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Article

Influence of Habitat Factors on the Yield, Morphological Characteristics, and Total Phenolic/Flavonoid Content of Wild Garlic (Allium ursinum L.) in the Republic of Serbia

by
Stefan V. Gordanić
1,
Aleksandar Ž. Kostić
2,
Đorđe Moravčević
2,
Sandra Vuković
2,
Sofija Kilibarda
2,
Ana Dragumilo
1,
Željana Prijić
1,
Milan Lukić
1 and
Tatjana Marković
1,*
1
Institute for Medicinal Plants Research “Dr Josif Pančić”, Belgrade, Tadeuša Košćuška 1, 11000 Belgrade, Serbia
2
Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(2), 118; https://doi.org/10.3390/horticulturae11020118
Submission received: 25 December 2024 / Revised: 16 January 2025 / Accepted: 18 January 2025 / Published: 22 January 2025
(This article belongs to the Section Vegetable Production Systems)
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Abstract

:
Allium ursinum L. (Alliaceae) is a perennial geophyte known for its medicinal properties. This study examines the yield, morphological characteristics, and bioactive component composition of A. ursinum across forty-three different habitats in Serbia, focusing on the relationship between these factors and habitat conditions. Data on habitat locations and soil conditions were gathered from previous studies, while climate parameters were estimated using meteorological data from the Republic Hydrometeorological Institute of Serbia. Cluster analysis identified five habitat clusters, with the first and third clusters representing 88% of the sampled habitats. Fresh leaf yield H1:39.46–H15:564.83 g m⁻2 was correlated with morphological parameters grouped into two clusters. A positive correlation was found between habitat conditions, particularly soil type and altitude. Spectrophotometric quantification of phenolics (1.47–2.49 mg FAE g−1) and flavonoids (0.27–0.82 mg QE g−1) identified five clusters, with soil type being the key factor influencing bioactive component concentration. A. ursinum displayed significant adaptability, thriving in higher altitudes and fertile soils, which enhanced yield and morphological traits, though inversely related to bioactive components. These findings support sustainable cultivation and conservation practices for A. ursinum.

1. Introduction

Allium ursinum L., commonly known as wild garlic or ramsons, is a perennial genotype belonging to the family Alliaceae [1]. In A. ursinum, both the leaves and bulbs are utilized in the pharmaceutical and food industries [2]. The leaves are particularly valued for their medicinal properties, which are attributed to their content of sulphur and phenolic compounds (especially phenolic acids), saponins, and carotenoids [2,3,4]. They exhibit high antioxidant potential, due to the presence of phenolic compounds [5]. Consumption of the leaves offers numerous health benefits, including treatment for cardiovascular diseases, elevated cholesterol levels, cancer-related illnesses, diabetes, obesity, gastrointestinal disorders, and various inflammatory conditions [2]. According to previous research [6,7], A. ursinum also contains a number of valuable mineral components (K, Ca, Mg, Fe, Zn, Cu, B, Ni, Na, and Mn). Given these benefits, the demand for A. ursinum is steadily increasing, as it is utilized as a spice or flavoring agent in a wide variety of food products and is also employed in teas and tinctures for health purposes [2].
Currently, the raw material for A. ursinum, both in nutrition and pharmaceuticals, is primarily sourced from wild habitats, raising additional concerns. It has been observed that plants adapt to varying habitat conditions by altering their morphological and physiological traits [8], which supports both species and ecological resilience. Researchers [9,10,11] have extensively studied these characteristics across different biological and ecological habitats to gain a better understanding of their adaptive mechanisms. The nutritional quality and yield of many other crops used to be influenced by habitat factors such as elevation, light, and temperature [12,13,14]. Therefore, it is essential to investigate how changes in environmental factors affect the nutritional quality of A. ursinum due to specific morphological, physiological, and biochemical responses to different habitats [15]. Previous research [16,17,18,19,20] shows that A. ursinum displays distinct morphological, physiological, and biochemical adaptations, depending on habitat variations. These adaptations have been explored to understand differences in quality across various A. ursinum populations in detail [2]. To the best of our knowledge, no studies have yet investigated this relationship in A. ursinum populations across different habitats. Understanding this connection is critical for the effective cultivation and utilization of the species. Linking the morphological and chemical characteristics of A. ursinum with habitat conditions would facilitate the development of sustainable cultivation methods and the conservation of natural habitats. Furthermore, investigating novel bioactive compounds in A. ursinum and promoting ecologically sustainable agricultural practices could enhance the application of this species in healthcare and industry, while simultaneously supporting the preservation of biodiversity.
The objectives of this study were to (I) investigate the relationship between habitat conditions and the yield, morphological characteristics, and bioactive compounds of A. ursinum populations across forty-three different habitats in the Republic of Serbia and (II) identify potential habitat factors that positively affect the quality of raw materials. These findings will provide a foundation for selecting optimal locations and enhancing environmental conditions for the cultivation and conservation of this important medicinal plant species, particularly in light of the growing threats to its natural habitats.

2. Materials and Methods

2.1. Research Area

2.1.1. Soil Properties in the Habitat

Information on 43 distinct habitats of wild A. ursinum populations across the Republic of Serbia was collected and analyzed [7]. Table 1 and Figure 1 present precise data on the localities and distribution within the region.

2.1.2. Climate Conditions in Habitat

Data on multi-year climate parameters (temperature and precipitation) from 1990 to 2020 in the habitats of A. ursinum were obtained by approximating the data of nearby Meteorological Stations (Figure 2; Table 2) of the Republic Hydrometeorological Institute of Serbia (RHMZS) [22], following the methodology of Kolić [23]. Specifically, for every 100 m increase in altitude, summer temperatures decrease by 0.75 °C, winter temperatures by 0.30 °C, and temperatures in spring and autumn decrease in line with the average annual thermal gradient of 0.56 °C. Precipitation levels were estimated using Schreiber’s method, which suggests that annual precipitation increases by approximately 54 mm for every 100 m rise in altitude [24].

2.2. Collection of Plant Material

At each habitat (Table 1; Figure 1), plant material was collected in 2022 within a radius of 5–50 m while the plants were in the same phenological phase, specifically before flowering (Table 3). The pre-flowering phase of A. ursinum has previously been described [25] as the period with the most favorable chemical composition for this plant species, as it occurs under conditions of maximum light exposure due to the absence of developed overhead shading.
For the yield analysis, collection of fresh plant material occurred according to the previously established “square meter method” [26]. For the analysis of morphological parameters, 30 whole fresh plants were sampled, while for the analyses of bioactive compounds, an additional 20 fresh leaves were collected.

2.3. Yield Analysis

Yield assessments were performed immediately after the collection of plant material, ensuring consistency in timing. Specifically, within each habitat population of A. ursinum, as demonstrated in Figure 3, four randomly selected squares (P = 1 m2) were analyzed [27] to determine the yield of fresh leaves per unit area (g m−2) and the number of leaves per unit area (m−2). All individual plants and leaves within each square were counted, and the entire plants were sampled to determine yield accurately. The plant samples were washed and then weighed using a field scale (Kern HDB 10K10N, Balingen, Germany).
In the laboratory, harvested plant material was dried at 40 °C in a forced air convection oven (SANIO Laboratory Convection Oven MOV-212F Dry Heat Sterilizer, Osaka, Japan), following the drying procedure outlined by Tomšik et al. [28], to preserve their edible and nutritional qualities. The final data were expressed as the yield of air-dried leaves per unit area (g m−2).

2.4. Morphological Analysis

On the day of sampling, morphological analyses were performed on 30 plants of A. ursinum, including measurements of total plant length (cm), habitus height (cm), bulb diameter (mm), bulb weight (g−1), number of leaves per plant, and leaf dimensions (length and width, in cm per plant). The measurements of leaf area, leaf area by square, and average plant width were determined using the ‘ImageJ’ software, version 2.14.0 [29] and an analytical balance (KERN ABJ, Balingen, Germany).

2.5. Analysis of Bioactive Compounds

2.5.1. Extraction Procedure

One gram of fresh leaf samples was immersed in 2 × 5 mL of 80% acetone and subjected to vigorous shaking for two intervals of 90 min in plastic cuvettes, shielded from light, at ambient temperature. The ratio of fresh sample to solvent was maintained at 1:10. The separation of the precipitate from the supernatant was achieved by filtering the extracts through appropriate filter paper. Subsequently, the supernatants were stored at 4 °C until further analysis [30].

2.5.2. Determination of Bioactive Compounds

Total Phenolic Content (TPC)

The Folin-Ciocalteu (FC) method was utilized for the quantification of total phenolic content (TPC), as previously reported [31]. TPC quantification was based on a calibration curve generated using ferulic acid (FA) as a standard, due to its significance as one of the predominant phenolic compounds present in Allium species [32,33]. The results were reported as milligrams of ferulic acid equivalents (FAEs) (g−1) of fresh weight (FW).

Total Flavonoid Content (TFC)

The total flavonoid content (TFC) was assessed using the spectrophotometric method [34]. TFC quantification was performed utilizing a calibration curve constructed with quercetin (Q) as the standard, and the results were expressed as milligrams of quercetin equivalents (QEs) (g−1) of fresh weight (FW).

2.6. Statistical Analysis

Quality indicators were analysed using one-way analysis of variance (ANOVA), while significant differences were assessed using Duncan’s multiple range test. The relationship between the yield and morphological traits of A. ursinum populations and habitat factors was assessed using cluster analysis, Pearson correlation, and PCA. Statistical analyses were performed using the XLSTAT and SPSS v22.0 software packages (SPSS Inc., Chicago, IL, USA), with statistical significance set at p < 0.05.

3. Results and Discussion

3.1. Research Area

3.1.1. Soil Properties in the Habitat

According to the previous studies [7,21], eight soil types predominated in the examined habitats (Table 1), belonging to the order of automorphic soils and classes: humus accumulative (chernozem, rendzina, and ranker), cambic (eutric cambisol, distric cambisol, and red soil), and eluviated–illuvial (luvisol). Additionally, the hydromorphic soil order includes the gleysol class (eugley) [35]. In general, differences in soil types across Serbia, as presented in the table, result from a combination of climatic, geological, hydrological, and biological factors [36]. Geological factors, such as the type of substrate, directly influence soil fertility. For instance, luvisols and eutric cambisols, known for their nutrient richness, provide favorable conditions for wild garlic growth [37]. In contrast, rendzina and ranker, which are typically found in mountainous areas with poorer substrates, impose limitations on the growth of this plant species [38,39,40]. Climatic factors, including precipitation and temperature, also shape soil properties. Fertile soils are more common in lower, temperate regions, whereas cooler, higher-elevation areas promote faster drainage and result in less fertile soil layers [41]. Biological factors, such as vegetation, enhance soil fertility, with forest ecosystems supporting richer soil that promotes wild garlic growth [42]. Hydrological factors, including water retention, also play a significant role, as wild garlic prefers moist habitats [43]. These combined influences shape soil types and affect the distribution and quality of wild garlic habitats in Serbia. However, the soils exhibited a wide pH range (strongly acidic to slightly alkaline), were rich in humus, had high total nitrogen reserves, and were well supplied with available potassium, while available phosphorus levels were generally low [7]. The total content of biogenic elements, particularly Fe and Mn (1.19–8.98% and 270–5980 mg kg⁻1, respectively), varied significantly, depending on soil type and parent substrate. In most soils, Zn and Cu concentrations were below the maximum permissible limits, with exceptions observed in some mountainous regions. These exceptions may result from lower pH values, which increase the solubility and mobility of these elements, reducing their binding to soil particles and enhancing their availability in the soil solution [16]. The concentrations of other analyzed elements varied by habitat (Al > Ca > Mg > V > Na > Ni > Cr > Pb > As > B > Co > Cr (VI) > Sb > Hg > Cd), with some soils containing certain elements above allowable limits [6,7,21]. Fertile habitats, such as those with eutric cambisols (H32, H35, H39), generally had higher levels of nitrogen, phosphorus, potassium, and humus, providing optimal conditions for wild garlic growth. In contrast, drier, nutrient-poor, and acidic soils, such as those with rendzinas and rankers (H18, H36), posed significant limitations. Wild garlic prefers soils with neutral pH values (6.0–7.0), which support better growth. The observed variability in soil directly influences the distribution and quality of wild garlic habitats in Serbia, as confirmed by statistical analyses.

3.1.2. Climatic Conditions in the Habitat

In general, the Republic of Serbia is characterized by a moderately continental climate, with steppe features predominant in the northern and southeastern regions, while mountainous climate prevails at higher altitudes. This climatic variation is influenced by a complex interplay of factors, including topography, large-scale air pressure distribution, terrain exposure, the presence of river systems, vegetation, urbanization, and numerous other elements (RHMZS). Measured and approximated values for temperature and precipitation at meteorological stations near the studied habitats (Table 2) reveal that the mean annual temperature ranges from 7.2 °C in Raška Sjenica to 13.2 °C in Belgrade, where populations of A. ursinum are located at lower elevations. According to data from RHMZS, the average multi-year air temperature (1961–1990) for areas up to 300 m in altitude is 10.9 °C, for areas between 300 and 500 m it is approximately 10.0 °C, and for altitudes exceeding 1000 m, it is around 6.0 °C. Precipitation amounts vary from 597.1 mm in Zrenjanin to 1031.8 mm in Zlatibor (Figure 2). The RHMZS indicates that annual precipitation totals generally increase with altitude. In lower regions, precipitation per year ranges from 540 to 820 mm, while areas above 1000 m typically receive between 700 and 1000 mm. Some mountain peaks in southwestern Serbia experience even higher precipitation, reaching up to 1500 mm. The approximated meteorological parameters align partially with the data from the RHMZS. Discrepancies, as noted by the RHMZS, are attributed to the degree of continentality influenced by significant geographical determinants that characterize essential synoptic situations, including the Alps, the Mediterranean Sea and the Gulf of Genoa, the Pannonian Plain, the Morava Valley, the Carpathians, and the Rhodope Mountains, along with hilly and mountainous regions featuring valleys and plateaus. Notably, the prevailing meridional positioning of river basins and the lowland area in the northern part of the country facilitate the deep penetration of polar air masses southward, resulting in pronounced fluctuations in meteorological parameters, as demonstrated in this study.

3.1.3. Relationship Between the Habitat of A. ursinum and Climatic and Soil Parameters

The habitats of A. ursinum were classified into five distinct clusters (C1–C5) (Figure 4), each reflecting specific combinations of climatic and soil factors. The first group (C1) consists of twenty-three habitats, the second group (C2) contains four habitats, and the third group (C3) encompasses fourteen habitats, while the fourth (C4) and fifth (C5) groups each contain one habitat. Cluster C1 includes the largest number of similar habitats (53%), followed by cluster C3 (33%). Cluster C1, including habitats such as H37 and H8, is likely representative of moderate ecological conditions, while cluster C2, encompassing habitats H9 and H28, likely corresponds to more extreme or specific conditions, including variations in soil pH, increased organic matter content, or microclimatic differences. Dendrogram analysis revealed significant dissimilarity between clusters, particularly between C1 and C5, while habitats within the same cluster exhibited homogeneity with similar ecological characteristics. This suggests that key factors such as precipitation, temperature, and soil texture play a critical role in the formation of these clusters and in determining the species’ potential adaptability. Clusters with greater heterogeneity, such as C3, may indicate transitional zones between different ecological conditions, offering valuable insights into the adaptive capacity of A. ursinum and ecological gradients across its habitats. Soil data analysis for habitats in clusters C1 and C3 revealed that they are characterized by acidic soils (pH-KCl: x ¯ = 5.54; 5.08), with a good supply of nitrogen (N: x ¯ = 0.23; 0.29%), humus ( x ¯ = 4.75; 5.75%), and potassium (K2O: x ¯ = 21.2; 23.4 mg/100 g), but a poor supply of phosphorus (P2O5: x ¯ = 5.2; 8.4 mg/100 g). Moreover, the presence of certain elements, such as Al, Ni, Cr, Pb, As, B, Co, Cr (VI), Hg, and Cd, exceeded permissible limits in most habitats, as previously reported [6,7]. Since clusters C1 and C3 encompass the majority of the studied habitats (88%), it can be concluded that A. ursinum thrives in diverse climatic conditions, where annual precipitation varies significantly (max–min = 435 mm), as well as temperature (max–min = 6 °C), demonstrating the species’ ability to adapt to a wide range of ecological environments.

3.2. Morphology and Yield

The outcomes of morphological analysis revealed significant variations between the examined populations of A. ursinum. Total plant fresh weight (g per plant), total plant length (cm) (Figure 5), height of the shoot (cm), bulb diameter (mm) (Figure 6), number of leaves per plant, yield of fresh leaves (g) (Figure 7), average leaf length per plant (cm), and average leaf width per plant (cm) (Figure 8) were found to be within the ranges of 0.9–8.32 g per plant, 17.77–33.11 cm, 10.45–22.76 cm, 3.1–9.28 mm, 1.0–2.07 leaves, 0.35–3.81 g, 8.2–17.02 cm, and 2.08–5.02 cm, respectively. Similar intervals for studied morphological parameters in A. ursinum plants, including 1.10–11.97 g of total plant weight, 20.62–42.70 cm for total plant length, 0.9–7.72 mm of bulb diameter, 1.03–2.07 number of leaves per plant (Figure 9), 0.31–4.93 g of fresh leaf weight per plant, 8.53–16.19 cm for leaf length, and 2.91–6.05 cm for leaf width, were recently reported [21]. However, Todorović et al. [42] recorded slightly higher values for the average weight of total plant (12.5 g), leaf length (12.6 cm), and leaf width (0.36 cm). Additionally, considerable variability in leaf size, precisely in length (7–35.3 cm) and width (2.2–6.6 cm), was observed in another study [17], and it was attributed to the influence of ecological factors.
The fresh plant mass varied significantly across habitats, ranging from a minimum of 0.90 g (H15), characteristic of plants growing in less favorable conditions, to 8.32 g (H1), where the habitat conditions were evidently more favorable. Similar differences were observed in plant height, which was the lowest at H15 (17.77 cm) and the highest at H18 (33.11 cm), indicating different growth strategies, depending on the availability of light, moisture, and nutrients [43]. The height of the aboveground part of the plant also reflects adaptation to habitat conditions, ranging from 10.45 cm (H15) to 22.76 cm (H18), which may be related to competition for light in denser plant communities [44]. Bulb diameter, an important parameter affecting nutrient storage capacity, was the smallest at H18 (3.10 mm) and the largest at H35 (9.28 mm), indicating better resource accumulation in plants with larger bulbs, typical of fertile and stable habitats. The number of leaves per plant ranged from 1 (H12, H15) to 2.07 (H1 and H19), with plants having more leaves often associated with better photosynthetic efficiency and greater growth potential [45]. Leaf mass exhibited similar trends, varying from 0.35 g (H15) to 3.81 g (H1), with plants having larger leaves potentially possessing a better capacity for energy conversion from sunlight [44]. Average leaf length was the smallest at H15 (8.20 cm) and the largest at H9 (17.02 cm), while average leaf width varied from 2.08 cm (H15) to 5.02 cm (H20). These parameters directly influence the plant’s ability to capture light and efficiently utilize resources [43]. A particularly interesting parameter, “Bulb weight: total leaf weight” ratio (Table 4), which represents the ratio of bulb weight to total leaf weight, showed significant variation, ranging from 1.0:0.5 (H15) to 8.0:5.9 (H35). This ratio reflects different resource allocation strategies between vegetative and reproductive growth, with plants having a higher bulb weight percentage potentially possessing greater regeneration and survival capacity under stressful conditions [45]. These results are in line with previous studies by Đorđević et al. [43] and Todorović et al. [44], showing that habitat factors, such as light, moisture, and soil quality, significantly influence the morphological characteristics of A. ursinum plants. For example, Jakovljević et al. [45] observed that plants growing in shaded areas had lower leaf mass and a lower bulb-to-leaf ratio compared to those from more illuminated habitats, further confirming the species’ adaptive capabilities. These data highlight the key ecological interactions shaping plant morphology and enabling their survival in diverse conditions.
The outcomes of the yield of fresh A. ursinum ranged widely (Figure 9). The ratio of fresh to dry A. ursinum at the studied habitats ranged from 8.20 (H5) to 13.40 (H43), with an average ratio of 10.23, indicating a high water content in the fresh plant material (Table 4), which is in agreement with earlier research [46]. However, significant variability in water content (min/max = 61.2%) was observed, which is assumed to be influenced by habitat conditions, as confirmed by a previous study [19]. For a clearer understanding of the similarities among populations in morphological parameters and yield through cluster analysis, the habitats of A. ursinum were grouped into two clusters. The first cluster (C1) included six habitats (H1, H16, H19, H32, H37, and H20), while the second cluster (C2) encompassed the remaining studied habitats that exhibited similar parameters in the analysis (Figure 10). Morphological and physiological traits have been attributed [38,39] to the adaptability of the species. The results of this study reflect the findings mentioned above. The fresh leaf yields of A. ursinum vary from 39.46 g/m2 at the H15 habitat to 564.83 g/m2 at the H1 habitat, indicating the significant impact of habitat factors such as soil type, moisture, and other ecological factors on the productivity of this plant. Higher-yielding habitats, like H1 (564.83 g/m2), H19 (485.46 g/m2), and H20 (526.24 g/m2), likely provide more favorable growth conditions, including fertile soils and optimal moisture levels. Conversely, lower-yielding habitats, like H15 (39.46 g/m2), H33 (54.51 g/m2), and H39 (84.03 g/m2), suggest limited conditions, such as lower moisture and poorer soil quality, which can reduce plant productivity [41]. However, H39 is characterized by a rich luvisol and high precipitation (1031.8 mm), both favorable for plant growth. This discrepancy suggests that soil quality and moisture alone do not fully explain the lower yield observed at H39. These differences highlight the adaptive ability of A. ursinum to grow under varying conditions, with better resource competition in habitats with higher yields. Understanding these factors can aid in optimizing cultivation conditions for A. ursinum and improving agricultural practices [42]. These data also emphasize the importance of ecological interactions in shaping the morphological and physiological characteristics of A. ursinum, which are crucial for its growth and survival [37,38].

3.3. Total Phenolic (TPC) and Flavonoid (TFC) Content

Significant differences were observed among habitats regarding the content of total phenolics (TPC) and flavonoids (TFC) (Figure 11). The TPC ranged from 1.47 to 2.49 mg FAE g−1 FW, while TFC varied between 0.27 and 0.82 mg QE g−1 FW, indicating a high degree of variability (min/max: approximately 60% for phenolics and around 33% for flavonoids). Cluster analysis resulted in the grouping of habitats based on examined bioactive components into five clusters. The first cluster (C1) included six habitats, the second (C2) comprised fourteen habitats, the third (C3) contained eighteen habitats, the fourth (C4) consisted of four habitats, and the fifth (C5) included only one habitat (H18), which exhibited the highest content of bioactive components (Figure 12).
It was suggested by a previous study [25] that the variability in phenolic content of A. ursinum leaves is influenced by the timing of harvest, specifically the physiological stage at which the leaves are collected. For example, in the mentioned study, the phenolic content in leaves harvested in March ranged from 38.1 to 47.7 mg/100 g. While this variability may explain differences in phenolic content in some studies, it is not a relevant factor for the present study, as all plant samples were collected at the same physiological stage of A. ursinum. This is in line with previous findings [47,48,49], which reported that approximately 60% of the variability in phenolic content (ranging from 642.95 to 1069.51 mg GAE kg⁻1) was attributed to ecological factors as being the main contributors to this variation. Studies on the TPC and TFC of A. urisunum have shown phenolic levels ranging from 1.42 to 1.98 mg g−1 FAE and flavonoid levels from 0.35 to 1.28 mg g−1 QE, where soil properties were found to play a significant role in these differences [30]. Further research [50] indicated variations in phenolic content (1027.77–2111.11 mg GAE/100 g) and flavonoid content (13.75–20.00 mg QE/g) in dried A. ursinum leaves, which were linked to the habitat (ecotype) effects. These findings align with earlier studies [51], which also reported variability in phenolics (138.41–186.18 mg GAE/100 g) and flavonoids (75.49–110.65 mg CE/100 g). These results support the findings of the current research (Figure 11). Moreover, it was highlighted [52] that there was a significant correlation between environmental parameters and phenolics content in plants, while variations in phenolic levels were attributed [53] to the synthesis of other non-phenolic compounds (such as carbohydrates, terpenes, etc.) or the formation of complexes between individual compounds, under the influence of ecological factors. Pedoclimatic factors have a considerable impact on the production of most plant species, playing a crucial role in their adaptation to environmental conditions [54,55]. These findings were further confirmed by the results of this study. By comparing the TPC/TFC across different habitats (Figure 11), it was determined that soil factors play the most significant role in the observed differences in their levels. In general, H18 stands out, with the highest concentrations of phenolic and flavonoid compounds among all 43 populations of A. ursinum, indicating specific ecological conditions that favor the synthesis of these metabolites. The high values may result from a favorable microclimate, nutrient-rich soil composition, or the presence of biotic factors such as microorganisms and insects that induce stress in the plant [18,25]. Habitat H18 exhibits a high nitrogen content (0.33%), good phosphorus availability (6.32 mg/100 g), and an alkaline pH (7.64 in water, 7.17 in KCl). The humus content of 6.61% ensures soil fertility, while rendzina, rich in calcium, contributes to the synthesis of secondary metabolites, making this site unique, compared to others.
Earlier research [56] indicates that soil composition can have a substantial impact on the content of phenolic compounds in plants.
This finding is supported by other researchers [57,58,59], who state that secondary metabolites accumulate most when biogenic elements are in low availability, consistent with the data from this study. Nunez-Ramirez et al. [60] highlight the fact that antioxidant activity and the concentration of total phenolics are primarily influenced by nitrogen content in the soil. It was also noted [61] that elevated phenolic concentrations are associated with higher nitrogen and organic matter content in the absence of certain mineral elements. According to other research [62], the availability of biogenic elements such as Ca, Mn, Fe, and Zn in the soil negatively correlates with phenolic content, as protein synthesis increases while the synthesis of secondary metabolites decreases.
Given the ample supply of nitrogen and organic matter in the studied soils [7], it is assumed that the elemental composition of the soil is the primary factor influencing the varied production of phenolic compounds in plants. The outcomes of a previous study [30] emphasize soil type as the main determinant of the variability observed in these compounds. Due to differing physical and chemical properties, soils possess varying water retention capacities, leading to differences in the content of phenolic compounds in wild garlic grown on different substrates. Based on these findings, it can be concluded that the content of phenolics and flavonoids primarily depends on the soil in which the plant grows, with a lesser influence from climatic factors.
Generally, variations in the content of phenolics and flavonoids across sites may result from multiple factors, including microclimatic changes, soil type, geographical location, and genetic differences among plants. For example, sites with higher phenolic and flavonoid content may be more exposed to higher UV radiation intensity, which could stimulate the synthesis of these protective compounds in plants [63]. Similar trends have been observed in studies that investigated variations in the chemical composition of plants across different geographical locations [64].
For a better interpretation of the results, particularly when considering specific sites with high phenolic content (such as H1, H13, and H18), factors such as soil type, climatic conditions, and the presence of certain plant and animal species that may influence the chemical composition of the plants should be taken into account. Similar studies have shown that specific ecological factors can directly impact the concentration of phenolics and flavonoids, as well as their potential for pathogen protection and resistance [65].

Correlation and Principal Component Analysis (PCA) of Habitat Factors, Yield, Morphological Traits, and Bioactive Compounds in A. ursinum

Pearson correlation analysis was conducted to investigate the relationships and correlations between various habitat parameters, including fundamental climatic and soil factors, and the morphological traits, yield, and bioactive components of A. ursinum (phenolics and flavonoids) (Figure 13).
The yield of fresh and dried leaves exhibits a very high positive correlation (r = 0.975), indicating their interdependence and consistent growth under similar conditions. A strong association between the weight of fresh leaves per plant and yields (r > 0.6) highlights the importance of individual plant biomass for overall yield, while a weaker correlation with the number of leaves per plant (r = 0.151−0.270) suggests that the quality of individual leaves may be more critical than their quantity. Chemical parameters, such as flavonoids, show negative correlations with agronomic traits, including the number of leaves per plant (r = −0.403) and the yield of fresh leaves (r = −0.326), indicating a potential trade-off between secondary metabolite production and plant growth. Phenolics, although weakly associated with most morphological parameters, exhibit a moderate positive correlation with flavonoids (r = 0.282), which may suggest shared metabolic pathways. Soil quality has emerged as a key factor. Humus content strongly correlates with nitrogen (r = 0.982), phosphorus (r = 0.941), and potassium (r = 0.917) concentrations, emphasizing the importance of organic matter in enhancing soil fertility. Conversely, soil pH values (pH-H2O and pH-KCl) have a weaker impact on agronomic traits, but their negative correlation with flavonoids (r = −0.270) suggests that soil acidity may influence plant metabolism. Among trace elements, cadmium, chromium, and nickel exhibit strong intercorrelations (r > 0.9), potentially pointing to shared contamination sources in the soil. Meanwhile, elements such as calcium, magnesium, and manganese show moderate positive correlations with yields (r ≈ 0.3), underscoring their importance in plant physiological processes. Ecological factors, such as temperature and precipitation, also affect plant growth and yield. The negative correlation between temperature and yield (r = −0.416) indicates potentially adverse effects of higher temperatures, while a weak positive correlation between precipitation and yield (r ≈ 0.07) suggests that water availability may be a limiting factor in arid conditions (Figure 13).
For a simplified visualization of these relationships, PCA was performed, incorporating the elemental composition of the soil from the aforementioned study [7] (Figure 14). The PCA analysis identified key variables and relationships among the samples based on their morphological, ecological, and physiological characteristics. The use of the 1–3 principal component analysis (PCA) plan instead of the 1–2 plan may be justified by the need to capture additional variance from the third principal component (PC3), offering clearer separation of traits, highlighting specific relationships, providing orthogonal insights, revealing important trait loadings, and identifying clusters or anomalies relevant to the study’s objectives. The first principal component (PC1, 18.23%) emphasizes the importance of morphological variables, while the third component (PC3, 9.74%) does not significantly reflect the influence of habitat factors and secondary metabolites. The variables that contribute most to the variation include morphological traits such as “leaf area per square meter” and “yield of fresh leaves per unit area”, which strongly influence the PC1 component, highlighting the significance of leaf area and yield. Variables such as “altitude”, “phenolics”, and “flavonoids” have a dominant impact on the F3 component, indicating a connection to elevation and adaptive plant responses.
Habitats associated with a high number of leaves per unit area include H1, H3, H4, and H5, while habitats with higher concentrations of phenolics, characteristic of higher altitudes, include H35, H36, and H41. Distinct habitats H18 and H21 exhibit extreme or specific characteristics, which may be linked to unique ecological conditions, as previously mentioned in the text.
Relationships between key variables, such as “altitude” and “phenolics”, show a strong positive correlation, indicating stimulation of phenolics synthesis at higher altitudes. The variables “phenolics” and “yield of air-dried leaves per unit area” are independent, suggesting that the accumulation of secondary metabolites is not directly related to the quantitative yield of biomass. Additionally, “altitude” and “yield of air-dried leaves per unit area” show a moderate correlation, indicating the ecological impact of the habitat on dry biomass.
In general, the analysis clearly highlights the significance of morphological and ecological habitat factors in the variation among the samples. Variables related to yield and leaf area dominate the overall variation, while altitude influences the accumulation of secondary metabolites. These results provide a basis for the identification of genotypes more resistant to stress conditions and for improving plant selection in agricultural systems.

4. Conclusions

The results of this study suggest that populations of A. ursinum grow in diverse habitats across varying elevations, with different soil types and climatic conditions that influence both the morphological characteristics and bioactive compound yield of this species. Common to all habitats are well-drained soils rich in organic matter and high moisture levels, with shaded or semi-shaded environments.
The data analysis reveals a positive correlation between elevation and both the morphological parameters and yield of A. ursinum, with the exception of leaf width per plant, which demonstrated a positive correlation. In contrast, elevation exhibited a negative correlation with other yield-related and morphological traits.
Regarding soil, 88% of the studied habitats were classified into two out of five clusters, indicating that A. ursinum prefers acidic soils rich in humus, nitrogen, and potassium, although phosphorus availability is limited. Notably, the presence of other elements, such as Ca, Zn, Ni, Cu, As, Cd, and Pb, was observed, and these elements exhibited a positive correlation with leaf yield and bioactive compound content.
In terms of climatic parameters, A. ursinum shows significant adaptability, thriving in habitats with a wide range of mean annual temperature (max–min = 6 °C), and precipitation (max–min = 435 mm). However, climatic factors were found to have a weak correlation with both yield and bioactive compound content.
These findings advance our understanding of the habitat conditions that influence the yield and quality of A. ursinum, offering valuable insights for its future cultivation and conservation. The ecological parameters identified in A. ursinum habitats facilitate the more efficient establishment of cultivation practices, which can contribute to preventing the degradation of natural populations.

Author Contributions

Conceptualization, T.M., A.Ž.K. and Đ.M.; methodology, T.M., A.Ž.K. and Đ.M.; research: S.V.G. and T.M.; software, T.M. and S.V.G.; validation, A.Ž.K., Đ.M. and T.M.; formal analysis, S.V.G., A.D., S.K. and S.V.; investigation S.V.G.; resources, T.M., M.L. and A.Ž.K.; data curation, S.V.G. and A.D.; writing—original draft preparation, S.V.G., A.D. and A.Ž.K.; writing—review and editing, Ž.P., M.L. and Đ.M.; visualization, S.V.G. and Ž.P.; supervision, Ž.P. and T.M.; project administration, A.Ž.K. and M.L.; funding acquisition, M.L. and A.Ž.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by joint funding from the Ministry of Science and Technology of the Republic of Serbia (Grants: 451-03-66/2024-03/200003 and 451-03-65/2024-03/200116).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Distribution of studied habitats of Allium ursinum populations in the Republic of Serbia (author: Stefan Gordanić).
Figure 1. Distribution of studied habitats of Allium ursinum populations in the Republic of Serbia (author: Stefan Gordanić).
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Figure 2. Description of average microclimate air variables in each region during a 30-year period (1991–2020), based on data from the Republic Hydrometeorological Institute of Serbia (author: Stefan Gordanić).
Figure 2. Description of average microclimate air variables in each region during a 30-year period (1991–2020), based on data from the Republic Hydrometeorological Institute of Serbia (author: Stefan Gordanić).
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Figure 3. Analysis of yield of fresh leaves of Allium ursinum using the “square meter method”.
Figure 3. Analysis of yield of fresh leaves of Allium ursinum using the “square meter method”.
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Figure 4. Cluster analysis of climate and soil parameters in studied habitats of A. ursinum populations in Serbia.
Figure 4. Cluster analysis of climate and soil parameters in studied habitats of A. ursinum populations in Serbia.
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Figure 5. A. ursinum at studied habitats in Serbia: (A) total plant fresh weight (g−1); (B) total plant length (cm). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
Figure 5. A. ursinum at studied habitats in Serbia: (A) total plant fresh weight (g−1); (B) total plant length (cm). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
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Figure 6. A. ursinum plants in studied habitats of Serbia: (A) height of habitus (cm); (B) bulb diameter (mm). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
Figure 6. A. ursinum plants in studied habitats of Serbia: (A) height of habitus (cm); (B) bulb diameter (mm). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
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Figure 7. A. ursinum in studied habitats in Serbia: (A) number of leaves per plant; (B) yield of fresh leaves per plant (g−1). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
Figure 7. A. ursinum in studied habitats in Serbia: (A) number of leaves per plant; (B) yield of fresh leaves per plant (g−1). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
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Figure 8. Leaf dimensions in Allium ursinum plants from studied habitats in Serbia: (A) leaf length per plant (cm); (B) leaf width per plant (cm). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
Figure 8. Leaf dimensions in Allium ursinum plants from studied habitats in Serbia: (A) leaf length per plant (cm); (B) leaf width per plant (cm). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
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Figure 9. A. ursinum leaves per unit area in studied habitats in Serbia. (A) Number of leaves (m−2). (B) Yield of fresh leaves (g m−2). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
Figure 9. A. ursinum leaves per unit area in studied habitats in Serbia. (A) Number of leaves (m−2). (B) Yield of fresh leaves (g m−2). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
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Figure 10. Cluster analysis of A. ursinum habitats based on morphological parameters and yield.
Figure 10. Cluster analysis of A. ursinum habitats based on morphological parameters and yield.
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Horticulturae 11 00118 g011
Figure 11. Content of bioactive compounds in fresh leaves of Allium ursinum) from studied habitats in Serbia: (A) phenolic content (mg FAE/g); (B) flavonoid content (mg QE/g). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
Figure 11. Content of bioactive compounds in fresh leaves of Allium ursinum) from studied habitats in Serbia: (A) phenolic content (mg FAE/g); (B) flavonoid content (mg QE/g). Lowercase letters indicate significant differences (p < 0.05) between each fragmentation grade according to Duncan’s new multiple-range test (DMRT), with different letters showing significant differences and the same letters indicating no significant difference.
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Horticulturae 11 00118 g012
Figure 12. Cluster analysis of bioactive compounds of Allium ursinum in studied habitats in Serbia.
Figure 12. Cluster analysis of bioactive compounds of Allium ursinum in studied habitats in Serbia.
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Horticulturae 11 00118 g013
Figure 13. Pearson correlation analysis based on correlation coefficients of A. ursinum traits related to vegetative morphometric characteristics, yield, and bioactive components.
Figure 13. Pearson correlation analysis based on correlation coefficients of A. ursinum traits related to vegetative morphometric characteristics, yield, and bioactive components.
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Horticulturae 11 00118 g014
Figure 14. PCA Analysis of morphological, ecological, and physiological characteristics of A. ursinum: key variables and habitat relationships.
Figure 14. PCA Analysis of morphological, ecological, and physiological characteristics of A. ursinum: key variables and habitat relationships.
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Table 1. Habitats (H) of A. ursinum in the Republic of Serbia.
Table 1. Habitats (H) of A. ursinum in the Republic of Serbia.
Habitat
[7]		GPSElevation
a.s.l. (m)		Soil Type
[21]
NE
H144°43′46.1″21°01′46.0″70Chernozem
H244°36′34.0″20°12′02.8″77Eutric cambisol
H344°44′14.3″19°33′52.3″82Euglei
H444°44′04.4″19°37′20.1″83Euglei
H544°44′41.2″20°08′46.5″83Eutric cambisol
H644°33′01.5″20°23′54.4″130Eutric cambisol
H744°14′08.9″20°15′44.2″139Luvisol
H845°10′03.1″19°29′10.6″200Eutric cambisol
H945°07′44.5″19°31′58.8″216Eutric cambisol
H1044°06′10.7″21°28′29.2″221District cambisol
H1144°34′43.1″19°22′44.5″222Luvisol
H1245°09′14.8″19°34′56.4″264Eutric cambisol
H1344°14′53.6″20°23′55.3″266Eutric cambisol
H1444°15′46.9″20°20′45.1″268Eutric cambisol
H1544°39′02.0″20°24′23.0″277Eutric cambisol
H1645°07′45.2″21°22′09.1″287Ranker
H1744°36′17.8″20°33′18.2″302Luvisol
H1843°48′30.5″20°00′41.8″346Rendzina
H1943°54′13.2″20°12′04.6″359Rendzina
H2044°10′58.4″20°32′47.6″386Luvisol
H2143°54′17.7″20°13′20.5″405Eutric cambisol
H2243°14′37.0″22°06′06.9″502District cambisol
H2344°05′57.4″20°27′13.8″508Eutric cambisol
H2444°17′57.7″20°32′08.2″529District cambisol
H2544°28′11.5″20°34′17.0″550Luvisol
H2643°24′42.5″21°22′56.1″660Ranker
H2743°50′33.6″20°17′35.4″662Ranker
H2844°08′31.2″20°30′29.1″675District cambisol
H2943°10′45.7″21°32′52.7″684Luvisol
H3044°06′54.2″19°55′19.2″737Redness
H3144°06′48.1″20°08′58.8″748Luvisol
H3243°54′86.3″20°73′88.4″756District cambisol
H3343°34′01.8″19°20′09.5″776Luvisol
H3443°24′07.7″21°56′58.3″793Luvisol
H3544°08′31.3″20°33′20.3″814Luvisol
H3644°10′21.6″19°37′11.1″823Rendzina
H3743°55′22.6″20°73′64.9″837District cambisol
H3843°12′17.6″22°09′12.2″854District cambisol
H3943°43′58.2″19°42′36.8″996Luvisol
H4043°54′03.4″19°22′41.0″1021District cambisol
H4143°36′24.9″19°54′36.3″1053Rendzina
H4244°07′47.9″20°32′40.1″1061Rendzina
H4344°07′34.1″19°45′25.8″1211Rendzina
Table 2. Meteorological stations of the Republic Hydrometeorological Institute of Serbia (RHMZS) with altitudes located near the natural habitats of A. ursinum.
Table 2. Meteorological stations of the Republic Hydrometeorological Institute of Serbia (RHMZS) with altitudes located near the natural habitats of A. ursinum.
Meteorological StationsNearby Habitats of
A. ursinum Populations
PlaceElevation a.s.l. (m)
Zrenjanin80H16
Sremska Mitrovica82H3; H4; H8; H9; H12
Loznica121H11
Ćuprija123H10
Beograd132H1; H2; H5; H6; H15; H17; H25
Valjevo176H7; H13; H14; H24; H30; H36; H43
Kragujevac185H20; H23; H27; H28; H31; H35; H42
Niš202H22; H26; H29; H38
Kraljevo215H32; H34; H37
Požega310H18; H19; H21
Zlatibor1028H39; H40; H41
Sjenica1038H33
Table 3. Sampling schedule and locations of plant material based on elevation.
Table 3. Sampling schedule and locations of plant material based on elevation.
Elevation a.s.l. RangeSampling PeriodHabitats of
A. ursinum
<500March2nd weekH1–H8
3rd weekH9–H14
4th weekH15–H21
>500April1st weekH22–H30
2nd weekH31–H38
4th weekH39–H43
Table 4. Variation in bulb weight and total leaf weight ratio across different habitats of A. ursinum.
Table 4. Variation in bulb weight and total leaf weight ratio across different habitats of A. ursinum.
HabitatsBulb Weight: Total Leaf WeightHabitatsBulb Weight: Total Leaf Weight
H12.0: 1.1 ± 0.235H232.0: 1.1 ± 0.233
H22.2: 1.4 ± 0.249H242.9: 1.6 ± 0.244
H31.8: 1.0 ± 0.221H252.3: 1.4 ± 0.229
H42.3: 1.2 ± 0.239H262.8: 1.7 ± 0.240
H53.1: 1.9 ± 0.218H272.1: 1.3 ± 0.244
H62.2: 1.5 ± 0.228H282.7: 1.8 ± 0.246
H72.1: 1.2 ± 0.237H293.0: 1.8 ± 0.223
H82.0: 1.0 ± 0.213H303.7: 2.3 ± 0.234
H93.5: 1.8 ± 0.233H313.1: 2.0 ± 0.258
H102.3: 1.3 ± 0.247H321.6: 1.2 ± 0.202
H111.5: 0.9 ± 0.242H331.1: 0.7 ± 0.221
H121.5: 0.7 ± 0.240H343.8: 2.4 ± 0.235
H132.4: 1.3 ± 0.221H358.0: 5.9 ± 0.250
H143.2: 1.8 ± 0.215H367.0: 4.5 ± 0.220
H151.0: 0.5 ± 0.227H373.5: 2.3 ± 0.239
H162.0: 1.0 ± 0.245H384.3: 1.7 ± 0.242
H171.6: 1.1 ± 0.230H391.9: 1.3 ± 0.228
H183.3: 2.1 ± 0.241H404.4: 3.1 ± 0.223
H195.4: 2.8 ± 0.237H412.0: 1.2 ± 0.229
H207.0: 4.2 ± 0.250H423.4: 2.0 ± 0.233
H212.8: 1.7 ± 0.228H435.7: 4.0 ±0.237
H222.8: 1.8 ± 0.225
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MDPI and ACS Style

Gordanić, S.V.; Kostić, A.Ž.; Moravčević, Đ.; Vuković, S.; Kilibarda, S.; Dragumilo, A.; Prijić, Ž.; Lukić, M.; Marković, T. Influence of Habitat Factors on the Yield, Morphological Characteristics, and Total Phenolic/Flavonoid Content of Wild Garlic (Allium ursinum L.) in the Republic of Serbia. Horticulturae 2025, 11, 118. https://doi.org/10.3390/horticulturae11020118

AMA Style

Gordanić SV, Kostić AŽ, Moravčević Đ, Vuković S, Kilibarda S, Dragumilo A, Prijić Ž, Lukić M, Marković T. Influence of Habitat Factors on the Yield, Morphological Characteristics, and Total Phenolic/Flavonoid Content of Wild Garlic (Allium ursinum L.) in the Republic of Serbia. Horticulturae. 2025; 11(2):118. https://doi.org/10.3390/horticulturae11020118

Chicago/Turabian Style

Gordanić, Stefan V., Aleksandar Ž. Kostić, Đorđe Moravčević, Sandra Vuković, Sofija Kilibarda, Ana Dragumilo, Željana Prijić, Milan Lukić, and Tatjana Marković. 2025. "Influence of Habitat Factors on the Yield, Morphological Characteristics, and Total Phenolic/Flavonoid Content of Wild Garlic (Allium ursinum L.) in the Republic of Serbia" Horticulturae 11, no. 2: 118. https://doi.org/10.3390/horticulturae11020118

APA Style

Gordanić, S. V., Kostić, A. Ž., Moravčević, Đ., Vuković, S., Kilibarda, S., Dragumilo, A., Prijić, Ž., Lukić, M., & Marković, T. (2025). Influence of Habitat Factors on the Yield, Morphological Characteristics, and Total Phenolic/Flavonoid Content of Wild Garlic (Allium ursinum L.) in the Republic of Serbia. Horticulturae, 11(2), 118. https://doi.org/10.3390/horticulturae11020118

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