responses to S. pratensis extracts contributes to evaluating their potential role in future agricultural applications.

2. Materials and Methods

2.1. Plant Material Collection and Preparation of Water Extracts

The aboveground biomass of S. pratensis was collected during the full flowering stage (phenological stage 6/65 [32]) in July of 2022 from meadows in Osijek–Baranja County, Croatia. Healthy fresh plants without visible damage and signs of disease were selected, shade-dried for 72 h, and then oven-dried at 40 °C for an additional 72 h. Dried biomass was subsequently chopped and ground with electronic mill to fine powder that passed through a 1 mm sieve. The obtained powder was stored in paper bags in a cool, dark environment until further analysis.

The preparation of S. pratensis water extracts followed the procedure of Norsworthy [33], with slight modifications. A total of 100 g of dry plant biomass was extracted in 1000 mL of distilled water at room temperature (21 ± 2 °C) for 24 h. The mixture was filtered through cloth to remove debris, and after that twice through filter paper to obtain a 10% concentration extract. Further dilutions with distilled water were performed to obtain a total of five different concentrations (1%, 2.5%, 5%, 7.5%, and 10%) for the bioassay. All extracts were prepared a day before the experiment and stored at 4 °C in the refrigerator for the duration of the experiment to prevent microbial contamination and minimize potential degradation of bioactive compounds.

2.2. Petri Dish Bioassay

Allelopathic potential of S. pratensis water extracts was evaluated under laboratory conditions using a Petri dish bioassay on four different vegetable crops. The test species included lettuce (Lactuca sativa L. ‘Majska kraljica’), radish (Raphanus sativus L. ‘Saxa 2’), tomato (Solanum lycopersicum L. ‘Cuor di Bue’), and carrot (Daucus carota L. ‘Nantes’), all of which were commercially obtained. These crops were selected based on their agricultural relevance, distinct physiological characteristics, and varying potential sensitivity to allelochemicals, allowing for a comprehensive evaluation of species-specific responses to S. pratensis extracts. Prior to the experiment, seeds were surface-sterilized in 1% sodium hypochlorite (NaOCl) solution for 10 min and rinsed thoroughly three times with distilled water.

The experiment was conducted using completely randomized design with four replications per treatment. Each replicate consisted of a separate sterilized Petri dish (90 mm diameter) lined with a double layer of filter paper, containing 25 seeds (radish, tomato) or 30 seeds (lettuce, carrot) of the species, evenly distributed. The filter paper was moistened with 3 mL of water extract in each concentration. For the control treatment, the filter paper was moistened with 3 mL of distilled water, which served as the solvent control. On the fourth day of the experiment, an additional 2 mL of extract or distilled water was added to each Petri dish in order to maintain moisture and prevent the seedlings from drying out. The seeds of test species were incubated at 21 ± 2 °C (light/dark, 12 h/12 h) for six (lettuce), eight (radish), and eleven (tomato, carrot) days. The incubation period varied due to natural differences in germination rates among species, and each was monitored until reaching a comparable early seedling stage.

Total germination percentage for each test species and repetition was calculated as G = [(germinated seeds/total seeds) × 100]. Root and shoot length and the fresh weight of seedlings were measured on the final day of the experiment. Seedling vigor index (SVI) was calculated as SVI = [(seedling length (cm) × percentage of germinated seeds)/100] [34]. The shoot-to-root ratio was calculated as Shoot-to-root ratio = Ls/Lr, where Ls is the shoot length (cm), and Lr is the root length (cm). The overall allelopathic potential (OAP) was determined as OAP = [(IR_a + IR_b)/2]/100, where IR_a is the percent inhibition of germination or growth parameter at the lowest applied concentration compared to control, and IR_b is percent inhibition of germination or growth parameter at the highest applied concentration compared to control. The OAP score was categorized as follows: 0–0.25—non-allelopathic; 0.26–0.5—moderately allelopathic; 0.51–0.75—highly allelopathic; 0.76–1.0—extremely allelopathic [35].

2.3. Determination of Total Phenolic and Flavonoid Content

Total phenolic content (TPC) in S. pratensis extracts was determined at five different concentrations using the Folin–Ciocalteu method [36], with gallic acid serving as the standard. In brief, 100 µL of each diluted extract concentration was combined with 1500 µL of distilled water and 100 µL of the Folin–Ciocalteu reagent in a test tube. After a 5 min incubation, 300 µL of sodium carbonate (Na₂CO₃) solution was added, and the mixture was vortexed. The test tube was then incubated in the dark at 37 °C for 60 min. Afterward, absorbance was measured at 765 nm. The total phenolic content was calculated on the basis of the calibration curve of gallic acid and was expressed as gallic acid equivalents (GAE) in mg per mL of S. pratensis extract. All measurements were performed in four replicates.

Total flavonoid content (TFC) in S. pratensis extracts was measured at five different concentrations using the aluminum chloride colorimetric assay [37], with quercetin serving as a standard. Briefly, 200 µL of each extract concentration was mixed with 100 µL of 4% aluminum chloride (AlCl₃) solution and 1700 µL of 96% ethanol. The mixture was incubated in dark at room temperature for 60 minutes, after which the absorbance was measured at 415 nm. The total flavonoid content was calculated based on calibration curve of quercetin and expressed as quercetin equivalents (QE) in mg per ml of S. pratensis extract. All measurements were performed in four replicates.

2.4. Statistical Analysis

The collected data was analyzed statistically using one-way ANOVA, and differences between treatment means of measured parameters were tested with the LSD test at a significance level of p < 0.05. The assumption of homogeneity of variance was tested using Levene’s test, while the Kolmogorov–Smirnov test was used to assess the normality of the distribution of the results. Pairwise Pearson correlation coefficients were calculated among selected numerical variables, and statistical significance (p < 0.01) was assessed for each correlation.

3. Results

Statistically significant effects of S. pratensis water extracts on seed germination were recorded for all tested species (Figure 1). In general, germination inhibition was observed at concentrations above 2.5%. For lettuce, radish, and carrot, the maximum reduction in germination did not exceed 25%, even at the highest concentration (10%). In contrast, tomato germination was unaffected at lower concentrations, with a significant reduction of 41.9% only at the highest extract concentration.

S. pratensis water extracts caused substantial decrease in root length of the test species (Figure 2). This effect became more pronounced as the concentration of water extract increased. Radish root was inhibited in all treatments, with reductions ranging from 30.9% to 77.2% compared to control. A similar trend was observed for lettuce and tomato, where the extracts significantly inhibited seedling root length in all treatments (from 14.6% to 79.8%), with the exception of the lowest concentration. For carrot, lower concentrations had no effect, while higher concentrations caused root length decrease of 68.3% to 89.5%.

Exposure to S. pratensis water extracts had varying effects on the shoot length of test species (Figure 3). Only the highest extract concentration significantly reduced lettuce and tomato shoot growth by 46.5% and 62.1%, respectively, whereas lower concentrations stimulated shoot length by approximately 30% compared to control. Positive effects of 1% extract concentration were also observed for radish and carrot, with an increase of 12.6% and 24.6%, respectively. However, concentrations greater than 2.5% negatively impacted shoot elongation, with reductions in shoot length up to 66.8% for radish and up to 66.2% for carrot.

A similar finding was noted for the fresh weight of the tested species, although the significant stimulatory effect of lower concentrations was not observed (Figure 4). Maximum reduction in lettuce fresh weight amounted to 31.1%, while the fresh weight of other test species was reduced up to around 55% compared to control.

Overall, the values of seedling vigor index (SVI) of all test species were significantly decreased in treatments with S. pratensis water extracts (Figure 5). The exception to this were the SVI values of lettuce and carrot at 1% extract concentration, and the SVI values of tomato at 1% and 2.5% concentrations. Generally, as the extract concentration increased, the seedling vigor of all test species decreased, with reductions exceeding 65% at the highest concentration. Across all concentrations, S. pratensis extracts had the strongest inhibitory effect on seedling vigor of radish.

The shoot-to-root ratio was significantly affected by different concentrations of S. pratensis extract and varied among the tested species (Figure 6). In lettuce and tomato, the ratio increased with rising extract concentrations, peaking at 10% for lettuce (up to 2.6 times higher than the control) and 7.5% for tomato (up to two times higher compared to control). In radish, the highest shoot-to-root ratio was observed at 1%. On the other hand, carrot exhibited relatively high shoot-to-root ratio across all treatments, with a steady upward trend observed at higher extract concentrations.

The overall allelopathic potential (OAP) was calculated to enable the comparison of species sensitivity and classification of allelopathic strength according to established thresholds. The OAP of S. pratensis water extracts ranged from 0.07 to 0.54 for germination and growth parameters of test species (Figure 7). For germination, OAP for all test species did not exceed 0.15, which is classified as non-allelopathic, as well as the OAP for shoot length and fresh weight of lettuce, tomato, and carrot (0.07 to 0.25). Moderate OAP was observed for shoot length and fresh weight of radish, while moderate-to-high OAP was observed for root length of all test species, particularly for radish (0.54). On average, radish was the most sensitive test species (OAP 0.31), while lettuce was the most tolerant (OAP 0.18) to S. pratensis water extracts.

Phytochemical analysis was performed to determine the total phenolic content (TPC) and total flavonoid content (TFC) in S. pratensis extracts (Figure 8). A significant concentration-dependent increase in TPC was observed across all S. pratensis extract concentrations. The lowest TPC was recorded at 1%, with a mean value of 0.315 mg GAE/mL of extract. This value more than doubled at 2.5% (0.797 mg GAE/mL) and continued to rise markedly, reaching 1.587 mg GAE/mL at 5%, 2.343 mg GAE/mL at 7.5%, and up to 3.1 mg GAE/mL at the 10% concentration. A similar trend was observed for total TFC, which increased from 0.036 mg QE/mL at 1% to 0.081 mg QE/mL at 2.5%, 0.162 mg QE/mL at 5%, 0.259 mg QE/mL at 7.5%, and 0.342 mg QE/mL at the 10% concentration.

A Pearson correlation analysis was conducted to assess the relationships between S. pratensis extract concentration, its phenolic and flavonoid content, and the growth parameters of the tested plant species. Extract concentration showed a very strong and statistically significant positive correlation with total phenolic content (r = 0.999, p < 0.01) and total flavonoid content (r = 0.999, p < 0.01). Similarly, phenolic and flavonoid contents were very strongly correlated (r = 0.998), indicating that these compounds varied together consistently (Figure 9).

In lettuce, extract concentration showed a very strong negative correlation with root length (r = −0.966), along with strong negative correlations with germination percentage (r = −0.821) and shoot length (r = −0.748), while a moderate correlation was found with fresh weight (r = −0.629) (Figure 9a). Similar trends were observed for TPC and TFC, which were also negatively correlated with germination and all growth parameters, particularly root length (r = −0.968 and −0.961, respectively). For radish, a statistically significant strong negative correlation (p < 0.01) was observed between extract concentration, TPC, and TFC and growth parameters (Figure 9b). The correlations were particularly high for root length (r = −0.922, −0.923, and −0.917), followed by shoot length (r = −0.901, −0.904, and −0.891), and fresh weight (r = −0.823, −0.826, and −0.809). In contrast, moderate negative correlations were found between germination percentage and extract concentration (r = −0.489), TPC (r = −0.491), and TFC (r = −0.482). For tomato, extract concentration, TPC, and TFC all showed statistically significant (p < 0.01) moderate-to-strong negative correlations with germination and growth parameters (Figure 9c). Root length exhibited a very strong negative correlation with these factors (r = −0.967 to −0.968), while both shoot length and fresh weight displayed strong negative correlations, ranging from r = −0.719 to −0.74 and r = 0.844 to −0.861, respectively. Germination percentage, however, was moderately negatively correlated with extract concentration (r = −0.525), TPC (r = −0.52), and TFC (r = −0.521). Extract concentration, TPC, and TFC all showed statistically significant (p < 0.01) strong-to-very-strong negative correlations with germination and growth parameters of carrot (Figure 9d). The strongest effects were observed for fresh weight (r = −0.899 to −0.903), followed by root length (r = −0.848 to −0.854) and shoot length (r = −0.837 to −0.843). Germination percentage was also negatively affected, showing strong negative correlations with extract concentration (r = −0.763), TPC (r = −0.768), and TFC (r = −0.758).

Correlation analysis revealed positive relationship between growth parameters of test species (Figure 9). For all four species, statistically significant (p < 0.001) very-strong to-strong positive relationships were found between shoot length and fresh weight (r = 0.744 to 0.945). Similarly, strong positive correlation was observed between root length and fresh weight (r = 0.849 to 0.852) and shoot and root lengths (r = 0.706 to 0.821) for radish, tomato, and carrot, while moderately positive correlation was found in lettuce (r = 0.542 and r = 0.686). Moderate-to-strong positive correlations were found between germination percentage and growth parameters for lettuce, tomato, and carrot (r = 0.495 to 0.824), while for radish, germination showed significant moderate positive correlation only with root length (r = 0.531).

4. Discussion

The results of our study indicated that S. pratensis water extracts exerted both stimulatory and inhibitory effects on the tested species. In general, as the extract concentration increased, a decrease in germination percentage, root and shoot length, and seedling fresh weight was observed, which is consistent with the findings of previous studies on S. pratensis extracts [29,31]. A concentration-dependent inhibitory effect was also observed by Bisio et al. [38], who reported that aerial part exudates from thirteen Salvia species significantly suppressed the germination and seedling growth of common poppy (Papaver rhoeas L.) and oat (Avena sativa L.) in both Petri dish and pot assays. According to Erez and Fidan [39], methanolic extracts of Salvia macrochlamys Boiss. & Kotschy significantly reduced the seed germination of purslane (Portulaca oleracea L.) at concentrations above 2.5%, achieving 90% inhibition at a 10% concentration. The authors determined that the concentration required to inhibit 50% of germination (I₅₀) was 3.2%. Similarly, the increase in concentration of S. officinalis extract resulted in substantial decrease in seedling length of alfalfa (Medicago sativa L.) and common sainfoin (Onobrychis viciifolia Scop.) [21]. Our study also showed that higher extract concentrations resulted in a reduction in seedling vigor index (SVI), indicating decreased seedling performance of all test species. As reported by Mirmostafaee et al. [12], the SVI of lettuce was decreased by over 20 essential oils from the Lamiaceae family, with S. officinalis and S. nemorosa showing inhibitory effects at concentration as low as 1 µL and 3 µL, respectively. Besides suppressing growth parameters, Salvia extracts have been reported to interfere with key physiological and biochemical processes in target species, suggesting multiple underlying allelopathic mechanisms [39,40,41]. At concentrations above 5%, S. macrochlamys extracts suppressed the increase in gibberellic acid (GA₃) and reduced abscisic acid (ABA) levels in P. oleracea seeds, while also inhibiting α-amylase activity [39]. Allelopathic compounds can also induce the production of reactive oxygen species (ROS), leading to oxidative stress and subsequent cellular damage [42,43]. Šućur et al. [40] reported that S. sclarea aqueous extracts significantly affected antioxidant enzyme activity in black nightshade (Solanum nigrum L.), with superoxide dismutase (SOD) and catalase (CAT) showing increased activity in both leaves and roots depending on concentration and exposure time. These enzymatic changes, along with elevated lipid peroxidation, indicated that allelochemicals induced oxidative stress, with S. nigrum root tissues being more sensitive than leaves.

The lower concentrations of S. pratensis extracts in our study generally had no effect on the germination and growth parameters; in fact, an increase in shoot length of all test species was recorded up to 30% compared to control. Stimulatory effects were also demonstrated by higher values of the SVI of lettuce, tomato, and carrot at 1% concentration. Positive effects of plant extracts have been documented in allelopathic studies. For example, oregano (Origanum vulgare L.) and Salvia tebesana Bunge, as well as the combination of S. ceratophylla and lavender (Lavandula angustifolia Mill.) volatiles stimulated root and shoot growth of lettuce [14]. Similarly, Bonea [20] found significant stimulatory effects of low concentration of S. officinalis extract on maize (Zea mays L.) shoot and root growth, while Erhatić et al. [44] reported significant increase in germination percentage of lettuce with chia (Salvia hispanica L.) and wormwood (Artemisia absinthium L.) extracts. Plant extracts with stimulatory effects have the potential to be exploited in agricultural production as biostimulants, enhancing seed germination and the growth of seedlings, as well as improving crop efficiency, productivity, and tolerance to various abiotic stresses, such as drought and salinity, by stimulating a range of physiological and molecular responses in plants [45,46,47,48]. Stimulatory activity of mugwort (Artemisia vulgaris L.) water extract at low concentrations has been demonstrated, with enhanced seed germination and seedling growth observed in several vegetable species [46], while foliar application of A. vulgaris infusions and macerates was shown to increase photosynthetic pigment and proline content in potato leaves [45]. Furthermore, Pannacci et al. [49] documented both the bioherbicidal and biostimulant effects of A. vulgaris extract—specifically, the inhibition of emergence and growth of redroot pigweed (Amaranthus retroflexus L.) without adverse effects on maize—suggesting its potential as an effective component of integrated weed management by enhancing crop competitiveness and reducing weed pressure.

Both total phenolic (TPC) and total flavonoid (TFC) contents in S. pratensis extract were notably high, increasing with extract concentration from 0.315 to 3.1 mg GAE/mL and from 0.036 to 0.342 mg QE/mL, respectively. Correlation analysis confirmed a very strong positive relationship between extract concentration and the content of these secondary metabolites (r = 0.999). Generalić et al. [50] reported TPC in ranges of 4.34 to 5.15 mg GAE/mL measured in S. officinalis extract. Variability in phenolic content can be attributed to differences in plant population, the parts of plants used, or the extraction procedure applied [3,29,51]. High phenolic and flavonoid contents in S. pratensis aboveground extracts were also reported by Gruľová et al. [29], who compared water and ethanolic extraction methods and demonstrated that the choice of solvent significantly influences the yield of bioactive compounds. In addition to aerial parts, the roots of S. pratensis are known to contain active compounds that contribute to the species’ overall phytochemical profile. Notably, methanolic extracts of the roots exhibited higher phenolic content, whereas the aerial parts contained greater amounts of flavonoids [28]. In our experiment, TPC, and TFC showed strong-to-very-strong negative correlations with growth parameters of test species, suggesting that these compounds may contribute to the observed phytotoxicity. Amani et al. [21] similarly observed a relationship between the increase in TPC and TFC contents and phytotoxicity of S. officinalis and Achillea wilhelmsii C. Koch extracts on O. viciifolia germination. In addition, rosmarinic acid, reported as the main phenolic compound in S. pratensis and other Salvia species [13,28,29], has been shown to exhibit inhibitory activity on both shoot and root length of lettuce, with an EC₅₀ of 196 µg/mL for root elongation [52]. According to Šćepanović et al. [5] higher doses of phenolic acids and their mixtures inhibited early growth of common ragweed (Ambrosia artemisiifolia L.), highlighting the potential of phenolic-rich plant extracts as natural bioherbicides and offering an eco-friendly alternative to conventional weed control methods [2,10,29].

The greatest inhibitory effect of extracts was observed on root length, which decreased by over 75% with the highest concentration in all test species. At the same time, the shoot-to-root ratio increased with rising concentration in all treatments for tomato, lettuce and carrot, indicating that the roots were more significantly affected by the extracts than the shoots. This is likely because the radicle is first to be exposed to toxic compounds upon its emergence during germination [29,38]. This observation was supported by very strong negative correlations between extract concentration and root length in lettuce (r = –0.966), radish (r = –0.922), tomato (r = –0.967), and carrot (r = –0.854). Furthermore, the overall allelopathic potential (OAP) calculated in our experiment was the highest for root length and ranged from 0.40 to 0.54, characterizing S. pratensis extract as moderately to highly allelopathic. The results are consistent with Tsytsiura [53], who reported that 50% of the donor species in the study showed an OAP value over 0.50 for root length, compared to 30% for shoot length.

The allelopathic effect of specific plant is influenced by multiple factors such as plant part, developmental stage of the donor species, content of allelochemicals, and extraction method [3,5,17,29,54]. Additionally, various abiotic and biotic effects, such as temperature, elevation, and seasonal variations, as well as stress conditions influence allelopathic performance of plants [55,56]. Equally important is the selection of test species, as the observed allelopathic effect may differ not only among species, but also among genotypes within the same species [7,22,33,57]. High allelopathic potential of S. pratensis extracts on germination and growth of weed species was previously recorded, with up to 100% inhibition rate [31]. In this research, however, only moderate OAP was observed for radish and was on average for 0.31, while for other test species, the OAP can be characterized as non-allelopathic (on average < 0.25) indicating species-specific response to the S. pratensis extract. Gruľová et al. [29] observed similar differences among model plants in their research, suggesting that dicot plant species, such as white mustard (Sinapis alba L.) and radish had greater sensitivity to S. pratensis extracts compared to monocot species, such as barley (Hordeum vulgare L.) and winter wheat (Triticum aestivum L.). While seed size is often associated with sensitivity to allelochemicals, with smaller seeds generally showing greater susceptibility to phytotoxic effects [7,58], this was not evident in our experiment. Furthermore, lettuce was the least sensitive species in our study, with the OAP of 0.18. Lettuce is usually one of the most commonly used species in allelopathy experiments, especially for screening large number of donor species, due to its sensitivity to allelochemicals [11,12,14,15]. However, Politi et al. [22] found considerable differences among lettuce varieties in their response to allelopathic potential, with some varieties being unaffected by the applied treatments. Beyond this, differences in seed morphology and physiology, along with species ability to metabolize allelochemicals, could explain variations in test species tolerance [38,59,60].

5. Conclusions

The results of the study demonstrated that S. pratensis water extracts exerted both stimulatory and inhibitory effects, which depended on the concentration and the tested species. Phytochemical analysis revealed that S. pratensis water extracts contain notable levels of total phenolics and flavonoids, which were positively correlated with extract concentration, and negatively correlated with growth parameters. The test species—lettuce, radish, tomato, and carrot—differed in their responses to water extracts, with lettuce showing the highest tolerance and radish being the most susceptible. These findings highlight the potential of S. pratensis extracts to influence plant growth in a species- and dose-dependent manner. Further research, including field trials and broader species testing, is needed to evaluate their practical relevance in sustainable crop management.