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Article

Nitrogen Fertilization and Glomus Mycorrhizal Inoculation Enhance Growth and Secondary Metabolite Accumulation in Hyssop (Hyssopus officinalis L.)

by
Saeid Hazrati
1,*,
Marzieh Mohammadi
1,
Saeed Mollaei
2,
Mostafa Ebadi
3,
Giuseppe Pignata
4 and
Silvana Nicola
4
1
Department of Agronomy and Plant Breeding, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz 5375171379, Iran
2
Phytochemical Laboratory, Department of Chemistry, Faculty of Sciences, Azarbaijan Shahid Madani University, Tabriz 5375171379, Iran
3
Department of Biology, Faculty of Sciences, Azarbaijan Shahid Madani University, Tabriz 5375171379, Iran
4
Department of Agricultural, Forest, and Food Sciences, Inhortosanitas Lab, University of Torino, 10095 Grugliasco, Italy
*
Author to whom correspondence should be addressed.
Nitrogen 2025, 6(3), 60; https://doi.org/10.3390/nitrogen6030060
Submission received: 5 June 2025 / Revised: 10 July 2025 / Accepted: 23 July 2025 / Published: 26 July 2025
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Abstract

Nitrogen (N) availability often limits primary productivity in terrestrial ecosystems, and arbuscular mycorrhizal fungi (AMF) can enhance plant N acquisition. This study investigated the interactive effects of N fertilization and AMF inoculation on N uptake, plant performance and phenolic acid content in Hyssopus officinalis L., with the aim of promoting sustainable N management in H. officinalis cultivation. A factorial randomized complete block design was employed to evaluate four AMF inoculation strategies (no inoculation, root inoculation, soil inoculation and combined root–soil inoculation) across three N application rates (0, 0.5 and 1,1 g N pot−1 (7 L)) in a controlled greenhouse environment. Combined root and soil AMF inoculation alongside moderate N fertilization (0.5 mg N pot−1) optimized N use efficiency, maximizing plant biomass and bioactive compound production. Compared to non-inoculated controls, this treatment combination increased N uptake by 30%, phosphorus uptake by 24% and potassium uptake by 22%. AMF colonization increased chlorophyll content and total phenolic compounds under moderate N supply. However, excessive N application (1 g N pot−1) reduced AMF effectiveness and secondary metabolite accumulation. Notably, AMF inoculation without N fertilization yielded the highest levels of anthocyanin and salicylic acid, indicating differential N-dependent regulation of specific biosynthetic pathways. The interaction between AMF and N demonstrated that moderate N fertilization (0.5 g N pot−1) combined with dual inoculation strategies can reduce total N input requirements by 50%, while maintaining optimal plant performance. These findings provide practical insights for developing N-efficient cultivation protocols in medicinal plant production systems, contributing to sustainable agricultural practices that minimize environmental N losses.

1. Introduction

Medicinal plants are a valuable source of bioactive compounds that have wide-ranging applications in the pharmaceutical, food and cosmetics industries [1,2,3]. The quality of these plants is significantly influenced by soil properties and nutrient management strategies [4,5].
Hyssop (Hyssopus officinalis) is an aromatic, perennial medicinal plant belonging to the Lamiaceae family. There are seven taxonomically recognized species among the approximately 70 described variants of this plant [6,7]. This hardy species naturally thrives in dry, rocky, calcareous soils with limited nitrogen (N) availability in Asia, Europe and Africa [7,8,9,10]. It has been traditionally used worldwide to treat various ailments, including dizziness, inflammation, spasms and infections, and as an antiseptic agent [11,12]. H. officinalis derives its therapeutic value from its rich phytochemical composition, particularly the essential oils found in its aerial parts, which exhibit soothing, expectorant and digestive stimulant properties [13].
The essential oil yield typically ranges from 0.3% to 1% on a dry weight basis. Its chemical profile is dominated by oxygenated monoterpenes, particularly beta-pinene, pinocamphone and isopinocamphone, as well as sesquiterpene hydrocarbons and various phenolic compounds, such as polyphenols, flavonoids, phenolic acids and tannins. The oil also contains essential micronutrients, including vitamins C and E [6,10,14,15,16].
Effective N management is essential for cultivating medicinal plants as it directly affects the balance between growth and the production of secondary metabolites [17,18,19,20]. As it is a fundamental building block of amino acids, proteins and numerous secondary metabolites, N availability is directly linked to the biosynthetic pathways responsible for producing valuable phytochemicals. However, optimizing N management is a complex challenge that requires balancing vegetative growth with bioactive compound production. Research shows that controlled N stress can increase secondary metabolite production by activating plant defense pathways, although this generally reduces biomass accumulation [21]. This physiological trade-off is an adaptive mechanism whereby plants prioritize the production of protective secondary metabolites under nutrient-limited conditions.
The development of effective N fertilization strategies must consider economic feasibility and environmental sustainability. While insufficient N limits productivity, excessive application can lead to serious environmental consequences, including groundwater contamination through nitrate leaching, increased greenhouse gas emissions via nitrous oxide production and disruption to aquatic ecosystems through eutrophication [22,23]. Therefore, precise N management is essential for achieving optimal phytochemical yields while minimizing environmental impact and production costs.
Understanding this growth–defense trade-off is fundamental to developing evidence-based fertilization protocols that maximize the therapeutic value of medicinal plants. The challenge lies in identifying the optimal N supply level that enhances secondary metabolite production without severely compromising plant vigor and overall yield potential [24,25].
The effect of chemical and biological fertilization on hyssop yield and phytochemical traits has been targeted by only a few studies. Application of N and organic fertilizers significantly improved yield of flowers and percent and amount of active ingredient in H. officinalis [5,26,27]. A wide range of research in different regions of the world has proved that biological fertilizers can replace or supplement chemical fertilizers. N availability not only affects plant growth, yield and physiology, but also significantly influences the composition and diversity of soil microbial communities. Mycorrhizal fungi populations, in particular, respond dynamically to N fertilization regimes, with implications for plant–microbe interactions and the efficiency of nutrient cycling. Strategic fertilization approaches can manipulate the composition and activity of these communities to improve plant performance and secondary metabolite production [28].
The economic optimization of N fertilization requires the integration of multiple factors, including fertilizer costs, inoculation expenses, yield responses and the quality of secondary metabolites. To develop predictive models for such optimization, it is necessary to understand the dose–response relationships between N inputs and both growth and bioactive compound outputs, as well as the temporal dynamics of these responses under different environmental conditions [3].
Arbuscular mycorrhizal fungi (AMF) belong to the Glomeromycota phylum. These beneficial fungi form a mutually positive relationship with the root systems of around 90% of vascular plants worldwide. These fungi enhance the efficiency with which plants take up N and reduce the loss of nutrients from soil–plant systems. These beneficial microorganisms are a vital part of sustainable N management strategies, as they can improve plant growth and potentially influence the production of secondary metabolites by altering nutrient acquisition patterns. However, the intricate interplay between N fertilization, AMF colonization and secondary metabolism requires thorough investigation to establish effective management protocols [29,30,31,32]. However, it is important to acknowledge that controlled pot experiments, while valuable for studying specific plant–AMF interactions, may not fully replicate the complex multi-fungal networks that naturally occur in field conditions. The simplified experimental conditions in pot studies may limit the expression of the full potential of mycorrhizal symbiosis and N cycling dynamics that would be observed in natural or field agricultural systems. Among beneficial soil microorganisms, AMF are ecologically important and have considerable potential in sustainable crop management strategies that optimize N cycling [33,34,35]. AMF inoculation can significantly improve various plant growth parameters, such as leaf number and width, as well as shoot and root fresh and dry weights, often through enhanced N acquisition [36]. Recent research with Catalpa bungei has confirmed that mycorrhizal inoculation improves N and phosphorus uptake efficiency [37]. The symbiotic association of mycorrhizal fungi and the use of organic fertilizer significantly increased the dry weight, essential oil yield, citral accumulation and total phenol content of Melissa officinalis, demonstrating the synergistic effects of biological and chemical N sources [38]. A study on Stevia revealed that applying mycorrhizae under drought stress conditions resulted in notable increases in chlorophyll index, plant height, plant dry weight and leaf area compared to the control group. Furthermore, the use of mycorrhizae under stressful conditions was found to increase the content of N, potassium, glucose, fructose and total sugar [39].
Mycorrhizal fungi enhance N use efficiency through symbiotic relationships with plants, making them valuable candidates for sustainable N management in agricultural systems. Understanding these fungal–plant interactions is essential for developing effective strategies to optimize N cycling and reduce chemical fertilizer dependency [40]. In order to produce healthy medicinal and food products in sustainable, low-input agricultural systems—particularly for valuable medicinal plants such as hyssop—it is essential to examine the effect of biological and chemical fertilizers on plant performance, product quality and N cycling dynamics within the soil–plant–atmosphere continuum.

2. Materials and Methods

The present experiment was conducted during spring and summer of 2021 in the research greenhouse of the Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran (35°84′ N, 51°81′ E, 1215 m above sea level).
A factorial experiment was arranged in a randomized complete block design with three replications to evaluate two main factors: mycorrhizal fungi inoculation in four levels [M1: control (no inoculation), M2: root inoculation, M3: soil inoculation and M4: combined root and soil inoculation)] and nitrogen (N) fertilization in three levels [control (N1), 0.5 g N pot−1 (N2: equivalent to 40 kg N ha−1) and 1.0 g N pot−1 (N3: equivalent to 80 kg N ha−1)]. One-month-old seedlings of hyssop (Hyssopus officinalis L. subsp. angustifolius (Bieb.) Arcang.) obtained from the Zarin Giyah Urmia company were transplanted into pots on 24 April. A total of 72 pots were used, with three replicates per treatment, and in each replication, there were 2 pots for each treatment. Pots with a capacity of 7 L were filled with sterile soil, which was sterilized by autoclaving at 121 °C for 60 min to eliminate pathogens and weed seeds. Hyssop seedlings were then planted in the pots. Soil samples were collected randomly to analyze their physical and chemical properties before seedling transplantation (Table 1). A mixture of commercial arbuscular mycorrhizal fungi (AMF) inoculum, consisting of Glomus mosseae, Rhizophagus intraradices (syn. G. Intraradices) and G. etunicatum, was obtained from the Zist Fanavar Pishtaz Varian Company in Tehran, Iran, under the name ‘Mycoroot’. This formulation contained approximately 120 viable spores per gram. It was produced as a powder containing spores, hyphae and root fragments in a sterile vermiculite carrier medium. For root inoculation, at the time of transplanting, 20 g of AMF inoculum was applied directly to the root zone of each seedling by carefully dusting the inoculum powder onto the root surface and into the surrounding area. For soil inoculation, 20 g of AMF inoculum per pot was thoroughly mixed into the top 10 cm of the potting soil mixture prior to planting. Mechanical mixing ensured the inoculum was evenly distributed throughout the root zone.
Irrigation was triggered when soil water depletion reached 30% of field capacity, ensuring consistent soil moisture conditions throughout the study period.
N fertilization treatments were conducted using urea [CO(NH2)2] as the N source (Shiraz Petrochemical Company, Shiraz, Iran). Treatments were applied in two split applications, containing 46% N by weight as an organic compound, starting one month after plant establishment, with one month between applications. The N fertilizer was mixed with the soil twice during the growth period, at a depth of 2 cm. The first fertilization was performed on the 30 May (36 days after transplantation), and the second on the 15 July (81 days after transplantation). Throughout the experiment, the plants were closely monitored daily for any signs of pests, pathogens and weeds. Throughout the experiment, plants were maintained under controlled greenhouse conditions and regularly monitored for optimal growth and development.
Plants were harvested at full flowering stage on 21 August (approximately 120 days after transplanting). Various parameters were evaluated, including morphological and yield traits (plant height, shoot fresh and dry weight, root fresh and dry weight, root volume and extract percentage); three plants were used to determine physiological traits (chlorophyll a, chlorophyll b and total chlorophyll) and phytochemical traits (total phenol, flavonoid, anthocyanin and phenolic acid content).
Plant height was measured from the ground to the top of the highest inflorescence. Plants were harvested by cutting the stems 5 cm above ground. Fresh weight was determined immediately using a digital balance (BBI41, Boeco, Hamburg, Germany). For dry weight determination, plant samples were dried in an oven (BINDER D. 78532, Tuttlingen, Germany) at 60 °C for 48 h until constant weight was achieved.

2.1. Extraction of Extract Content

The powdered samples (1 g) were mixed with 10 mL of methanol (0.1% HCl). The mixture was vortexed thoroughly and subjected to an ultrasonic device (frequency 30 kHz, nominal power 100 W) (SONICA 220EP S3, Soltec srl, Milan, Italy) for 30 min. After extraction, the sample was vortexed again and centrifuged at 2795× g for 10 min. The supernatant was then placed in an oven at 50 °C for 48 h to obtain the dried extract. Finally, the dry extract was weighed, and the extract percentage was calculated in three replicates per treatment.

2.2. Chlorophyll Content

A total of 0.2 g of frozen leaf sample was extracted with 80% acetone in a porcelain mortar, then the resulting extract was transferred to a falcon using a funnel and filter paper, the filter paper was washed with solvent, and the extract was made up to a volume of 15 mL. Then the optical absorption of chlorophyll a and b was read at wavelengths of 663 and 645 nm by a spectrophotometer (T80+, PG Instruments Ltd., Lutterworth, UK) and expressed in mg g−1 of fresh weight in three replicates per treatment [41].

2.3. Determination of Total Phenolic Content

To measure the total phenol content, 500 μL of Folin–Ciocalteau reagent (10% v/v) was added to 100 μL of the extract at a concentration of 4 mg/mL, and after 5 min, 500 μL of sodium carbonate (7% w/v) was added. The resulting solution was kept in the dark for 2 h. Then the optical absorbance of the samples was read by spectrophotometer (T80+, PG Instruments Ltd., Lutterworth, UK) at a wavelength of 765 nm, and the results were expressed in mg of gallic acid per g of dry extract in three replicates per treatment [42].

2.4. Determination of Total Flavonoid Content

To measure the total flavonoid content, the method of Chang et al. [43] was used. For this purpose, 100 μL of aluminum chloride solution (2% w/v) and 200 μL of potassium acetate solution (1 M) were added to 250 μL of extract (1 mg/mL), and after 10 min, the optical absorbance of the samples was read by spectrophotometer (T80+, PG Instruments Ltd., Lutterworth, UK) at a wavelength of 426 nm, and the results were expressed in mg of quercetin per g of dry extract in three replicates per treatment.

2.5. Determination of Total Anthocyanin Content

To measure the anthocyanin content, 0.1 g of frozen leaf sample was ground and extracted with 3 mL of methanol (0.1% HCl), and after vortexing, it was stored in a refrigerator in the dark for 24 h at 4 °C. After that, it was centrifuged for 10 min at 2795× g and then its absorbance was measured by spectrophotometer (T80+, PG Instruments Ltd., Lutterworth, UK) at wavelengths of 530 and 657 nm, and the results were expressed in mg g−1 fresh weight in three replicates per treatment [44].

2.6. Isolation and Identification of Phenolic Acids

Phenolic acids were isolated and identified using a high-performance liquid chromatography (HPLC) Waters 2695 system (Milford, CT, USA). HPLC analysis was performed on a Welch Ultisil XB-C18 reversed-phase column (150 mm × 4.6 mm, 5 μm particle size) with an injection volume of 20 μL (500 ppm concentration). The mobile phases consisted of solvent A [HPLC-grade methanol (Merck, Darmstadt, Germany)] and solvent B [ultrapure water acidified with 0.1% (v/v) formic acid (Merck, Darmstadt, Germany)]. The gradient elution program began with an initial ratio of solvent A to solvent B of 30:70 (v/v), which increased linearly to 100% solvent A over 38 min at a rate of 1.84% per minute (a 70% to 100% increase over 38 min). This was followed by a 7 min isocratic hold at 100% solvent A, with the flow rate maintained at 0.5 mL/min and UV detection performed at 280 nm. A 20 µL sample with a concentration of 500 ppm was injected into the HPLC system. The total analysis time for each sample was 45 min. Phenolic compounds were identified by comparing the retention times of each peak with those of authentic standard compounds. Quantification of the phenolic acids was performed based on multilevel external calibration curves. The method validation parameters for the studied phenolic compounds and their respective calibration curves are presented in Figure S1 [45].

2.7. Assessment of Arbuscular Mycorrhizal Fungal Spore Extraction and Root Colonization

The wet sieving method was used to extract Glomus spores from soil samples in accordance with established protocols [46]. The soil samples (10 g) were suspended in 500 mL of distilled water and stirred vigorously to ensure homogeneity. This suspension was then decanted sequentially through stacked sieves with gentle rinsing in between each one: first through a 1000 µm sieve, then a 250 µm sieve and finally a 45 µm sieve. The fraction retained by the 45 µm sieve, which contained the target spores, was backwashed into a Petri dish using sterile distilled water. The spores were then carefully collected under a stereomicroscope (40× magnification) using a micropipette. The spores were washed three times in sterile water to remove debris, after which they were either processed immediately for downstream applications or stored at 4 °C in sterile water for short-term preservation. Bright-field microscopy (Olympus CH-2 microscope, Olympus Corporation, Tokyo, Japan)was employed to examine and capture images of the Glomus spores at varying magnifications.
To evaluate the presence of AMF in the roots, the trypan blue staining method [47] was employed. The root samples were cleared in 10% KOH at 90 °C for 10–15 min, rinsed with distilled water, acidified in 1% HCl for 2–3 min and stained with 0.05% trypan blue in lactic glycerol at 90 °C for 30 min. After destaining, the fungal structures (hyphae, arbuscules and vesicles) could be observed under a light microscope. Root colonization percentage was determined using the gridline intersection method. Briefly, the stained roots were mounted on slides and the number of intersections between the microscope eyepiece grid and the AMF structures was counted. Colonization percentage was calculated as follows: (number of infected intersections/total intersections examined) × 100.

2.8. Shoot Nitrogen, Phosphorus and Potassium Content

After the oven drying, the shoot samples (0.5 g) were finely ground using an electric mill. Total N was determined by the Kjeldahl method, as described by Novamsky et al. [48]. This process involved digestion of the samples with sulfuric acid/hydrogen peroxide, followed by distillation with sodium hydroxide and, finally, titration of the ammonia-containing solution. The results were expressed as a percentage of total N on a dry weight basis. The extract P content was measured by changing the color in reacting molybdate–vanadate by spectrophotometer (Lambda EZ210, Granite Quarry, NC, USA) at a wavelength of 420 nm. Similarly, K phosphate was used to prepare the standard P. The content of K was read using the flame diffusion method utilizing a flame photometer in mg g−1 of the extract. For this reason, the samples were dried and placed in a 500 °C oven and converted into ash. Following the addition of HCl and the heating and addition of distilled water, the content of K was read using a flame photometer [49]. It was determined based on K standards (KCl). All nutrient contents were expressed as mg g−1 leaf dry weight in three replicates per treatment.

2.9. Data Analysis

Statistical analyses were performed using SAS software version 9.4. Prior to analysis, all data were tested for normality and transformed as necessary. Treatment means were compared using the least significant difference (LSD) test at p ≤ 0.01. Graphs of the data were generated using Microsoft Excel 2016.

3. Results

3.1. Fresh and Dry Biomass Production and Plant Height of Shoots

The fresh and dry weight of H. officinalis shoots was significantly influenced by both N application and mycorrhizal inoculation (p < 0.01) (Table S1). In mycorrhiza-inoculated plants, the fresh shoot weight ranged from 16.03 to 20.45 g per plant, with M4 treatment giving the highest values. In contrast, non-inoculated plants showed the lowest fresh weight values (Figure 1a). N fertilization resulted in fresh shoot weights ranging from 16.55 to 18.87 g per plant, with N2 treatment giving the highest values. The effects of N2 (0.5 g N pot−1) and N3 (1 g N pot−1) treatments were statistically comparable, while plants without N application had the lowest fresh weight values (Figure 1c). Both mycorrhizal inoculation (p < 0.01) and N application (p < 0.05) significantly increased shoot dry weight. Among the mycorrhizal treatments, combined root and soil inoculation with mycorrhiza produced the highest dry weight (3.24 g per plant), although this was not statistically different from root inoculation with mycorrhiza (3.17 g per plant) and soil inoculation with mycorrhiza (2.92 g per plant). Regarding N application, the N2 treatment gave the highest dry weight values. Although the N3 treatment increased shoot dry weight compared to the control, this difference was not statistically significant (Figure 1b).
N fertilization and mycorrhizal inoculation significantly influence the height of H. officinalis plants (p < 0.05) (Table S1). Analysis of treatment interaction effects showed that the root and soil inoculation with mycorrhiza combined with 1 g N pot−1 produced the tallest plants (42.5 cm), representing a 32% increase over the control plants. While treatments of non-inoculation with 1 g N pot−1, soil inoculation with mycorrhiza without N application and soil inoculation with mycorrhiza combined with 1 g N pot−1 did not significantly affect plant height compared to the control, all other treatment combinations resulted in significant height increases (Figure 2a).

3.2. Fresh and Dry Weight of Roots

The interaction between mycorrhizal inoculation and N application significantly influenced both fresh root weight (p < 0.05) and dry root weight (p < 0.01) in H. officinalis (Table S1). The non-inoculation with 1 g N pot−1 treatment gave the highest fresh root weight with an increase of 40% compared to the control treatment (Figure 2b).
Root dry weight was maximized in the root and soil inoculation with mycorrhiza combined with 0.5 g N pot−1 treatment, with an increase of 34% compared to the control. Most treatments showed statistically similar effects on root dry weight, with the notable exceptions of non-inoculation with 1 g N pot−1 and soil inoculation with mycorrhiza combined with 1 g N pot−1, which resulted in decreased root dry weight compared to the control. The highest root dry weight was observed in the root and soil inoculation with mycorrhiza combined with 0.5 g N pot−1 treatment, while non-inoculation with 1 g N pot−1 produced the lowest values (Figure 2c).

3.3. Extract Content

The extract content was significantly influenced by the interaction between mycorrhizal inoculation and N fertilization (p < 0.01) (Table S2). Treatment with soil inoculation with mycorrhiza combined with 1 g N pot−1 showed the highest extract content (18.76%), representing a 100% increase compared to the lowest content observed in the control (9.36%). With the exception of non-inoculation with 0.5 g N pot−1, all other treatments showed significant increases compared to the control (Figure 2d).

3.4. Nitrogen, Phosphorus and Potassium Content

Combining mycorrhizal treatments with N fertilization significantly influences the uptake of N, phosphorus and potassium in H. officinalis (Table S2). The best results were achieved using the combined root and soil inoculation method, which produced higher N and potassium concentrations at all N application levels. Maximum nutrient accumulation was achieved through the combined application of N fertilization and mycorrhizal inoculation treatments. A significant interaction was observed between mycorrhizal inoculation and N application regarding plant N content. All inoculated treatments showed substantial improvement compared to the non-inoculated control. Soil inoculation with mycorrhizae combined with 1 g of N per pot yielded the highest N concentration (3.06% leaf dry weight), which was a 30% increase on the control treatment (2.35% leaf dry weight) (Figure 3a).
The accumulation of phosphorus was significantly affected by the interaction between mycorrhizal inoculation and N fertilization. Combined root and soil inoculation with 0.5 g of N per pot produced the highest phosphorus content (0.266% of leaf dry weight), which was 24% higher than the lowest recorded value in the non-inoculated treatment with 1 g of N per pot (0.20% of leaf dry weight). However, the non-inoculated treatment with 1 g of N per pot showed no significant improvement compared to the control treatment (0.21% leaf dry weight). In contrast, all other mycorrhizal treatments demonstrated significant increases (Figure 3b).
The uptake of potassium was significantly affected by the interaction between mycorrhizal inoculation and N application. Root inoculation with mycorrhizae in the absence of N resulted in the highest potassium content (3.85% leaf dry weight), 22% higher than the control (3.15% leaf dry weight). Conversely, the lowest potassium values were observed in non-inoculated plants that received the highest N dose (2.85% leaf dry weight per g of N per pot), suggesting that high N levels may have an antagonistic effect in the absence of mycorrhizal symbiosis (Figure 3c).

3.5. Mycorrhizal Colonization Percentage

The percentage of mycorrhizal colonization was significantly influenced by the interaction between nitrogen application and the mycorrhizal inoculation method (p < 0.05) (see Table S2). The highest rate of colonization (70.66%) was achieved in plants that received both root and soil mycorrhizal inoculation in combination with moderate nitrogen application (0.5 g N per pot). This treatment produced statistically similar results to those observed in plants treated with soil inoculation and high N application. In contrast, the lowest percentage of colonization was observed in plants treated with root inoculation alone and no N supplementation (Figure 3d). In the stained root segments, the mycorrhizal fungi were clearly visible as intraradical spores (Figure 4).

3.6. Chlorophyll a and b and Total Chlorophyll Content

Chlorophyll a content was significantly influenced by the interaction between mycorrhizal inoculation and N application (p < 0.01) (Table S2). All treatments showed significant increases compared to the control treatment. The highest chlorophyll a content was recorded in soil inoculation with mycorrhiza combined with 1 g N pot−1 (M3N3, 3.14 mg g−1 fresh weight), showing a 49% increase compared to the control treatment, which had the lowest content (2.1 mg g−1 fresh weight) (Figure 5a).
Chlorophyll b content showed a significant response to the interaction between mycorrhizal inoculation and N application (p < 0.05) (Table S2). All treatments showed significant increases compared to the control. The highest chlorophyll b content was observed in root and soil inoculation with mycorrhiza combined with 1 g N pot−1 (1.34 mg g−1 fresh weight), representing an increase of 131% compared to the control treatment (0.58 mg g−1 fresh weight) (Figure 5b).
Total chlorophyll content was significantly influenced by the interaction between mycorrhizal inoculation and N fertilization (p < 0.01) (Table S2). Treatment with soil inoculation with mycorrhiza combined with 1 g N pot−1 gave the highest total chlorophyll content (4.46 mg g−1 fresh weight), showing a 66% increase compared to the control (2.68 mg g−1 fresh weight). The effect of N was particularly notable; under non-inoculated conditions, application of 1 g N pot−1 resulted in the highest total chlorophyll content (4.14 mg g−1 fresh weight), representing a 54% increase compared to the control. Mycorrhizal inoculation also significantly increased total chlorophyll content. All inoculation treatments without N application showed significant increases in total chlorophyll content, with treatment with root and soil inoculation with mycorrhiza and without N application achieving the highest value (4.17 mg g−1 fresh weight) (Figure 5c).

3.7. Total Phenol and Flavonoid Content

The interaction between mycorrhizal inoculation and N application significantly influenced the total phenol content (p < 0.05) (Table S2). Treatment with combined root and soil inoculation with 0.5 g N pot−1 showed the highest total phenolic content, with an increase of 12.5% compared to the control. Under non-inoculated conditions, the application of 1 g N pot−1 resulted in the highest increase in total phenolic content compared to the control, although this increase was not statistically different from the 0.5 g N pot−1 application. The combined root and soil mycorrhizal inoculation without N application showed the most pronounced effect on total phenolic content (Figure 5d).
The total flavonoid content showed a significant response to the interaction between mycorrhizal inoculation and N application (p < 0.05) (Table S2). Treatment with combined root and soil inoculation with 0.5 g N pot−1 yielded the highest total flavonoid content, demonstrating a 33% increase compared to the control. Under non-inoculation conditions, the application of 0.5 g N pot−1 resulted in a significant increase in flavonoid content. Notably, all three mycorrhizal inoculation treatments without N application induced significant increases in total flavonoid content (Figure 5e).

3.8. Anthocyanin Content

The interaction between mycorrhizal inoculation and N application significantly influenced the anthocyanin content in H. officinalis (p < 0.05) (Table S2). Plants treated with both root and soil mycorrhizal inoculation combined with no N application showed the highest anthocyanin content. In contrast, the lowest anthocyanin content was observed in plants receiving both types of mycorrhizal inoculation and the highest N rate.
In non-inoculated plants, N applications of both 0.5 and 1 g pot−1 significantly reduced anthocyanin content compared to the control. When applied individually without N supplementation, neither root inoculation nor soil inoculation of mycorrhiza showed significant effects on anthocyanin levels. While combined root and soil inoculation of mycorrhiza resulted in a significant increase in anthocyanin content, root inoculation alone did not increase levels compared to the control. Soil inoculation showed a slight increase in anthocyanin content, although this increase was not statistically significant (Figure 5f).

3.9. Phenolic Acid Content

HPLC analysis of H. officinalis extract revealed six main phenolic compounds including gallic acid, salicylic acid, rosmarinic acid, meta-coumaric acid, caffeic acid and protocatechuic acid. The interaction between mycorrhizal inoculation and N fertilization significantly influenced all the phenolic acids analyzed (p < 0.01, except for caffeic acid: p < 0.05) (Table S3). For gallic acid, the highest content was observed in soil inoculated with moderate N treatment, while the lowest was found in non-inoculated soil with high N treatment, which was statistically similar to the control (Figure 6a). For salicylic acid, the maximum was found in the combined root and soil inoculation with no N treatment, while the minimum was found without N and soil inoculation treatment. In particular, mycorrhizal inoculation increased salicylic acid content, whereas increasing N levels generally decreased its production (Figure 6b).
The rosmarinic acid content reached its maximum in the combined inoculation with high N treatment, showing an increase of 72% compared to the control, while N application had no significant effect in the absence of mycorrhizal inoculation (Figure 6c). Meta-coumaric acid showed its highest concentration in the control and the lowest in the high N root inoculation treatment, with treatment combinations generally showing a decreasing trend in content (Figure 6d). Caffeic acid reached its maximum content in root inoculation with moderate N treatment, while the minimum was recorded in the control treatment, showing a moderate response to different treatment combinations (Figure 6e). Protocatechuic acid was highest in the control treatment and lowest in root inoculation with moderate N and combined inoculation with high N treatments, with a general decreasing trend with treatment combinations (Figure 6f).

4. Discussion

Our study demonstrates that the combined application of mycorrhizal inoculation and N fertilization significantly increases the growth of H. officinalis and the production of its secondary metabolites through multiple complementary mechanisms. Results showed that the combination of dual mycorrhizal inoculation (root and soil) with N application (1 g N pot−1) maximized plant height in H. officinalis. This synergistic effect can be attributed to the complementary mechanisms of the two treatments. N promotes plant growth by triggering physiological processes, such as increased chlorophyll production. These metabolic improvements promote overall cellular metabolism and biomass accumulation, thereby increasing plant vigor and productivity. The growth enhancement observed in our study likely reflects the fundamental role of N in optimizing photosynthetic capacity. This synergistic relationship between mycorrhizal colonization and N availability aligns with the concept that optimal plant nutrition requires both adequate nutrient supply and efficient nutrient uptake mechanisms [37].
The growth-promoting effects of mycorrhizal fungi involve hormonal synthesis, particularly of gibberellins, and enhanced nutrient uptake via extended hyphal networks [50]. Our results showed that combining root and soil inoculation with 1 g of nitrogen per pot produced the best results (42.5 cm; a 32% increase over the control group), whereas mycorrhizal inoculation alone showed no significant improvement in height.
The observed increase in H. officinalis height under high N conditions is consistent with previous research. Hazrati et al. [3] reported that increased plant height under elevated N correlated with increased soil N availability and subsequent plant uptake. This relationship between N availability and plant height further validates our findings and suggests that optimizing both mycorrhizal colonization and N fertilization may be a valuable strategy for improving H. officinalis cultivation. The synergistic effects of mycorrhizal inoculation and optimal N fertilization appear to be mediated through enhanced nutrient uptake efficiency and improved plant–water relations [51].
Mycorrhizal associations improve water use efficiency and positively affect various plant growth parameters, including shoot fresh and dry weight, leaf number, leaf area and plant height [52,53]. In addition, N fertilization significantly affects aerial growth, particularly leaf area development and photosynthetic pigment synthesis [50].
Research on Catalpa bungei seedlings showed significant growth responses to N application, both with and without mycorrhizal inoculation. Medium-level N fertilizer application increased the mycorrhizal benefits to plant growth. Compared to non-inoculated seedlings, there was an increase of 30% in plant height, 12% in basal diameter and 76% in total biomass [37]. These findings are consistent with numerous studies showing significantly higher shoot and root biomass in mycorrhizal plants, particularly when combined with appropriate fertilization [53,54,55].
Notably, the M1N3 and M3N3 treatments, which involved excessive N concentrations, reduced root dry weight. This suggests that high N levels may disrupt the optimal functioning of mycorrhizae. This phenomenon may be attributed to the disruption of the symbiotic relationship between plants and fungi in environments with high N levels. Under these conditions, plants rely less on mycorrhizal nutrient acquisition, which could result in reduced investment in root biomass [56]. Furthermore, excessive N availability can cause an imbalance in carbon allocation, resulting in plants allocating more resources towards above-ground growth at the expense of root development [57].
Our results showed that N fertilization combined with mycorrhizal inoculation increased chlorophyll a, chlorophyll b and total chlorophyll content in H. officinalis. N fertilization increases soil and leaf N content, photosynthetic pigment concentration, light energy use efficiency and the maximum quantum yield of photosystem II [58]. Previous studies have consistently shown a strong correlation between plant N concentration and chlorophyll content [59,60].
This relationship was demonstrated in hyssop, where inoculation with species of AMF, including those in the Glomus genus (G. fasiculatum, R. Intraradices (G. Intraradices) and G. mosseae), increased the levels of chlorophyll a and b by 15% and 24%, respectively, in the first year, and by 34% and 31% in the second year, compared to control plants. Mycorrhizal treatment was found to be more effective than chemical fertilizer (NPK), which increased the level of chlorophyll a by 31% in the second year [5].
The mycorrhizal colonization rate is an important indicator of close symbiosis [61], which reflects the affinity between fungi and host plants. In general, the total colonization rates under two N levels (0.5 and 1 g) were higher than 60%, indicating that the root system of H. officinalis could form a good symbiotic system with AMF. Our research showed that the highest colonization percentages were achieved by combining root and soil inoculation with mycorrhiza and application of 0.5 g N, which promoted the growth of H. officinalis seedlings, but excessive N could inhibit mycorrhizal colonization and seedling growth. The efficacy of mycorrhizal associations is significantly influenced by fertilization practices, with appropriate phosphate and N levels being crucial determinants of mycorrhizal benefits. Studies with Salvia miltiorrhiza have shown that low N applications significantly increase mycorrhizal colonization rates. Specifically, the presence of mycorrhizal fungi alters the nutrient requirements of S. miltiorrhiza, shifting the primary limiting factor from phosphorus in non-mycorrhizal plants to N in mycorrhizal plants [62].
Mycorrhizal colonization rate serves as a critical indicator of symbiotic effectiveness, reflecting the interdependence between fungal symbionts and host plants [24]. These fungal associations are highly sensitive to environmental nutrient status. While moderate N levels promote mycorrhizal colonization and plant growth, excessive N supplementation inhibits both colonization efficiency and plant development [37,63]. The colonization rate of root systems by mycorrhizal fungi, a key indicator of fungal activity, is influenced by several factors, including root system morphology and structure, root exudate composition and quantity, chemical fertilizer applications (especially phosphorus and N), and heavy element concentrations [64].
The integrated application of nutrient sources improves plant growth indices through improved nutrient availability across developmental stages and improved soil physico-chemical properties. This comprehensive approach promotes optimal shoot growth while increasing root longevity [65]. The resulting increase in root growth and surface area provides expanded colonization opportunities.
The study demonstrated that mycorrhizal inoculation significantly increased the uptake of essential nutrients, including N, P, K, Ca and Mg [66].
A study on Mentha arvensis investigating the effects of the G. fasciculatum on essential oil yield and nutrient uptake revealed that fungal inoculation led to significant increases in the uptake of N, P and K [67]. Other studies have indicated that the mycorrhizal fungus G. mosseae enhances the uptake of N, P, K, water and minerals compared to the control [68]. Additionally, a recent study on Catalpa bungei demonstrated that mycorrhizal inoculation can improve N and P uptake [37]. The hyphae of AMF play a crucial role in enhancing nutrient and water uptake by forming beneficial pathways within the soil. AMF enhance N assimilation in plants by stimulating the activity of nitrate reductase enzymes. The increased N status in mycorrhizal plants results from the enhanced transport of N through AMF hyphae, which simultaneously elevates phosphorus concentrations. This is particularly crucial in low N conditions, as phosphorus is required for nitrate reductase phosphorylation [69]. Additionally, phosphatase enzymes play a key role in the absorption, assimilation and metabolism of phosphorus. AMF contribute to improved phosphatase activity, facilitating the release of organically bound phosphorus, thereby enhancing its availability for uptake and transport [70]. Therefore, the promotion of symbiotic plant growth by AMF is closely related to improved plant nutrition, enhanced growth and increased yield. This is in accordance with the results of this study.
Our research showed that the combined application of mycorrhiza and N resulted in a significant increase in phenol and flavonoid compound concentrations. Plant N acquisition, whether from organic or chemical sources, plays a fundamental role in plant growth, development and secondary metabolite production, particularly under conditions of nutrient deficiency and environmental stress. This relationship is particularly pronounced in purslane, where stress conditions induce increased accumulation of phenolics and flavonoids contributing to improved stress tolerance [71].
The beneficial effects of mycorrhizal associations on the synthesis of phenolic and flavonoid compounds have been extensively documented in several medicinal plant species. Notable examples include studies on Calendula officinalis [72], Hypericum perforatum [73] and Ocimum basilicum [74]. These secondary metabolites, phenolics and flavonoids, play essential roles in mediating cell–environment interactions and regulating various enzymatic activities in plants [75].
The biosynthesis of phenolic compounds is regulated by both genetic and environmental factors, with nutrition playing a crucial role. Increased N accessibility through mycorrhizal associations significantly improves the photosynthetic capacity of plants, leading to increased availability of carbon skeletons for the biosynthesis of phenolic compounds and flavonoids [76]. In a study investigating the effects of multiple mycorrhizal species on beetroot physiology and gene expression, multi-species inoculation was found to maximize anthocyanin and chlorophyll content. These findings suggest that increased mycorrhizal diversity enhances plant physiological and phytochemical traits [40].
AMF have a significant effect on plant secondary metabolism. Studies have shown that mycorrhizal inoculation under reduced fertilization conditions increases anthocyanin concentrations in strawberry fruit. This combination of reduced nutrient availability and soil microbial inoculation promotes the development of healthier fruits with increased antioxidant compounds and improved nutritional quality [77]. Similar increases in phenolic compound, flavonoid and anthocyanin accumulation following mycorrhizal inoculation have been documented in Ipomoea purpurea [78], supporting our current findings.
Anthocyanin biosynthesis is modulated by several environmental factors, including light, temperature and N availability. N acts as a key regulator of anthocyanin synthesis and accumulation by modulating transcription factors involved in the biosynthetic pathway. In particular, low N conditions promote anthocyanin accumulation, while high N levels have an inhibitory effect. N primarily affects genes involved in nutrient transport and metabolism through the regulation of carbon–N metabolism, enzyme catalysis and binding, and signal transduction pathways. Recent research has identified nine candidate genes specifically related to anthocyanin synthesis, providing fundamental insights into the role of N in regulating anthocyanin biosynthesis [79]. The stimulatory effect of low N conditions on anthocyanin accumulation has been well documented in several species, including Malus domestica [80] and Arabidopsis [81]. These findings collectively highlight the potential of mycorrhizal-assisted cultivation as a sustainable strategy for enhancing secondary metabolite production in H. officinalis, offering promising applications for pharmaceutical and nutraceutical industries.
Our research showed that combining mycorrhizae with N significantly increased the concentrations of gallic, rosmarinic and caffeic acids, bringing them up to maximum levels. Studies of Salvia miltiorrhiza under mycorrhizal treatment with varying N and phosphorus levels produced comparable quantitative results. Without phosphorus application, the concentrations of rosmarinic acid and salvianolic acid B increased by 43.9% and 50.9%, respectively, in mycorrhizal plants compared to non-mycorrhizal plants. However, moderate N application increased the accumulation of phenolic acids in mycorrhizal plants, whereas high phosphorus levels decreased the concentration of rosmarinic acid by 34.3%. The mycorrhizal benefit coefficient was highest without phosphorus application and decreased with increasing phosphorus levels. This suggests that N supplementation rather than phosphorus enhances the production of phenolic compounds via mycorrhizae. These results are directly comparable to those of our own study, since both investigations used AMF inoculation combined with N fertilization, measuring similar phenolic acid compounds. This demonstrates consistent enhancement of secondary metabolite production across different plant species within the same family [62]. A study conducted by Engel et al. [82] found that the presence of three different mixtures of AMF led to an increase in the content of rosmarinic acid and lithospermic acid A isomer in Melissa officinalis. However, in Majorana hortensis, the levels of these compounds were reduced under the same conditions.
N availability plays a fundamental role in the biosynthesis of secondary metabolites, especially phenolic acids. Low N conditions enhance N uptake by both symbiotic organisms and plant root systems, affecting secondary metabolite biosynthetic pathways and leading to increased phenolic acid accumulation in plant tissues [62]. Mycorrhizal inoculation has been shown to promote the accumulation of specific phenolic compounds, including salvianolic acid in Salvia miltiorrhiza [83] and both caffeic and rosmarinic acids in Ocimum basilicum [84]. Rosmarinic acid biosynthesis occurs via the shikimate–phenylpropanoid pathway, with phenylalanine and tyrosine as precursors. N, which is essential for amino acid synthesis, enhances phenylalanine and tyrosine production and subsequently promotes the accumulation of rosmarinic acid in plant tissues [61].
Secondary metabolites composition and concentration are modulated by biological, abiotic and agronomic factors [85]. The interaction between N and phosphorus levels can significantly affect the plant–mycorrhizal relationship, which can have both positive and negative effects on plant health [62]. The observed reduction in salicylic acid, coumaric acid and catechic acid levels is consistent with the carbon–nutrient balance hypothesis, which postulates an inverse relationship between nutrient availability and carbon-based secondary metabolite concentrations [86].

5. Conclusions

In this study, we found that the combination of mycorrhizal inoculation and N application significantly influenced various growth and biochemical parameters of the plant. The highest plant height, as well as the fresh and dry weights of both aerial parts and roots, were observed with this treatment. Additionally, the maximum values for traits such as extract percentage, chlorophyll a, chlorophyll b, and total chlorophyll were recorded in the treatment involving mycorrhizal inoculation and 0.5 g of N. The highest levels of total phenolic content, total flavonoids, and colonization percentage were achieved in the treatment with both root and soil inoculation of mycorrhiza and N application. Furthermore, the highest accumulation of anthocyanins and salicylic acid occurred under root and soil inoculation with mycorrhiza without N, but a reduction in their content was observed with the addition of N. The highest concentrations of gallic acid, caffeic acid and rosmarinic acid were observed with mycorrhiza and N treatment. In contrast, the levels of coumaric acid and catechic acid decreased with mycorrhizal inoculation and N application, with the highest concentrations found in the control treatment. In conclusion, it can be asserted that the inoculation of mycorrhizal fungi combined with low N levels represents a viable strategy for promoting sustainable and organic agriculture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nitrogen6030060/s1.

Author Contributions

Conceptualization, S.H.; methodology, S.H., M.M., S.M. and M.E.; validation, S.H., M.M. and S.N.; formal analysis, S.H., M.M., S.M. and M.E.; investigation, S.H., M.M., S.M. and S.N.; resources, S.H. and G.P.; data curation, S.H., M.M., S.M. and M.E.; writing—original draft preparation, S.H., M.M. and M.E.; writing—review and editing, S.H., M.M., S.N., S.M., M.E. and G.P.; visualization, S.H., G.P. and S.N.; supervision, S.H.; project administration, S.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Azarbaijan Shahid Madani University.

Data Availability Statement

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

Acknowledgments

The support of Azarbaijan Shahid Madani University is greatly appreciated.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Ochatt, S.; Alan, A.R.; Bhattacharya, A.; Hano, C.; Kiselev, K.V.; Marconi, P.L.; Otoni, W.C.; Park, S.Y.; Tang, K.X.; Weathers, P.J. Secondary metabolites: A boon from plants, the best chemist in nature: Preface from the editors. Plant Cell Tissue Organ Cult. 2022, 149, 1–6. [Google Scholar] [CrossRef]
  2. Selwal, N.; Rahayu, F.; Herwati, A.; Latifah, E.; Suhara, C.; Suastika, I.B.K.; Mahayu, W.M.; Wani, A.K. Enhancing secondary metabolite production in plants: Exploring traditional and modern strategies. J. Agric. Food Res. 2023, 14, 100702. [Google Scholar] [CrossRef]
  3. Hazrati, S.; Mousavi, Z.; Mollaei, S.; Sedaghat, M.; Mohammadi, M.; Pignata, G.; Nicola, S. Optimizing nitrogen fertilization to maximize yield and bioactive compounds in Ziziphora clinopodioides. Agriculture 2024, 14, 1690. [Google Scholar] [CrossRef]
  4. Bistgani, Z.E.; Siadat, S.A.; Bakhshandeh, A.; Pirbalouti, A.G.; Hashemi, M.; Maggi, F.; Morshedloo, M.R. Application of combined fertilizers improves biomass, essential oil yield, aroma profile, and antioxidant properties of Thymus daenensis Celak. Ind. Crops Prod. 2018, 121, 434–440. [Google Scholar] [CrossRef]
  5. Aghaei, K.; Pirbalouti, A.G.; Mousavi, A.; Badi, H.N.; Mehnatkesh, A. Effects of foliar spraying of L-phenylalanine and application of bio-fertilizers on growth, yield, and essential oil of hyssop [Hyssopus officinalis L. subsp. angustifolius (Bieb.)]. Biocatal. Agric. Biotechnol. 2019, 21, 101318. [Google Scholar] [CrossRef]
  6. Fathiazad, F.; Hamedeyazdan, S. 2011. A review on Hyssopus officinalis L.: Composition and biological activities. Afr. J. Pharm. Pharmacol. 2011, 5, 1959–1966. [Google Scholar]
  7. Guerrini, A.; Sacchetti, G.; Echeverria Guevara, M.P.; Paganetto, G.; Grandini, A.; Maresca, I.; Menghini, L.; Di Martino, L.; Marengo, A.; Tacchini, M. Wild Italian Hyssopus officinalis subsp. Aristatus (Godr.) Nyman: From morphological and phytochemical evidences to biological activities. Plants 2021, 10, 631–647. [Google Scholar]
  8. Borrelli, F.; Pagano, E.; Formisano, C.; Piccolella, S.; Fiorentino, A.; Tenore, G.C.; Izzo, A.A.; Rigano, D.; Pacifico, S. Hyssopus officinalis subsp. aristatus: An unexploited wild-growing crop for new disclosed bioactives. Ind. Crops Prod. 2019, 140, 111594. [Google Scholar]
  9. Rosato, A.; Maggi, F.; Cianfaglione, K.; Conti, F.; Ciaschetti, G.; Rakotosaona, R.; Fracchiolla, G.; Clodoveo, M.L.; Franchini, C.; Corbo, F. Chemical composition and antibacterial activity of seven uncommon essential oils. J. Essent. Oil Res. 2019, 30, 233–243. [Google Scholar] [CrossRef]
  10. Venditti, A.; Bianco, A.; Frezza, C.; Conti, F.; Bini, L.M.; Giuliani, C.; Bramucci, M.; Quassinti, L.; Damiano, S.; Lupidi, G.; et al. Essential oil composition, polar compounds, glandular trichomes and biological activity of Hyssopus officinalis subsp. aristatus (Godr.) Nyman from central Italy. Ind. Crops Prod 2015, 77, 353–363. [Google Scholar] [CrossRef]
  11. Kizil, S.; Toncer, O.; Ipek, A.; Arslan, N.; Saglam, S.; Mahmood Khawar, K. Blooming stages of Turkish hyssop (Hyssopus officinalis L.) affect essential oil composition. Acta Agric. Scand. Sec B–Soil. Plant Sci. 2008, 58, 273–279. [Google Scholar]
  12. Jangi, F.; Ebadi, M.; Ayyari, M. Evaluation of qualitative characteristics of hyssop (Hyssopus officinalis L.) in infrared drying. Irani. J. Med. Aroma. Plants Res. 2019, 35, 876–887. [Google Scholar]
  13. Kazazi, H.; Rezaei, K.; Ghotb-Sharif, S.J.; Emam-Djomeh, Z.; Yamini, Y. Supercriticial fluid extraction of flavors and fragrances from Hyssopus officinalis L. cultivated in Iran. Food Chem. 2007, 105, 805–811. [Google Scholar] [CrossRef]
  14. Torikov, V.E. Medicinal Value of Vegetable, Fruit and Berry, Field Plants and Wild Plants; Publishing House of the Bryansk State Agricultural Academy: Bryansk, Russia, 2013. [Google Scholar]
  15. Sharifi-Rad, J.; Quispe, C.; Kumar, M.; Akram, M.; Amin, M.; Iqbal, M.; Koirala, N.; Sytar, O.; Kregiel, D.; Nicola, S.; et al. Hyssopus essential oil: An update of its phytochemistry, biological activities, and safety profile. Oxidative Med. Cell. Longev. 2022, 2022, 1155–1164. [Google Scholar] [CrossRef]
  16. Srivastava, A.; Awasthi, K.; Kumar, B.; Misra, A.; Srivastava, S. Pharmacognostic and pharmacological evaluation of Hyssopus officinalis L. (Lamiaceae) collected from Kashmir Himalayas, India. Pharmacogn. J. 2018, 10, 690. [Google Scholar] [CrossRef]
  17. Anas, M.; Liao, F.; Verma, K.K.; Sarwar, M.A.; Mahmood, A.; Chen, Z.L.; Yeng, X.-P.; Liu, Y.; Li, Y.-R. Fate of nitrogen in agriculture and environment: Agronomic, eco-physiological and molecular approaches to improve nitrogen use efficiency. Biol. Res. 2020, 53, 47. [Google Scholar] [CrossRef]
  18. Cassman, K.G.; Dobermann, A. Nitrogen and the future of agriculture: 20 years on. Ambio 2022, 51, 17–24. [Google Scholar] [CrossRef]
  19. Strzemski, M.; Płachno, B.J.; Mazurek, B.; Kozłowska, W.; Sowa, I.; Lustofin, K.; Załuski, D.; Rydzik, Ł.; Szczepanek, D.; Sawicki, J. Morphological, anatomical, and phytochemical studies of Carlina acaulis L. cypsela. Int. J. Mol. Sci. 2020, 21, 9230. [Google Scholar] [CrossRef]
  20. Du, L.; Zhong, H.; Guo, X.; Li, H.; Xia, J.; Chen, Q. Nitrogen fertilization and soil nitrogen cycling: Unraveling the links among multiple environmental factors, functional genes, and transformation rates. Sci. Total Environ. 2024, 951, 175561. [Google Scholar] [CrossRef]
  21. Heimler, D.; Romani, A.; Ieri, F. Plant polyphenol content, soil fertilization and agricultural management: A review. Eur. Food Res. Technol. 2017, 243, 1107–1115. [Google Scholar] [CrossRef]
  22. Skubij, N.; Dzida, K. Essential oil composition of summer savory (Satureja hortensis L.) cv. Saturn depending on nitrogen nutrition and plant development phases in raw material cultivated for industrial use. Ind. Crops Prod. 2019, 135, 260–270. [Google Scholar] [CrossRef]
  23. Skubij, N.; Dzida, K.; Jarosz, Z.; Pitura, K.; Jaroszuk-Sierocińska, M. Nutritional value of savory herb (Satureja hortensis L.) and plant response to variable mineral nutrition conditions in various phases of development. Plants 2020, 9, 706. [Google Scholar] [CrossRef]
  24. Wang, J.; Qin, X.; Xu, S.; Zhao, M.; Shu, P.; Xu, F. Nitrogen availability affects stem development and response to differential root-zone drought stress in Catalpa bungei. Environ. Exp. Bot. 2021, 186, 104429. [Google Scholar] [CrossRef]
  25. Cui, L.J.; Liu, X.Y.; Lin, J.; Shi, K.M. Effects of arbuscular mycorrhizal fungi on roots growth and endogenous hormones of Phoebe zhennan under salt stress. J. Nanjing For. Univ. 2020, 44, 119–124. [Google Scholar]
  26. Naderi, G.; Madani, H.; Farahani, E. The assessment effects of bio and chemical fertilizers on vegetative growth and essential oil of Hyssop (Hyssopus officinalis L.). Appl. Ecol. For. Sci. 2015, 1, 1–4. [Google Scholar] [CrossRef]
  27. Ghanbari-Odivi, A.; Fallah, S.; Carrubba, A. Optimizing Hyssop (Hyssopus officinalis L.) cultivation: Effects of different manures on plant growth and essential oil yield. Horticulturae 2024, 10, 894. [Google Scholar] [CrossRef]
  28. Pichon, N.A.; Cappelli, S.L.; Soliveres, S.; Mannall, T.; Nwe, T.Z.; Hölzel, N.; Klaus, V.H.; Kleinebecker, T.; Vincent, H.; Allan, E. Nitrogen availability and plant functional composition modify biodiversity-multifunctionality relationships. Ecol. Lett. 2024, 27, e14361. [Google Scholar] [CrossRef]
  29. Whitman, T.; Neurath, R.; Perera, A.; Chu-Jacoby, I.; Ning, D.; Zhou, J.; Nico, P.; Pett-Ridge, J.; Firestone, M. Microbial community assembly differs across minerals in a rhizosphere microcosm. Environ. Microbiol. 2018, 20, 4444–4460. [Google Scholar] [CrossRef]
  30. Mollavali, M.; Perner, H.; Rohn, S.; Riehle, P.; Hanschen, F.S.; Schwarz, D. Nitrogen form and mycorrhizal inoculation amount and timing affect flavonol biosynthesis in onion (Allium cepa L.). Mycorrhiza 2018, 28, 59–70. [Google Scholar] [CrossRef]
  31. Smith, S.E.; Smith, F.A. Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant nutrition and growth. Mycologia 2012, 104, 1–13. [Google Scholar] [CrossRef]
  32. Ferrol, N.; Azcón-Aguilar, C.; Pérez-Tienda, J. Arbuscular mycorrhizas as key players in sustainable plant phosphorus acquisition: An overview on the mechanisms involved. Plant Sci. 2019, 280, 441–447. [Google Scholar] [CrossRef]
  33. Barea, J.; Pozo, M.; López-Ráez, J.; Aroca, R.; Ruíz-Lozano, J.; Ferrol, N.; Azcón, R.; Azcón-Aguilar, C. Arbuscular Mycorrhizas and their Significance in Promoting Soil-Plant Systems Sustainability Against Environmental Stresses; CRC Press: Boca Raton, FL, USA, 2013; pp. 353–387. [Google Scholar]
  34. Igiehon, N.O.; Babalola, O.O. Biofertilizers and sustainable agriculture: Exploring arbuscular mycorrhizal fungi. Appl. Microbiol. Biotechnol. 2017, 101, 4871–4881. [Google Scholar] [CrossRef]
  35. Pozo, M.J.; Zabalgogeazcoa, I.; de Aldana, B.R.V.; Martinez-Medina, A. Untapping the potential of plant mycobiomes for applications in agriculture. Curr. Opin. Plant Biol. 2021, 60, 102034. [Google Scholar] [CrossRef]
  36. Bi, Y.; Zhang, J.; Song, Z.; Wang, Z.; Qiu, L.; Hu, J.; Gong, Y. Arbuscular mycorrhizal fungi alleviate root damage stress induced by simulated coal mining subsidence ground fissures. Sci. Total Environ. 2019, 652, 398–405. [Google Scholar] [CrossRef]
  37. Chen, W.; Mou, X.; Meng, P.; Chen, J.; Tang, X.; Meng, G.; Xin, K.; Zhang, Y.; Wang, C. Effects of arbuscular mycorrhizal fungus inoculation on the growth and nitrogen metabolism of Catalpa bungei CA Mey. under different nitrogen levels. Front. Plant Sci. 2023, 14, 1138184. [Google Scholar]
  38. de Assis, R.M.A.; Carneiro, J.J.; Medeiros, A.P.R.; de Carvalho, A.A.; da Cunha Honorato, A.; Carneiro, M.A.C.; Bertolucci, S.K.V.; Pinto, J.E.B.P. Arbuscular mycorrhizal fungi and organic manure enhance growth and accumulation of citral, total phenols, and flavonoids in Melissa officinalis L. Ind. Crops Prod. 2020, 158, 112981. [Google Scholar]
  39. Haji Mohammadi, A.; Zarghami, R.; Kashani, A.; Heydari Sharifabad, H.; Nour Mohammadi, G. The effect of mycorrhizas under water deficit conditions on some morphophysiological and biochemical traits in greenhouse cultivation of Stevia Tissue culture plantlets. J. Plant Res. 2023, 36, 16–28. [Google Scholar]
  40. Yadav, V.K.; Kumar, D.; Jha, R.K.; Bairwa, R.K.; Singh, R.; Mishra, G.; Singh, J.P.; Kumar, A.; Vinesh, B.; Yang, R.; et al. Effects of AMF inoculation and nitrogen application on nitrogen mineralization of coastal saline soil. J. Nanjing Univ. Sci. Technol. 2023, 45, 145–152. [Google Scholar]
  41. Arnon, D.I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949, 24, 1–15. [Google Scholar] [CrossRef]
  42. McDonald, S.; Prenzler, P.D.; Antolovich, M.; Robards, K. Phenolic content and antioxidant activity of olive extracts. Food Chem. 2001, 73, 73–84. [Google Scholar] [CrossRef]
  43. Chang, C.C.; Yang, M.H.; Wen, H.M.; Chern, J.C. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food. Drug Anal. 2002, 10, 178–182. [Google Scholar]
  44. Sutharut, J.; Sudarat, J. Total anthocyanin content and antioxidant activity of germinated colored rice. Int. Food Res. J. 2012, 19, 215–221. [Google Scholar]
  45. Babaei-Rad, S.; Mumivand, H.; Mollaei, S.; Khadivi, A. Postharvest UV-B and UV-C treatments combined with fermentation enhance the quality characteristics of Capparis spinosa L. fruit, improving total phenols, flavonoids, anthocyanins, phenolic acids, and antioxidant activity. Food Chem. 2025, 483, 144306. [Google Scholar] [CrossRef]
  46. Walker, C.; Schüßler, A.; Vincent, B.; Cranenbrouck, S.; Declerck, S. Anchoring the species Rhizophagus intraradices (formerly Glomus intraradices). Fungal Syst. Evol. 2021, 8, 179–201. [Google Scholar] [CrossRef]
  47. Vierheilig, H.; Schweiger, P.; Brundrett, M. An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots. Physiol. Plant. 2015, 154, 423–434. [Google Scholar] [CrossRef]
  48. Novamsky, I.; Van Eck, R.; Van Schouwenburg, J.C.H.; Walinga, I. Total nitrogen determination in plant material by means of the indophenol blue method. Neth. J. Agric. Sci. 1974, 22, 3–5. [Google Scholar] [CrossRef]
  49. Chapman, H.D.; Pratt, P.F. Methods of analysis for soils, plants and waters. Soil Sci. 1962, 93, 68. [Google Scholar] [CrossRef]
  50. Mulyadi; Jiang, L. Combined application of Arbuscular Mycorrhizal Fungi (AMF) and nitrogen fertilizer alters the physicochemical soil properties, nitrogen uptake, and rice yield in a polybag experiment. Agriculture 2023, 13, 1364. [Google Scholar] [CrossRef]
  51. Zhang, B.; Chang, S.X.; Anyia, A.O. Mycorrhizal inoculation and nitrogen fertilization affect the physiology and growth of spring wheat under two contrasting water regimes. Plant Soil 2016, 398, 47–57. [Google Scholar] [CrossRef]
  52. Jabborova, D.; Annapurna, K.; Al-Sadi, A.M.; Alharbi, S.A.; Datta, R.; Zuan, A.T.K. Biochar and arbuscular mycorrhizal fungi mediated enhanced drought tolerance in Okra (Abelmoschus esculentus) plant growth, root morphological traits and physiological properties. Saudi J. Biol. Sci. 2021, 28, 5490–5499. [Google Scholar] [CrossRef]
  53. Zhao, R.; Guo, W.; Bi, N.; Guo, J.; Wang, L.; Zhao, J.; Zhang, J. Arbuscular mycorrhizal fungi affect the growth, nutrient uptake and water status of maize (Zea mays L.) grown in two types of coal mine spoils under drought stress. Appl. Soil Ecol. 2015, 88, 41–49. [Google Scholar] [CrossRef]
  54. Moradtalab, N.; Hajiboland, R.; Aliasgharzad, N.; Hartmann, T.E.; Neumann, G. Silicon and the association with an arbuscular-mycorrhizal fungus (Rhizophagus clarus) mitigate the adverse effects of drought stress on strawberry. Agronomy 2019, 9, 41. [Google Scholar] [CrossRef]
  55. Das, D.; Basar, N.U.; Ullah, H.; Salin, K.R.; Datta, A. Interactive effect of silicon and mycorrhizal inoculation on growth, yield and water productivity of rice under water-deficit stress. J. Plant Nutr. 2021, 44, 2756–2769. [Google Scholar] [CrossRef]
  56. Johnson, N.C.; Diane, L.; Corkidi, R.L.; Egerton-Warburton, L.M.; Allen, E.B. Nitrogen enrichment alters mycorrhizal allocation at five mesic to semiarid grasslands. Ecology 2003, 84, 1895–1908. [Google Scholar] [CrossRef]
  57. Treseder, K.K. A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and atmospheric CO2 in field studies. New Phytol. 2004, 164, 347–355. [Google Scholar] [CrossRef]
  58. Berghetti, A.L.P.; Araujo, M.M.; Tabaldi, L.A.; Turchetto, F.; Tassinari, A.; Bernardy, D.; Griebeler, A.M.; Barbosa, F.M.; Aimi, S.C.; Brunetto, G. Effects of nitrogen fertilization on the growth and on photochemical efficiency in plants of Handroanthus heptaphyllus. J. Plant Nutr. 2021, 44, 2464–2475. [Google Scholar] [CrossRef]
  59. Skudra, I.; Ruza, A. Effect of nitrogen and sulphur fertilization on chlorophyll content in winter wheat. Rural. Sustain. Res. 2017, 37, 29–37. [Google Scholar] [CrossRef]
  60. Chowdhury, T.; Chowdhury, A.H.; Oingyue, W.; Enyoh, C.E.; Wang, W.; Islam Khan, S. Nutrient uptake and pharmaceutical compunds of Aloe vera as in fluenced by integration of in organic fertilizer and poultry manure in soil. Heliyon 2021, 7, e07464. [Google Scholar] [CrossRef]
  61. Wang, Y.X.; Li, Q.; Shen, Y.K.; Yang, Q.; Zhang, J.B.; Wang, Y.H.; Song, X.Z. Effects of nitrogen deposition on arbuscular myorrhizal fungal colonization and glomalin-related soil protein of Chinese fir. Acta Ecol. Sin. 2021, 41, 194–201. [Google Scholar] [CrossRef]
  62. Pu, C.; Yang, G.; Li, P.; Ge, Y.; Garran, T.A.; Zhou, X.; Shen, Y.; Zheng, H.; Chen, M.; Huang, L. Arbuscular mycorrhiza alters the nutritional requirements in Salvia miltiorrhiza and low nitrogen enhances the mycorrhizal efficiency. Sci. Rep. 2022, 12, 19633–19641. [Google Scholar] [CrossRef]
  63. Yang, S.; Shi, Z.; Zhang, M.; Li, Y.; Gao, J.; Wang, X.; Liu, D. Stoichiometry of carbon, nitrogen and phosphorus in shrub organs linked closely with mycorrhizal strategy in northern China. Front. Plant Sci. 2021, 12, 687347. [Google Scholar] [CrossRef]
  64. Al-Karaki, G.N.; Williams, M. Mycorrhizal mixtures affect the growth, nutrition, and physiological responses of soybean to water deficit. Acta Physiol. Plant. 2021, 43, 75. [Google Scholar] [CrossRef]
  65. Azeez, J.O.; Van Averbeke, W.; Okorogbona, A.O.M. Differential responses in yield of pumpkin (Cucurbita maxima L.) and nightshade (Solanum retroflexum Dun.) to the application of three animal manures. Bioresour. Technol. 2010, 101, 2499–2505. [Google Scholar] [CrossRef]
  66. Sun, M.; Yuan, D.; Hu, X.; Zhang, D; Li, Y. Effects of mycorrhizal fungi on plant growth, nutrient absorption and phytohormones levels in tea under shading condition. Not. Bot. Horti Agrobot. Cluj-Napoca 2020, 48, 2006–2020. [Google Scholar]
  67. Gupta, M.L.; Prasad, A.; Ram, M.; Kumar, S. Effect of the vesiculararbuscular mycorrhizal (VAM) fungus Glumus fasciculatum on the essential oil yield related characters and nutrient acquisition in the crops of different cultivars of menthol mint (Mentha arvensis) under field conditions. Bioresour. Technol. 2002, 81, 77–79. [Google Scholar] [CrossRef]
  68. Nautiyal, C.S.; Chauhan, P.S.; DasGupta, S.M.; Seem, K.; Varma, A.; Staddon, W.J. Tripartite interactions among (Paenibacillus lentimorbus) NRRL B-3048, (Piriformospora indica) DSM 11827, and (Cicer arietinum L.). World J. Microbiol. Biotechnol. 2010, 26, 1393–1399. [Google Scholar] [CrossRef]
  69. Caravaca, F.; Diaz, E.; Barea, J.M.; Azcón-Aguilar, C.; Roldan, A. Photosynthetic and transpiration rates of Olea europaea subsp. sylvestris and Rhamnus lycioides as affected by water deficit and mycorrhiza. Biol. Plant. 2003, 46, 637–639. [Google Scholar]
  70. Egamberdieva, D.; Wirth, S.J.; Alqarawi, A.A.; Abd-Allah, E.F.; Hashem, A. Phytohormones and beneficial microbes: Essential components for plants to balance stress and fitness. Front. Microbiol. 2017, 8, 2104. [Google Scholar] [CrossRef]
  71. Montoya-García, C.O.; Volke-Hallera, V.H.; Trinidad-Santosa, A.; Villanueva-Verduzco, C. Change in the contents of fatty acids and antioxidant capacity of purslane in relation to fertilization. Sci. Hortic. 2018, 234, 152–159. [Google Scholar] [CrossRef]
  72. Kheyri, Z.; Moghaddam, M.; Farhadi, N. Inoculation efficiency of different mycorrhizal species on growth, nutrient uptake, and antioxidant capacity of Calendula officinalis L.: A Comparative Study. J. Soil Sci. Plant Nutr. 2022, 22, 1160–1172. [Google Scholar] [CrossRef]
  73. Lazzara, S.; Militello, M.; Carrubba, A.; Napoli, E.; Saia, S. Arbuscular mycorrhizal fungi altered the hypericin, pseudohypericin, and hyperforin content in flowers of Hypericum perforatum grown under contrasting P availability in a highly organic substrate. Mycorrhiza 2017, 27, 345–354. [Google Scholar] [CrossRef]
  74. Hristozkova, M.; Gigova, L.; Geneva, M.; Stancheva, I.; Velikova, V.; Marinova, G. Influence of mycorrhizal fungi and microalgae dual inoculation on basil plants performance. Gesunde Pflanz. 2018, 70, 99–107. [Google Scholar] [CrossRef]
  75. Hassanpouraghdam, M.B.; Vojodi Mehrabani, L.; Badali, R.; Aazami, M.A.; Rasouli, F.; KaKaei, K.; Szczepanek, M. Cerium Oxide Salicylic Acid Nanoparticles’ (CeO2: SA-NPs) foliar application and in-soil animal manure use influence the growth and physiological responses of Aloe vera L. Agronomy 2022, 12, 731. [Google Scholar] [CrossRef]
  76. Rahimi, A.; Siavash Moghaddam, S.; Ghiyasi, M.; Heydarzadeh, S.; Ghazizadeh, K.; Popovic-Djordjevic, J. The influence of chemical, organic and biological fertilizers on agrobiological and antioxidant properties of Syrian Cephalaria (Cephalaria syriaca L.). Agriculture 2019, 9, 122. [Google Scholar] [CrossRef]
  77. Lingua, G.; Bona, E.; Manassero, P.; Marsano, F.; Todeschini, V.; Cantamessa, S.; Copetta, A.; D’Agostino, G.; Gamalero, E.; Berta, G. Arbuscular mycorrhizal fungi and plant growth-promoting pseudomonads increases anthocyanin concentration in strawberry fruits (Fragaria x ananassa var. Selva) in conditions of reduced fertilization. Int. J. Mol. Sci. 2013, 14, 16207–16225. [Google Scholar] [CrossRef]
  78. Rashidi, S.; Yousefi, A.R.; Pouryousef, M.; Goicoechea, N. Effect of three Species of mycorrhizal fungus on photosynthesis, growth and secondary metabolites content of Ipomoea purpurea L. Irani. J. Weed Sci. 2020, 16, 99–114. [Google Scholar]
  79. Zhang, Z.; Cai, B.; Guo, Y.; Na, T.; Guo, Y. The impact of different nitrogen levels on the tuber yield and anthocyanin synthesis of purple potatoes. Agriculture 2024, 14, 125. [Google Scholar] [CrossRef]
  80. An, J.P.; Wang, X.F.; Li, Y.Y.; Song, L.Q.; Zhao, L.L.; You, C.X.; Hao, Y.J. EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 act in a regulatory loop that synergistically modulates ethylene biosynthesis and anthocyanin accumulation. Plant Physiol. 2018, 178, 808–882. [Google Scholar] [CrossRef]
  81. Liang, J.; He, J. Protective role of anthocyanins in plants under low nitrogen stress. Biochem. Biophys. Res. Commun. 2018, 498, 946–953. [Google Scholar] [CrossRef]
  82. Engel, R.; Szabo, K.; Abranko, L.; Rendes, K.; Fuzy, A.; Takacs, T. Effect of arbuscular mycorrhizal fungi on the growth and polyphenol profile of marjoram, lemon balm, and marigold. J. Agric. Food Chem. 2016, 64, 3733–3742. [Google Scholar] [CrossRef]
  83. Chen, M.; Yang, G.; Liu, D.; Li, M.; Qiu, H.; Guo, L.; Huang, L.; Chao, Z. Inoculation with Glomus mosseae improves the growth and salvianolic acid b accumulation of continuously cropped Salvia miltiorrhiza. Appl. Sci. 2017, 7, 692. [Google Scholar] [CrossRef]
  84. Toussaint, J.P.; Smith, F.A.; Smith, S.E. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza 2007, 17, 291–297. [Google Scholar] [CrossRef]
  85. Li, Y.; Kong, D.; Fu, Y.; Sussman, M.R.; Wu, H. The effect of developmental and environmental factors on secondary metabolites in medicinal plants. Plant Physiol. Biochem. 2020, 148, 80–89. [Google Scholar] [CrossRef]
  86. Ormeño, E.; Fernandez, C. Effect of soil nutrient on production and diversity of volatile terpenoids from plants. Curr. Bioact. Compd. 2012, 8, 71–79. [Google Scholar]
Nitrogen 06 00060 g001
Figure 1. Effect of N fertilization and mycorrhiza inoculation on the fresh (a,c) and dry weight (b,d) of shoots in H. officinalis. M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
Figure 1. Effect of N fertilization and mycorrhiza inoculation on the fresh (a,c) and dry weight (b,d) of shoots in H. officinalis. M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
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Nitrogen 06 00060 g002
Figure 2. Effect of mycorrhizal inoculation and N fertilization on plant height (a), root fresh weight (b), root dry weight (c) and extract content (d) of H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
Figure 2. Effect of mycorrhizal inoculation and N fertilization on plant height (a), root fresh weight (b), root dry weight (c) and extract content (d) of H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
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Nitrogen 06 00060 g003aNitrogen 06 00060 g003b
Figure 3. Effect of mycorrhiza inoculation and N fertilization on nitrogen (a), phosphorus (b), potassium (c) and colonization (d) in H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
Figure 3. Effect of mycorrhiza inoculation and N fertilization on nitrogen (a), phosphorus (b), potassium (c) and colonization (d) in H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
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Figure 4. Arbuscular mycorrhizal fungus (Glomus sp) infection in H. officinalis root.
Figure 4. Arbuscular mycorrhizal fungus (Glomus sp) infection in H. officinalis root.
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Nitrogen 06 00060 g005aNitrogen 06 00060 g005b
Figure 5. Effect of mycorrhiza inoculation and N fertilization on chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), total phenolic (d), total flavonoid (e) and anthocyanin content (f) in H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
Figure 5. Effect of mycorrhiza inoculation and N fertilization on chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), total phenolic (d), total flavonoid (e) and anthocyanin content (f) in H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
Nitrogen 06 00060 g005aNitrogen 06 00060 g005b
Nitrogen 06 00060 g006aNitrogen 06 00060 g006b
Figure 6. Effect of mycorrhiza inoculation and N fertilization on phenolic acid [(gallic acid (a), salicylic acid (b), rosmarinic acid (c), meta-coumaric acid (d), caffeic acid (e) and protocatechuic acid (f)] content in H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
Figure 6. Effect of mycorrhiza inoculation and N fertilization on phenolic acid [(gallic acid (a), salicylic acid (b), rosmarinic acid (c), meta-coumaric acid (d), caffeic acid (e) and protocatechuic acid (f)] content in H. officinalis. Treatments: M1 (no inoculation), M2 (root inoculation), M3 (soil inoculation), M4 (combined root and soil inoculation); N1 (0 g N pot−1), N2 (0.5 g N pot−1), N3 (1 g N pot−1). Means (columns) and standard errors (vertical bars) of three replicates are depicted. Means with the same letter are not significantly different according to the LSD test.
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Table 1. Properties of the experimental soil in the pot at the start of the experiment.
Table 1. Properties of the experimental soil in the pot at the start of the experiment.
ItemUnitSoil
Texture-Sandy loam
Organic carbon(%)0.51
EC(ds/m)1.36
pH-8.20
Total N (%)0.05
Available P(mg kg−1)7.80
Available K(mg kg−1)162
CaCO3(%)4.50
Available Mg(mg kg−1)9.84
Available Fe(mg kg−1)8.10
Available Mn(mg kg−1)6.22
Available Zn(mg kg−1)1.12
Available Cu(mg kg−1)1.05
Available B(mg kg−1)0.48
Cation-exchange capacity (CEC)(cmol kg−1)9.5
Bulk density(g cm−3)1.35
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Hazrati, S.; Mohammadi, M.; Mollaei, S.; Ebadi, M.; Pignata, G.; Nicola, S. Nitrogen Fertilization and Glomus Mycorrhizal Inoculation Enhance Growth and Secondary Metabolite Accumulation in Hyssop (Hyssopus officinalis L.). Nitrogen 2025, 6, 60. https://doi.org/10.3390/nitrogen6030060

AMA Style

Hazrati S, Mohammadi M, Mollaei S, Ebadi M, Pignata G, Nicola S. Nitrogen Fertilization and Glomus Mycorrhizal Inoculation Enhance Growth and Secondary Metabolite Accumulation in Hyssop (Hyssopus officinalis L.). Nitrogen. 2025; 6(3):60. https://doi.org/10.3390/nitrogen6030060

Chicago/Turabian Style

Hazrati, Saeid, Marzieh Mohammadi, Saeed Mollaei, Mostafa Ebadi, Giuseppe Pignata, and Silvana Nicola. 2025. "Nitrogen Fertilization and Glomus Mycorrhizal Inoculation Enhance Growth and Secondary Metabolite Accumulation in Hyssop (Hyssopus officinalis L.)" Nitrogen 6, no. 3: 60. https://doi.org/10.3390/nitrogen6030060

APA Style

Hazrati, S., Mohammadi, M., Mollaei, S., Ebadi, M., Pignata, G., & Nicola, S. (2025). Nitrogen Fertilization and Glomus Mycorrhizal Inoculation Enhance Growth and Secondary Metabolite Accumulation in Hyssop (Hyssopus officinalis L.). Nitrogen, 6(3), 60. https://doi.org/10.3390/nitrogen6030060

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