The Effect of Arbuscular Mycorrhizal Fungi on Plant Development and Accumulation of Phenolics in the Flower Heads of Meadow Arnica (Arnica chamissonis Less.)

Abstract

Meadow arnica is a valuable medicinal plant, used in both the pharmaceutical and cosmetic industries. The aim of the study was to determine the influence of arbuscular mycorrhizal fungi (AMF) on the development, yield, and quality of flower heads (raw material) of meadow arnica grown in an organic farming system. The inoculation of plants with AMF improved the mass of above- and underground organs, including the mass of raw material, as well as the content of chlorophylls and general sugar in the leaves, followed by enhanced starch storage in the roots. The content of phenolics in the raw material was determined using high-performance liquid chromatography (HPLC). The following flavonoids were assessed here: cynaroside, rutin, hyperoside, cosmosiin, astragalin, and diosmetin, as well as the phenolic acids: neochlorogenic, chlorogenic, caffeic, ferulic, rosmarinic, cichoric, 3,4-di-O-caffeoylquinic, and 1,5-dicaffeoylquinic acids. The contents of these substances were higher in non-inoculated plants than in inoculated ones, which contradicts most studies conducted to date on medicinal and aromatic plants. Nevertheless, the results are interesting primarily because of the beneficial developmental changes in inoculated plants, as evidenced by a significantly higher mass of arnica flower heads, more efficient uptake of mineral nutrients from the soil, and lower nitrogen levels in aboveground organs.

1. Introduction

Plants belonging to the Arnica genus (family Asteraceae) are distributed throughout North America, Europe, and Asia, and have also been introduced to other parts of the world. The genus comprises nearly 30 species, most of which are perennials growing in mountainous regions. Only one species, mountain arnica (Arnica montana L.), occurs naturally in Central Europe [1]. Its inflorescences (flower heads; Arnica flos) constitute the herbal raw material, listed in the European Pharmacopoeia [2], standardized for sesquiterpene lactone content (not less than 0.40%), and traditionally used as a remedy in subcutaneous bleeding, swelling, and rheumatic pain. The species, considered critically endangered, is legally protected in many countries, which precludes its collection from natural habitats. Attempts to cultivate it have so far proven unreliable [3]. To meet the demand for arnica products, another species that provides an equivalent raw material, i.e., meadow arnica (Arnica chamissonis Less.), has been successfully introduced into cultivation in Europe [4,5]. This species is native to the western regions of North America and Canada [6].

The flower heads of both Arnica species share a similar chemical composition, rich in sesquiterpene lactones and phenolics, primarily flavonoids and phenolic acids [7]. The sesquiterpene lactones are represented by pseudoguaianolides, helenalin, 11α,13-dihydrohelenalin, and their derivatives (e.g., chamissonolide) [8,9,10]. Among flavonoids, there are flavones such as apigenin, luteolin, hispidulin, and eupafolin, as well as flavonols including kaempferol, quercetin, 6-methoxykaempferol, and isorhamnetin, present both in free and glycosidically bound forms [8,11,12,13]. Within phenolic acids, caffeic acid and its esters with quinic, chlorogenic, and gallic acids are predominant [13,14,15,16]. Meadow arnica flower heads also contain small amounts of essential oil, in which α-pinene, cumene, p-cymene, germacrene, and spathulenol constitute the main substances [12,17,18].

Due to the wide range of biologically active compounds, arnica flower heads exhibit various pharmacological activities, including anti-inflammatory, antiseptic, antirheumatic, antioxidant, antibacterial, analgesic, anti-edema, and regenerative [12,13,18,19,20,21,22]. The raw material is used to prepare ethanolic tinctures applied externally (in the form of compresses, rubs, and ointments) for the treatment of skin and mucous membrane inflammation, as well as for edema, hematomas, bruises, or swellings. The arnica extracts stimulate blood circulation, strengthen blood vessels, accelerate healing, and promote the resorption of subcutaneous hemorrhages [12,13,18,19,20,21,22]. Such applications are well established in the pharmaceutical industry and are becoming increasingly common in the cosmetic sector. This trend aligns with the growing interest in ‘green cosmetics’, which emphasize the use of natural, plant-based ingredients and environmentally friendly production practices [23,24,25].

Given the growing interest of the herbal industry in meadow arnica, it is important to identify the factors determining its yield and the quality of the raw materials, which are not only related to the high content of biologically active compounds but also to the presence of harmful pesticide residues. Such raw materials can currently be obtained using organic farming methods. This production system relies on natural processes and environmental interactions to support plant growth and yield. Among these, root symbioses with AMF are one of the most effective. Currently, AMF inoculants are widely used in agricultural practice [26]. Their application provides numerous benefits, primarily by increasing root surface area, thereby enhancing water and mineral nutrient uptake, especially phosphorus. Inoculation of plants with properly selected AMF may increase their resistance not only to water deficiency, but also to salinity and pathogens [26,27,28,29,30]. As a result, it contributes to higher yields. Among medicinal and aromatic plants, it is considered to stimulate the accumulation of biologically active compounds, which is particularly important for the quality of herbal raw materials. This topic has recently been summarized by Yuan et al. [26], Zhao et al. [31], and Thockchom et al. [32].

The application of AMF seems promising for the genus Arnica. It was shown that the roots of wild-growing A. montana are colonized by a wide range of AMF, and such symbiosis enhances, or even determines its survival on nutrient-poor soils, which are typically occupied by the species [33]. So far, the studies on the impact of AMF on A. chamissonis are scarce [34]. It appears that this species, like mountain arnica, may be susceptible to forming AMF root symbiosis, especially in organic cultivation. In such symbiosis, through more efficient nutrient uptake, assimilation, and carbohydrate redistribution, plants may achieve more harmonious development, more abundant flowering, and, therefore, higher yields of flower heads. It also appears to influence the accumulation of biologically active compounds, including phenolics, which are considered indicators of plant stress.

The purpose of this work was to investigate the influence of inoculation of meadow arnica seedlings with AMF on the development, yield, and content of biologically active compounds in flower heads of the plants cultivated under an organic farming system.

2. Materials and Methods

2.1. Seedlings Production and AMF Inoculation

The seeds of meadow arnica (Arnica chamissonis L.) were purchased from the Jelitto Staudensamen GmbH (Schwarmstedt, Germany). The seedlings were produced in the greenhouse of the Warsaw University of Life Sciences (WULS-SGGW). The seeds were sown in the first ten days of January 2024 into garden boxes filled with peat substrate of pH 6. Six weeks after germination, the seedlings were transplanted into 96-cell seedling trays. Half of the seedlings were transplanted into the substrate inoculated with AMF (+AMF), and the other half into the substrate without the inoculum (−AMF; control). The AMF inoculum (Symbvit^®) was obtained from Symbiom Ltd. (Lanškroun, Czech Republic). The composition of the mycorrhizal inoculum is presented in detail by Bączek et al. [35].

2.2. Establishment of the Field Experiment

The field experiment was carried out at the WULS–SGGW Experimental Field (52.15988 N, 21.10056 E, 85 m a.s.l.) located in the suburbs of Warsaw, on alluvial soil. In April, well-rooted seedlings were planted out at a spacing of 40 cm × 30 cm, in four randomized replications per combination (−AMF and +AMF). The size of a single plot was 20 m² (12 plants per m²). The climatic conditions during the growing seasons are presented in

Table 1. The climatic parameters in the vegetative season of 2024.

	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	AVG
T (°C)	−1.9	−0.8	3.2	9.3	14.6	18.0	20.1	19.5	14.7	9.3	4.8	0.5	8.48
SH (h)	77.5	95.2	170.5	264.0	322.4	330.0	344.1	322.4	222.0	155.0	93.0	71.3	205.62
RF (mm)	47	42	48	52	71	71	89	68	62	48	48	49	58
AH (%)	83	81	75	66	65	64	67	67	72	78	85	84	74

Source: Warsaw Annual Weather Averages—Poland (https://pl.weatherspark.com, accessed on 24 March 2026); T—temperature; SH—sunshine hours; RF—rainfall; AH—air humidity.

. Soil parameters are shown in

Table 2. Soil parameters: pH, the content of main nutrients (mg/L), salinity (g KCl/L), and organic matter (%).

pH	NO3−	NH4+	P	K	Ca	Mg	Cl	Salinity	Organic Matter
5.8	64	6	34	81	666	129	87	0.55	2.28

. The Experimental Field was certified in accordance with the organic farming production rules by Ekogwarancja Ltd. (Dąbrowica, Poland).

2.3. Mycorrhizal Frequency and Intensity

Root samples were collected at the full-flowering stage of plant development (29 July 2024), separately for the −AMF and +AMF treatments. The colonization of meadow arnica roots was determined according to the protocol described in the earlier study by Sujkowska-Rybkowska et al. [36]. Randomly selected root pieces, collected from each combination (from 3 plants per treatment; 90 root fragments per plant) were mounted on slides after staining with aniline blue, and the following characteristics were recorded: presence or absence of AMF structures (arbuscules, hyphae and vesicles), as well as the mycorrhizal frequency (F%) and relative mycorrhizal intensity (M%) which were calculated using the MYCOCALC program (INRA, Dijon, France).

2.4. Plant Growth Parameters

At the full flowering stage, the following plant developmental traits were determined: the number of flowering shoots per plant, the length of the flowering shoots (cm), the number of flower heads per plant, fresh mass of a single flower head (g), fresh and dry mass (DM) of flower heads (g/plant), fresh and dry mass of the herb, i.e.,: green, not lignified shoots with the leaves and flowers (g/plant), fresh and dry mass of the roots (g/plant). The observations were conducted on 10 randomly selected plants per plot.

2.5. Plant Raw Materials Used for the Chemical Analysis

Harvesting of raw materials (leaves, roots, and flower heads) for chemical analysis was carried out during the full flowering stage. Fresh leaves were used to assess the contents of chlorophyll a, b, and carotenoids. In the fresh leaves and roots (separately), the general and reducing sugars, as well as starch contents, were analyzed, while in the dried leaves and roots, the contents of macro elements were determined. Dried flower heads, a herbal raw material, were analyzed for total flavonoid and phenolic acid contents, and for selected phenolic compounds using HPLC. The leaves and flower heads were dried at 35 °C, and the roots at 60 °C. All the chemical analyses were performed in triplicate, and the results were expressed as mean values.

2.6. Chlorophyll a, b, and Carotenoid Content

For the analysis, 0.50 g of fresh plant material was used. It was homogenized in a porcelain mortar with 80% (v/v) ethanol and a small amount of quartz sand. The resulting extract was filtered into a volumetric flask, and the filter was rinsed with 80% ethanol until the filtrate became colorless. The final volume was adjusted to 50 mL with 80% ethanol. Chlorophyll a, chlorophyll b, and carotenoid contents were determined according to the method by Lichtenthaler and Wellburn [37] with further modification [38]. Absorbance was measured at 470, 646, 652, and 663 nm against 80% ethanol as a blank using a Shimadzu UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan). All abovementioned pigment contents were expressed as mg per g of dry matter (mg/g DM).

2.7. General and Reducing Sugar Contents

For the analysis, 0.50 g of fresh plant material was used. The tissue was homogenized in a porcelain mortar with boiling 80% (v/v) ethanol. Samples were centrifuged for 5 min at 12,000 rpm using a Sigma 3K30 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The supernatant was transferred to volumetric flasks and adjusted to a final volume of 25 mL with 80% ethanol.

The general sugar content was determined according to the phenol–sulfuric acid method of Dubois et al. [39]. 100 μL of the ethanolic extract was mixed with 1 mL of 5% phenol solution, followed by the rapid addition of 5 mL of sulfuric acid (96%). The reaction mixture was incubated at room temperature for 20 min. The absorbance was measured at 490 nm using a Shimadzu UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan).

Reducing sugars were quantified according to the method described by Nelson [40]. 100 μL of the extract was mixed with 1 mL of copper reagent and heated in a boiling water bath (100 °C) for 20 min. After cooling 1 mL of arsenomolybdate reagent was added, and the solution was diluted to a final volume of 5 mL with distilled water. The absorbance was measured at 520 nm using a spectrophotometer.

Sugar concentrations were calculated from calibration curves prepared with standard glucose solutions. The results were expressed as mg glucose equivalents per g of dry matter (mg glucose/g DM). An 80% ethanol solution was used as the blank.

2.8. Starch Content

The analysis was performed using the pellet obtained after centrifugation during the preparation of extracts for sugar determination. The pellet was treated with 2.5 mL of 1 M sulfuric acid and incubated in a water bath for 20 min. Subsequently, 5 mL of distilled water was added twice, followed by centrifugation for 5 min at 12,000 rpm using a Sigma 3K30 centrifuge. The supernatant was transferred to volumetric flasks and brought to a final volume of 25 mL with distilled water. Starch content was determined using the anthrone method described by Samotus and Pałasiński [41]. Absorbance was measured at 625 nm against a reagent blank using a Shimadzu UV-1280 spectrophotometer. Starch content was expressed as mg per g of dry matter (mg/g DM).

2.9. Macroelements Content

The extraction solution was prepared according to the Spurway-Lawton [42] method. In the analysis, 0.40 g of powdered raw material was shaken (PAN, Warsaw, Poland) for 30 min with 100 mL of 2% acetic acid and a bit of activated carbon and filtered. Calcium and potassium content were determined using a flame photometer (Hornik Model 410, Hornik, Poznań, Poland). The content of NO₃⁻ was measured by the Foss FIAstar Analyzer 5000 (Foss, Hilleroed, Denmark) at a wavelength of 540 nm. Phosphorus content was determined spectrophotometrically using the vanadomolybdate colorimetric method. An aliquot of 10 mL of the obtained filtrate was transferred to a test tube and mixed with 2.5 mL of a vanadomolybdate reagent (a mixture of ammonium metavanadate and ammonium molybdate in a 1:1 ratio). After a 1 h reaction period, the intensity of the yellow complex formed between phosphate ions and the vanadomolybdate reagent was measured at 460 nm using a Shimadzu UV-1280 spectrophotometer. The blank solution consisted of 10 mL of 2% acetic acid and 2.5 mL of the vanadomolybdate reagent. The phosphorus concentration was calculated from a calibration curve prepared using standard solutions of KH₂PO₄. The results were expressed as g per 100 g of dry matter (g/100 g DM).

2.10. Total Content of Flavonoids

The total flavonoid content was determined according to the pharmacopoeial method described in FP VI [43]. For the analysis, 0.50 g of air-dried, powdered plant material was extracted under reverse column with acetone, 25% hydrochloric acid, and methenamine (0.5%) for 30 min. The extract was subjected to liquid–liquid extraction using ethyl acetate. An obtained extract was then reacted with aluminum chloride (2% w/v) reagent to form a flavonoid–aluminum complex. Absorbance was measured at 425 nm using a Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The flavonoid content was expressed as a percentage relative to quercetin (%).

2.11. Total Content of Phenolic Acids

The total phenolic acid content was determined according to the pharmacopoeial method described in FP VI [43]. For the analysis, 1.00 g of air-dried plant material was extracted twice with 25 mL of methanol for 30 min using a mechanical shaker. The obtained extract was reacted with hydrochloric acid solution (18 g/L), Arnov’s reagent, and sodium hydroxide solution (40 g/L), yielding a colored complex with phenolic acids. Absorbance was measured at 490 nm using a Shimadzu UV-1800 spectrophotometer. The phenolic acid content was expressed as a percentage relative to caffeic acid (%).

2.12. Analysis of Selected Flavonoids and Phenolic Acids by HPLC

Qualitative and quantitative analyses of flavonoids and phenolic acids were performed using a Shimadzu Prominence HPLC system (Shimadzu, Kyoto, Japan) equipped with an SIL-20AC HT autosampler, two LC-20AD pumps, a CTO-10AS VP column oven, and an SPD-M20A UV/VIS diode-array detector. Data acquisition and processing were carried out using LCsolution software version 1.21 SP1 (Shimadzu, Kyoto, Japan).

The analytical standards preparation and validation methodology were previously described by Bączek et al. [44]. Chromatographic separation was achieved on a Kinetex^® C18 column (2.6 µm, 100 Å, 100 mm × 4.6 mm i.d.; Phenomenex^®, Torrance, CA, USA) using a binary mobile phase consisting of water acidified with phosphoric acid (solvent A) and methanol (solvent B), at a flow rate of 1.2 mL/min. The column temperature was maintained at 45 °C, and the injection volume was 1 µL. The gradient elution program was as follows: 0.01 min, 40% B; 2 min, 50% B; 6 min, 100% B; 6.01 min, 40% B; 8 min, 40% B; 10 min, end of analysis. UV spectra were recorded over 190–515 nm.

1.00 g of air-dried raw material was extracted with 10 mL of methanol–water (50:50, v/v) at room temperature for 15 min in an ultrasonic bath. After filtration, the extract was diluted with methanol to a final volume of 10 mL and filtered through a cellulose syringe filter (Supelco^® Iso-Disc™, Nottingham, England, 25 mm diameter, 0.20 µm pore size) prior to HPLC analysis.

The following compounds were identified in the plant extracts (Figure 1): six flavonoids (cynaroside syn. luteolin 7-O-glucoside, rutin syn. quercetin 3-O-rutinoside, hyperoside syn. quercetin 3-O-galactoside, cosmosiin syn. apigenin 7-O-glucoside, astragalin syn. kaempferol 3-O-glucoside, diosmetin syn. luteolin 4′-methyl ether) and eight phenolic acids (neochlorogenic acid, chlorogenic acid, caffeic acid, ferulic acid, rosmarinic acid, cichoric acid, 3,4-di-O-caffeoylquinic acid, and 1,5-dicaffeoylquinic acids). Quantification was performed using external calibration, and results were expressed as mg per 100 g of dry matter (mg/100 g DM).

2.13. Statistical Analysis

Statistical analyses were performed using Statistica software (version 13.3; TIBCO Software, Palo Alto, CA, USA). Data were analysed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, with statistical significance set at α = 0.05. Results are presented as mean ± standard deviation (SD). Full statistical analysis is presented in Appendix A.

3. Results and Discussion

The process of mycorrhizal symbiosis is well described in the literature. It begins in the rhizosphere, and is regulated by both plant and fungal metabolites, including phytohormones, which coordinate partner recognition, colonization, and the formation of symbiotic structures. During the pre-symbiotic stage, root-released phenolics and 2-hydroxy fatty acids induce specific morphological responses in fungi, while strigolactones and cutin monomers stimulate hyphal formation on root surfaces [45,46,47]. The specificity of plant-AMF associations is largely established at this early stage. Following AMF colonization, host plant roots undergo physiological and metabolic adjustments that promote symbiosis and sustain the fungal partner. Further changes are observed in the root cortex cells, e.g., the number of mitochondria increases, and they migrate toward arbuscules (tree-like hyphal structures typical of AMF), which are the main sites of nutrient exchange between the fungus and the host plant [48]. These physiological changes are accompanied by metabolic shifts that influence the production of both primary and secondary metabolites [48,49].

3.1. The Effect of AMF Inoculation on Mycorrhizal Colonization of Arnica Roots

The analysis of meadow arnica roots, carried out in our study, revealed the presence of AMF in the samples collected from both inoculated and non-inoculated (control) plants (Figure 2). The presence of AMF in control plants can be considered as a natural phenomenon, as AMF are commonly found in all non-sterile soil substrates [50,51,52,53,54,55]. Moreover, in an organic farming system, avoiding chemical fertilizers (particularly phosphorus) and pesticides protects the fungal network, while moderate tillage allows mycorrhizal hyphae to persist. Thus, organic fields exhibit higher AMF species richness and diversity (roughly double in some studies) than do conventional fields [56]. In our studies, inoculating meadow arnica at an early developmental stage significantly increased mycorrhizal colonization parameters, i.e., frequency and intensity (Figure 3 and Figure 4). The additional use of AMF during seedling growth was more effective than the naturally occurring mycorrhiza alone. Generally, the introduction of AMF can effectively establish in soils with native AMF communities and improve plant growth, but this depends on the selection of AMF species used [57]. Heijne et al. [55] claim that root colonization levels depend on many factors, including the host plant, weather conditions, soil pH, and nutrient content. Studies carried out on the colonization of meadow arnica roots are relatively scarce. According to Zubek et al. [54], the mycorrhizal frequency of meadow arnica grown in a botanical garden, without AMF inoculation, reached 88%, while its intensity was 61.3%. However, the authors did not specify the factors that may have influenced the results.

3.2. The Effect of AMF Inoculation on Meadow Arnica Development and Mineral Uptake

In our work, inoculating plants with AMF improved their growth and increased the biomass of both the herb and the roots (

Table 3. Developmental characteristics of the plants.

Developmental Traits	−AMF	+AMF
Number of flowering shoots per plant	4.33 ± 1.25	7.00 ± 4.24
Length of flowering shoots (cm)	34.38 ± 3.62	36.67 ± 1.84
Number of flower heads per plant	29.00 ± 10.42	41.67 ± 10.87
Fresh mass of a single flower head (g)	0.50 ± 0.08	0.39 ± 0.07
Fresh mass of flower heads (g/plant)	14.47 ± 4.63	22.58 ± 5.59
Dry mass of flower heads (g/plant)	2.90 ± 0.93	6.08 * ± 0.94
Fresh mass of herb (g/plant)	116.67 ± 22.32	151.28 ± 10.21
Dry mass of herb (g/plant)	15.13 ± 2.92	25.09 * ± 1.83
Fresh mass of roots (g/plant)	32.42 ± 2.71	38.84 ± 4.75
Dry mass of roots (g/plant)	5.79 ± 0.50	8.22 ± 1.31

* α = 0.05.

). According to Schweiger and Müller [58], although mycorrhizae are morphologically restricted to underground organs, their physiological and metabolic alterations affect the entire plant. It is known that the symbiosis modifies root system architecture, particularly total root length, surface area, and volume [59]. These changes enhance nutrient and water uptake, leading to more effective plant growth and development [60,61]. In the present study, inoculated plants (+AMF) produced more flowering shoots per plant than did non-inoculated plants (−AMF), suggesting better branching and greater flowering potential under AMF colonization. The length of flowering shoots, however, was similar in both treatments, indicating that mycorrhiza affected shoot number rather than shoot elongation. Although the fresh mass of a single flower head (herbal raw material) was slightly lower in inoculated plants, their number per plant was distinctly higher, thus the total mass of the raw material was clearly higher (Table 3).

According to the literature, the yield of meadow arnica flower heads per hectare ranges from 100 to 600 kg [62,63,64] and depends on many factors, e.g., cultivation region, weather conditions, or fertilization [65,66,67,68,69]. It is worth noting that in our study, the yield of meadow arnica flower heads was over 700 kg per hectare in +AMF plants, whereas in the case of −AMF, it was less than 300 kg per hectare. The results obtained in the study are consistent with those of other authors on the application of AMF in the cultivation of chamomile, pot marigold, and African marigold [35,70,71].

In our study, inoculated plants showed slightly higher chlorophyll a and chlorophyll b content in leaves than did non-inoculated plants (

Table 4. The content of photosynthetic pigments and carbohydrates.

Plant Material	−AMF	+AMF
Leaves
Chlorophyll a (mg/g DM)	7.05 ± 0.28	7.73 * ± 0.11
Chlorophyll b (mg/g DM)	1.81 ± 0.10	2.31 ± 0.39
Carotenoids (mg/g DM)	1.51 ± 0.09	1.45 ± 0.06
General sugar (mg glucose/g DM)	154.56 ± 3.41	178.16 * ± 10.75
Reduced sugar (mg glucose/g DM)	92.74 * ± 1.77	27.11 ± 3.27
Starch (mg/g DM)	26.00 * ± 1.63	11.00 ± 1.22
Roots
General sugar (mg glucose/g DM)	289.10 ± 15.68	245.11 ± 26.40
Reduced sugar (mg glucose/g DM)	14.96 ± 1.43	15.04 ± 1.44
Starch (mg/g DM)	303.00 ± 11.14	408.00 * ± 10.40

* α = 0.05.

). This suggests enhanced light absorption capacity and improved photosynthetic apparatus efficiency under the applied treatment. Latef and Chaoxing [72] reported that the synthesis of chlorophylls was enhanced in Solanum lycopersicum when inoculated with AMF.

Significant differences in carbohydrate distribution between the leaves and roots of inoculated and non-inoculated meadow arnica were observed (Table 4). The leaves of +AMF plants were characterized by a markedly higher content of general sugars (178.16 mg glucose/g DM) compared to −AMF ones (154.56 mg glucose/g DM), which may be related to a stronger photosynthetic activity followed by the accumulation of soluble carbohydrates in the aboveground organs. However, the contents of reduced sugars and starch were lower in the leaves of inoculated plants (27.11 and 11.00 mg/g DM, respectively) than in non-inoculated ones (92.74 and 26.00 mg/g DM), suggesting that under mycorrhizal treatment, these carbohydrates were redirected to other metabolic pathways or exported to other organs (Table 4). In the roots, the pattern was different: −AMF plants showed slightly higher general sugar content (289.10 mg glucose/g DM) than +AMF (245.11 mg glucose/g DM), whereas reduced sugar levels were similar in both treatments (14.96 and 15.04 mg glucose/g DM). +AMF plants, however, accumulated more starch in the roots (408.00 mg/g DM) compared to −AMF plants (303.00 mg/g DM), indicating that mycorrhizal colonization favoured carbohydrate storage in underground organs, which may support both the root growth and fungal symbiont (Table 4). Carbohydrates play a key role in the symbiosis between plants and AMF, serving as the main carbon source for the fungal partner [73,74]. It is worth noting that AMF cannot perform photosynthesis and thus relies on the products provided by host plants [75,76]. Thus, roots colonized by AMF act as a strong carbon sink, redirecting assimilates from the shoots towards the mycorrhizal tissues. Carbohydrates may be absorbed directly from the host plant by the AMF and stored in vesicles as triacylglycerols in liposomes, anhydride sugars, or organic acids [77,78,79]. Products in those forms are difficult for host plants to utilize. Plants may allocate 10–20% of the carbon fixed by photosynthesis to fungal vesicles in exchange for nutrients and water [80]. It is also worth noting that under optimal growth conditions, soluble compounds, including reduced sugars, may not be stored in abundance in the plant. This phenomenon is more commonly observed under stressors such as drought, temperature extremes, or flooding [81], as the sugars play a significant role in regulating plant tolerance to abiotic stresses [82], serving as carbon reserves, scavengers of reactive oxygen species (ROS), and stabilizers of cell membranes [83,84]. On the other hand, the higher starch content in Arnica roots of +AMF plants may be explained by sugar transport from the shoot to the root, followed by its accumulation as starch. Similar observations were made by Gutjahr et al. [85] in Lotus japonicus starch mutants.

In our study, inoculation with AMF altered the uptake of macroelements in meadow arnica, with distinct patterns observed between leaves and roots (

Table 5. The content of macroelements (g/100 g DM).

Plant Material	−AMF	+AMF
Leaves
Phosphorous (P)	0.219 * ± 0.007	0.137 ± 0.019
Nitrate nitrogen (NO3)	1.071 * ± 0.105	0.487 ± 0.079
Nitrite nitrogen (N-NO2)	0.242 * ± 0.042	0.110 ± 0.016
Calcium (Ca)	0.321 ± 0.032	0.392 * ± 0.015
Potassium (K)	5.922 * ± 0.162	5.334 ± 0.176
Roots
Phosphorous (P)	0.085 ± 0.020	0.108 ± 0.015
Nitrate nitrogen (NO3)	0.198 ± 0.010	0.261 * ± 0.017
Nitrite nitrogen (N-NO2)	0.045 ± 0.012	0.059 ± 0.001
Calcium (Ca)	tr #	tr
Potassium (K)	2.043 ± 0.038	2.161 * ± 0.033

* α = 0.05, ^# trace amounts.

). Regarding the leaves, +AMF plants were characterised by approximately twofold lower phosphorus and nitrogen (nitrate and nitrite forms) content than in −AMF plants. This may indicate more efficient utilization or redistribution of these macroelements under AMF colonization. In contrast, calcium content in the leaves was slightly higher in inoculated plants in comparison to non-inoculated (0.392 and 0.321 g/100 g DM, respectively), whereas potassium content was found to be lower (5.334 and 5.922 g/100 g DM) (Table 5).

In the case of the roots, AMF inoculation visibly increased phosphorus levels, as well as nitrate and nitrite nitrogen, compared with non-inoculated plants, indicating improved nutrient acquisition under symbiotic conditions. However, calcium was detected only in trace amounts in both treatments, while potassium content was slightly higher in +AMF compared to −AMF (2.161 and 2.043 g/100 g DM, respectively) (Table 5). The phenomenon of enhanced mineral uptake under mycorrhization, especially for phosphorus, is widely documented in the literature [86,87,88,89,90,91]. Usually, fungal hyphae extend into the soil surrounding the roots, potentially modifying its structure, improving nutrient accessibility, and fertility [92,93,94]. It is also noted that under favourable environmental conditions and high nutrient availability, arbuscular mycorrhiza may have a limited impact on plant growth and development, whereas under various types of stress, their effect becomes much more pronounced [95,96,97]. Therefore, the use of AMF is more effective in organic farming systems, which rely on natural fertilizers with slower nutrient release, encouraging plants to form stronger symbiotic connections with AMF to acquire nutrients (specifically phosphorus and nitrogen), and making the inoculation more beneficial [56,98].

3.3. The Content and Composition of Phenolics in Arnica Flower Heads

In our experiment, the total content of flavonoids in meadow arnica flower heads was at a level of 0.57% in non-inoculated plants and 0.44% in inoculated ones. Six compounds were identified in the raw material: cynaroside, rutin, hyperoside, cosmosiin, astragalin, and diosmetin. Cynaroside was identified as the dominant compound here. There was a higher concentration of all the identified flavonoids in −AMF compared to +AMF (

Table 6. The total content of flavonoids (%) and the content of identified flavonoids (mg/100 g DM) in flower heads.

Flavonoids	−AMF	+AMF
Total content	0.57 * ± 0.01	0.44 ± 0.02
Cynaroside	132.13 * ± 1.75	47.82 ± 3.65
Rutin	23.16 * ± 0.47	9.57 ± 0.99
Hyperoside	30.71 * ± 0.66	13.01 ± 0.73
Cosmosiin	14.52 * ± 0.11	7.13 ± 0.32
Astragalin	11.51 * ± 0.22	9.48 ± 0.15
Diosmetin	33.09 * ± 1.91	23.33 ± 0.44
Sum	245.13 * ± 2.61	110.33 ± 6.24

* α = 0.05.

). These results seem to contradict most studies on the accumulation of biologically active compounds in medicinal and aromatic plants (MAPs) inoculated with AMF. Mycorrhizal inoculation is often associated with an increase, or at least no reduction, in the accumulation of flavonoids [35,58,99,100,101]. Thus, the results obtained in our work may indicate a distinctive metabolic response in meadow arnica and suggest that the symbiosis may modulate the production and/or distribution of flavonoids in a slightly different manner than previously described for other MAPs. When considering this phenomenon in the context of plant developmental stage and assimilate production in AMF-inoculated plants, it should be noted that flowering is an energy-intensive process that requires the transport of carbohydrates to the generative organs. During this transition, resources are often diverted away from secondary metabolism (such as the synthesis of certain defense-related phenolics) to prioritize growth and reproductive development. The premise that high sugar levels do not always lead to a corresponding increase in phenolic compounds, particularly during intense developmental phases such as flowering, is supported by several studies that indicate similar metabolic trade-offs among growth, storage, and defense [102,103].

In the case of phenolic acids, we observed that mycorrhizal inoculation modified both the total content and the qualitative profile of these compounds. Eight compounds were identified, all representing hydroxycinnamic acid derivatives, including neochlorogenic, chlorogenic, caffeic, ferulic, rosmarinic, cichoric, 4-di-O-caffeoylquinic, and 1,5-dicaffeoylquinic acids. Among these, ferulic acid was clearly dominant (

Table 7. The total content of phenolic acids (%) and the content of identified phenolic acids (mg/100 g DM) in flower heads.

Phenolic Acids	−AMF	+AMF
Total content	0.71 * ± 0.02	0.52 ± 0.01
Neochlorogenic acid	23.72 * ± 0.71	9.92 ± 0.87
Chlorogenic acid	96.53 * ± 0.74	39.95 ± 3.94
Caffeic acid	1.45 * ± 0.03	0.28 ± 0.04
Ferulic acid	293.29 * ± 13.37	129.57 ± 8.54
Rosmarinic acid	79.31 * ± 1.51	25.31 ± 2.11
3,4-Di-O-caffeoylquinic acid	3.67 * ± 0.05	2.15 ± 0.05
1,5-Dicaffeoylquinic acid	84.16 * ± 2.66	36.53 ± 3.09
Cichoric acid	58.1 * ± 1.58	23.77 ± 2.59
Sum	640.24 * ± 14.26	267.49 ± 21.07

* α = 0.05.

). Both the total content and the content of individual phenolic acids were higher in −AMF than in +AMF plants. A similar trend in phenolic acid accumulation was previously described by Wu et al. [104] in Salvia miltiorrhiza inoculated with AMF. However, this tendency was observed only when AMF communities were rich (with more than three or four AMF applied). In turn, studies on soybeans showed that the use of Funneliformis mosseae alone in monoculture regulates the expression of genes associated with the phenolic acid metabolic pathway, inhibiting their production [105].

The relationship between plant mycorrhization and phenolic acid content has been reported previously for many other MAPs, suggesting its species-dependency. For instance, Geneva et al. [106] reported a significant decrease in the total content of phenolic acids in the leaves of mycorrhizal Salvia officinalis; the study by Engel et al. [107] reported an increased content of rosmarinic and lithospermic acid A in Melissa officinalis, while both these compounds were diminished in Majorana hortensis. Li et al. [108] observed a significant change of ferulic acid under mycorrhizal inoculation in Citrus trifoliata. This relationship has also been documented for Echinacea purpurea, Salvia miltiorrhiza, Astragalus membranaceus, Curcuma longa, Citrus trifoliata, and Ipomoea purpurea [101,108,109,110,111,112].

It is worth noting that the content of phenolics in plants is influenced by many factors, consistent with their physiological roles closely linked to plant responses to various stressors, as these compounds are involved in protection, competition, adaptation, signalling, and defence. In addition, phenolic acids, as structural components of lignin, are incorporated into cell walls and reinforce them, for example, during pathogen attack [113,114]. Therefore, the content of phenolics in MAPs should be considered more as the combined effect of the plants’ genotype, age, climatic conditions, or biotic and abiotic factors of the habitat in which they grow than simply as a result of the agrosystems in which they are produced.

Nowadays, one of the main challenges in MAP cultivation is the instability of raw material quality, reflected primarily in low and unpredictable levels of biologically active compounds. Another important issue is the purity of the raw material. Herbal raw materials are expected to be free from toxic pesticide residues. Hence, more and more MAP crops are grown using organic farming methods. The implementation of AMF in their cultivation is widely recognised as beneficial here, as it can improve plant condition and, consequently, yields [32,110,115]. Numerous studies have shown that physiological changes in MAPs inoculated with AMF, such as increased chlorophyll content and improved photosynthetic efficiency, are also associated with secondary metabolite biosynthesis [116,117,118,119,120]. However, the molecular basis of AMF inoculation’s effect on the biosynthesis remains unclear, particularly given that this symbiosis involves complex signalling between host plants and fungi, is highly species-specific, and depends on many abiotic factors [116,121,122]. Pronounced reprogramming of the phenylpropanoid pathway in response to AMF colonisation has been reported in Vitis [123]. It is also worth noting that the positive effect of AMF fungi on this pathway is often attributed to increased phosphorus and nitrogen acquisition, which provides the nutritional basis for the intensification of secondary metabolism [93,124,125]. In general, secondary metabolites, including phenolics, play a dynamic role in plants’ adaptation to the environment, and are synthesized primarily as components of defence mechanisms [126]. Thus, colonization by AMF improves plant health by augmenting the overall defence system [127,128].

Despite the still limited knowledge about the interactions between AMF and MAPs plants, the available data, including the results of the present study on A. chamissonis, support the view that mycorrhizae may be important for the sustainable production of raw materials from MAPs. Nevertheless, to fully exploit the potential of AMF to increase the yield and quality of plant raw materials, further research integrating plant physiology, metabolomics, and agronomy is needed.

4. Conclusions

Our research has shown that inoculating meadow arnica with AMF at an early stage of plant growth significantly affects its development, resulting in increased mass of herbal raw material, i.e., flower heads. In terms of physiological traits, AMF inoculation increased leaf chlorophyll a and b content, accompanied by shifts in carbohydrate production and its relocation from leaves to roots, leading to enhanced starch storage in the roots. At the same time, AMF modified plant nutrient balance, increasing phosphorus and nitrogen content in the roots and decreasing their levels in the leaves, suggesting efficient acquisition and redistribution of these key nutrients within the plant. AMF inoculation significantly affected the phenolics content in meadow arnica flower heads. The content was higher in non-inoculated plants than in inoculated ones. Finally, the results demonstrated that AMF inoculation leads to a ‘growth-defence’ compromise, as evidenced by an increase in biomass followed by a decrease in phenolics in the herbal raw material. This result, although partly divergent from studies on other MAP species, appears closely related to improved physiological status and higher stress tolerance in inoculated plants, which may be interpreted as an indicator of better overall plant condition. However, the observed decrease in phenolics in flower heads affects the quality of the raw material. This highlights the need for further studies on the balance among mycorrhizae, plant productivity, and raw material quality.