Effects of Thinning Practices on Soil Properties and Arbuscular Mycorrhizal Fungi in Natural Pure Oriental Beech Forests

Abstract

Thinning intensities in Fagus orientalis Lipsky. stands may influence the soil properties, arbuscular mycorrhizal (AM) fungi symbiosis, and their interaction through soil quality enhancement. We aimed to investigate the impact of four thinning intensities—control (no thinning); moderate (15%), moderately intense (35%), and intense thinning (55%)—implemented five years ago in pure oriental beech forests. In this context, the percentage indicates the proportion of trees removed by each thinning intensity, based on the total number of trees before thinning. Our focus encompassed soil physical–chemical properties, AM fungi community composition, and root colonization. At the intense thinning sites, the soil organic carbon, total nitrogen, available potassium, AMF spore density, and root colonization increased by 209.7, 88.9, 115.8, 404.9, and 448.5%, respectively, when compared to the control sites. This suggests a potential rise in AMF spore density and root colonization—a vital aspect for natural regeneration. These findings highlight the importance of considering management practices in forest systems that can enhance the root system in a sustainable manner to improve plant performance, soil fertility, and symbiosis with AM fungi.

1. Introduction

Natural forest ecosystems provide unique habitats for endemic and native flora and fauna, necessitating maintenance activities to enhance the forest health and stability over time [1]. This is especially critical for natural forests with endemic species and those sensitive (also classified as bioindicators) to ecological conditions or environmental changes [2], such as slow-growing species vulnerable to open-field conditions (e.g., scorching–drying temperatures, early and late frosts, and dense living cover) [3]). In such conditions, soil properties and arbuscular mycorrhizal (AM) fungi symbiosis influenced by thinning intensity are vital for the optimal growth and forest stability [4].

Maintenance activities, from establishment to regeneration, are guided by the diverse biological and silvicultural characteristics of tree species [5], including the Oriental Beech (Fagus orientalis Lipsky.), reflecting the importance of managing forest resources effectively.

F. orientalis is native to Europe and Asia, and on both continents, it holds high ecological, silvicultural, and economic value. In Europe, its distribution range primarily includes regions of southeastern Europe, such as Bulgaria, Greece, and Turkey, while it is also found in the temperate forests of the Caucasus region, encompassing Armenia, Azerbaijan, Georgia, and Russia [6]. The distribution range of F. orientalis can vary due to abiotic stressors such as climate, habitat suitability, soil properties, forest management practices, and historical land use [7].

Thinning in forest management offers multiple benefits for soil fertility, tree root systems, and symbiotic relationships [8]. By selectively removing trees, thinning allows for more light to reach the forest floor, promoting the growth of understory vegetation that enriches the soil with organic matter and nutrients [9]. Reduced competition among trees for resources encourages better root development, leading to stronger root systems capable of deeper penetration into the soil [10]. This, in turn, fosters healthier symbiotic relationships with arbuscular mycorrhizal fungi, enhancing trees’ ability to absorb water and nutrients [11]. Additionally, thinning contributes to improved ecosystem health, resilience, and productivity by creating favorable conditions for nutrient cycling, root growth, and symbiotic interactions [1]. Thus, our main hypotheses were the following. (i) Thinning may improve the soil’s physical–chemical properties by improving the soil organic carbon, nutrient cycling, and soil total volume pores. According to Sharma et al. (2024) [12], we must expect distinct changes in soil organic carbon quality, quantity, biochemical composition, and soil porosity in response to thinning intensities. (ii) According to Souza et al. (2024) [1], management practices create distinct rhizobiomes with specific soil properties and specific pairings between AM fungi and host plants.

Our aim was to determine whether thinning intensities alter the soil physical–chemical properties, AM fungi community composition, and root colonization by AM fungi in monospecific stands of F. orientalis in Turkey. Based on the studies developed by Sharma et al. (2024) [3] and Souza et al. (2024) [1], we expected to find an increase in the soil organic carbon and soil exchangeable cations influenced by thinning intensities, especially intense thinning. Additionally, we expected that the improved rhizobiome in the F. orientalis rhizosphere would create an AM fungi–host pairing specificity. To our knowledge, this is the first study using the soil physical–chemical properties and AM fungi community structure (at the species level) in natural pure oriental beech forests, Turkey. To accomplish this, we performed a field study and sampled soil for four thinning intensities (control, moderate thinning, moderately intense thinning, and intense thinning) that are widely found in stands of F. orientalis, with a significant socioeconomic impact.

2. Material and Methods

2.1. Experimental Design Description

We conducted a field study with F. orientalis stands using a randomized block trial design with four thinning intensities as follows: (1) control (latitude: 41°24′25″ longitude: 32°25′36″); (2) moderate thinning (latitude: 41°24′11″ longitude: 32°25′20″); (3) moderately intense thinning (latitude: 41°24′08″ longitude: 32°25′13″); and (4) intense thinning (latitude: 41°24′39″ longitude: 32°25′44″), which were implemented five years ago in pure oriental beech forests in Bartın Province, Türkiye (

Table 1. Main description of the thinning intensities (control, moderate, moderately intense, and intense) with natural Fagus orientalis stands.

Descriptors	Control	Moderate Thinning	Moderately Intense Thinning	Intense Thinning
Stand type	Kncd2	Kncd2	Kncd2	Kncd2
Bonitet class	III	III	III	III
Elevation (m)	557	558	563	552
Exposure	South	South	South	South
Slope status	Middle slope	Middle slope	Middle slope	Middle slope
Canopy	0.8–0.9	0.7–0.8	0.6–0.7	0.5–0.6
Density	0.8	0.7	0.6	0.5
Stratification	Single layer	Single layer	Single layer	Single layer
Mix status	Pure beech	Pure beech	Pure beech	Pure beech
Average age (year)	58	56	56	58
Average diameter (cm)	26.7	27.4	28.5	29.3
Average height (m)	18.5	17.1	17.8	18.2
Average initial number of trees (number/ha)	1328.4	1216.7	1040.3	783.9
Average volume (m3/ha)	500.81	472.17	437.96	375.49
Average annual increment (m3/ha/year)	8.63	8.43	7.82	6.47
Soil texture
Sand (%)	71.14	65.28	62.50	52.97
Silt (%)	13.72	20.98	18.74	27.11
Clay (%)	15.14	13.74	18.76	19.92

). Thinning intensity was estimated in terms of the selective removal of trees (e.g., thinning was conducted from below, primarily removing thinner trees to reduce competition among the remaining thicker trees, with this approach aiming to improve the overall growth and quality of the forest stands) from the forest stands. The control involved no thinning, moderate thinning involved the removal of 15% of the trees from the initial stand, moderately intense thinning involved the removal of 35% of the trees from the initial stand, and intense thinning involved the removal of 55% of the trees from the initial stand [10]. Each thinning intensity was replicated in rectangular permanent plots (25 × 40 m). We delimited four blocks. F. orientalis was selected as the model plant due to its high ecological, silvicultural, and economic value in many countries of Europe and Asia. It covers an area of 1.7 million hectares in Turkey [6] The mean annual temperature of the experimental sites was +8.0 °C, and we registered a total annual precipitation of 1534 mm. The soil type in the experimental sites was classified as Ultisol [13].

2.2. Field Conditions and Soil Sampling

The field experiment took place in monospecific stands of F. orientalis within the Kumluca Forest Sub-District Directorate, under the Ulus Forestry Operation Directorate’s jurisdiction, Kirsinler, Ulus Distric, Bartin province, Turkey (41°24′03″–41°24′57″ N and 32°25′11″–32°25′54″ E) (Figure 1).

Soil samples were collected using a soil auger with a 6.5 diameter and were sampled at two soil depths, from 0 to 30 cm and from 30 to 60 cm. A total of 80 soil samples ((1 control + 3 thinning intensities) 4 × 10 sampling points per plot × 2 soil depths) were collected for the soil characterization and AM fungi assessment. The samples for the soil characterization were air-dried and passed through a 2 mm sieve, as described by Black (1965) [14]. Within each plot, samples were randomly collected near the rhizosphere of F. orientalis.

The grain diameters of the soils obtained from the study plots were determined utilizing the Bouyoucos hydrometer method, with soil classes subsequently being identified based on international grain diameter classifications [15]. Additionally, analyses were conducted to assess the bulk density, grain density, and soil pore volume [14]. Soil permeability was evaluated through methods outlined by Klute and Dirksen (1986) [16]. Furthermore, various parameters, including available water, soil field capacity (SFC), permanent wilting point (PWP), moisture equivalent, and infiltration, were determined following the methodology proposed by Tüzüner (1990) [17].

To assess the soil pH, the samples were immersed in a solution of 1 part soil to 2.5 parts pure water and left for 24 h for analysis. The organic carbon content was determined using the Walkley–Black wet combustion method [18]. For electrical conductivity, indicating soil salinity, the samples were mixed with a 1:5 ratio of soil to water in a mechanical mixer for 1 h and analyzed using an electrical conductivity device [19]. The lime content (CaCO₃) was quantified using the Scheibler calcimetry method [20], while the total nitrogen was assessed via the modified Kjeldahl method [14]. The available phosphorus was estimated as described by Olsen et al. (1954) [21] for alkaline soils. Finally, the available potassium content was determined according to Black (1965) [14].

2.3. Spore Isolation and Identification of AM Fungi

To ensure the peak of AM fungi spores in the soil, the sampling was conducted during July and August 2021 [22]. Soil samples designated for the AMF analysis were promptly refrigerated at +4 °C until analysis. AMF spores were isolated using the wet sieving method [23,24] and morphologically identified under a microscope [25]. Additionally, spore densities were quantified using a 50 g soil sample. In order to define the AM fungi community composition, the AMF species richness, Shannon–Wiener index, Simpson index, Hurlbert’s probability of intraspecific encounter, and Pielou index were analyzed.

2.4. Bioassay with Host Plant to Access Root Colonization by AMF

To assess the root colonization of AM fungi (AMF), we used a bioassay with Zea mays L. as a host plant in a greenhouse experiment with 30 replicates per thinning intensity. The soil samples were sieved (2 mm mesh) and then stored in polyethylene bags at +4 °C in dry conditions to prevent spore germination and spoilage, ensuring their viability as inoculum sources. To guarantee the QA/QC quality of the sterilized conditions, (i) Procholaraz solution was used to soak the seeds for 30 min and they were subsequently rinsed with sterile distilled water [26]; (ii) the pots utilized in the isolation process were disinfected with 10% formalin water; and (iii) the collected soil samples were diluted by mixing with sterile angular river sand at a 1:1 ratio, and corn seeds were sown one day after filling the pots with the diluted soil mixture.

The plants were cultivated under greenhouse conditions (23.5/18 °C day/night temperatures, 4000–6000 lux light intensity, and 12/12 photoperiod) over 10 weeks, receiving distilled water irrigation throughout. Upon the conclusion of the experimental period, root fixation and staining procedures were conducted according to Phillips and Hayman (1970) [27] on the harvested plant roots. To detect the presence of mycorrhizal fungus and quantify the extent of colonization, roots preserved in AFA (acetic acid–formaldehyde-ethyl alcohol) solution were stained with lactophenol blue. The dye solution was prepared by combining lactophenol blue with lactic acid (40 mL lactic acid + 60 mL lactophenol) [27]. The grid-line intersect method was employed to determine the percentage of colonization by AM fungi in the roots stained with lactophenol blue [28]. Samples of approximately 0.5 g were extracted from the stained capillary roots and cut into lengths ranging from 1 to 1.5 cm. These cut root segments were evenly distributed within a Petri dish divided into 1 square centimeter sections. Under a stereo microscope, the Petri dish containing the root segments was examined. During the examination, a button was depressed for each root segment intersecting the grid lines on the Petri dish. If an AM fungi propagule (hyphae, vesicle, and chlamydospore) was observed within that particular root segment, two buttons were simultaneously pressed.

2.5. Statistical Analyses

Before analysis, the datasets were tested for normality using the Shapiro–Wilk test. The soil properties, AM fungi indices, spore abundance, and root colonization were analyzed using one-way ANOVA with thinning intensity as a factor and sampling plots as random effects. Duncan’s test (p < 0.05) was used for post hoc comparisons. NMDS with Jaccard’s matrix was performed to assess the differences in the AM fungi community composition across different thinning intensities. To evaluate the similarities among the thinning intensities due to the soil physical–chemical properties, spores’ abundance, and root colonization by AM fungi, a principal component analysis (PCA) was carried out [29].

3. Results

Significant differences among the thinning intensities (df = 3), soil depths (df = 1), and their interaction (df = 3) were explored by using an explanatory two-way ANOVA. In this explanatory analysis, we did not find significant differences among soil depths (df = 1), plots (df =2), and sampling points (df = 2). They were not considered as sources of variation in the two-way ANOVA; thus, they were considered to be replicas, and the subsections show results from the one-way ANOVA considering the thinning intensities as the main source of variation in this study.

3.1. Thinning Intensities Influence on Soil Physical–Chemical Properties of a Ultisol Covered by Monospecific Stands of F. orientalis

We found significant differences among the different thinning intensities for all studied soil physical properties (p < 0.01), except the lime content (CaCO₃). The highest values of bulk density, grain density, soil pore volume, and soil permeability were found in plots with no thinning (control). By considering moderate thinning, we found the highest values of infiltration and electrical conductivity. Next, the highest values of P₂O₅ were observed in plots with moderately intense thinning. Finally, intense thinning showed the highest values of SFC, PWP, SAW, soil pH, SOC, total nitrogen, and K₂O₂. By comparing the results from the control with those from intense thinning, we found that the highest thinning intensity showed 4.92, 11.22, 16.56, 47.79, 48.62, and 31.25% lower bulk density, grain density, pore volume, permeability, infiltration, and electrical conductivity, respectively. On the other hand, we found that intense thinning showed 21.47, 13.90, 30.45, 5.86, 191.02, 140.00, 17.74, and 89.85% greater field capacity, permanent wilting point, available water, soil pH, soil organic carbon, total nitrogen, P₂O₅, and K₂O, respectively (

Table 2. Soil physical-chemical properties (mean ± standard deviation) as influenced by thinning intensities at monospecific stands of F. orientalis, Turkey.

Soil Properties	Control	MT	MIT	IT
Bulk density (g cm−3)	1.42 ± 0.01 a	1.39 ± 0.01 b	1.38 ± 0.01 c	1.35 ± 0.01 d
Grain density	2.85 ± 0.21 a	2.74 ± 0.24 a	2.56 ± 0.19 b	2.53 ± 0.15 b
Soil pore volume	80.69 ± 5.47 a	78.56 ± 6.24 a	72.43 ± 4.71 b	67.32 ± 5.35 b
SFC (%)	19.51 ± 0.89 d	20.53 ± 1.04 c	21.89 ± 1.86 b	23.70 ± 1.03 a
PWP (%)	10.57 ± 0.64 b	10.53 ± 0.49 b	11.82 ± 0.81 a	12.04 ± 0.97 a
SAW (%)	8.93 ± 0.33 c	10.50 ± 1.41 b	10.06 ± 1.27 b	11.65 ± 1.18 a
Soil permeability (mm/sa)	103.65 ± 19.01 a	85.60 ± 7.25 b	71.92 ± 5.15 c	55.15 ± 5.32 d
Infiltration	15.59 ± 2.57 b	15.95 ± 2.72 a	9.42 ± 2.98 c	8.01 ± 3.01 d
Soil pH (1:2.5, v:v)	4.09 ± 0.10 b	4.16 ± 0.15 b	4.32 ± 0.65 a	4.33 ± 0.12 a
CaCO3 (%)	1.08 ± 0.09 a	1.18 ± 0.15 a	1.17 ± 0.33 a	1.20 ± 0.24 a
Electrical conductivity (dS m−1)	0.16 ± 0.07 b	0.23 ± 0.11 a	0.16 ± 0.06 b	0.11 ± 0.04 c
SOC (%)	0.78 ± 0.36 d	1.37 ± 0.45 c	1.71 ± 0.85 b	2.27 ± 1.77 a
Total nitrogen (%)	0.05 ± 0.02 d	0.07 ± 0.03 c	0.10 ± 0.05 b	0.12 ± 0.08 a
P2O5 (kg/da)	2.48 ± 0.40 d	2.79 ± 0.65 c	3.25 ± 0.40 a	2.92 ± 0.34 b
K2O (kg/da)	13.80 ± 3.18 c	17.39 ± 4.75 b	18.77 ± 5.79 b	26.20 ± 13.2 a

The same lowercase letters represent no significant differences among thinning intensities by Duncan’s test (p < 0.05). MT = moderate thinning; MIT = moderately intense thinning; and IT = intense thinning. Bold cells represent the highest significant observed values. SFC = soil field capacity (%), PWP = permanent wilting point (%), SAW = soil available water (mm/sa), and SOC = soil organic carbon (%).

).

3.2. Thinning Intensities Influence Arbuscular Mycorrhizal Fungi Community Composition from Ultisol and Root Colonization by AM Fungi

The frequency of occurrence of spores from AM fungi varied significantly among the thinning intensities (p < 0.001). In total, we identified 16 AM fungi species, as follows: Acaulospora sp., A. dilatata, A. bireticulata, Dentiscutata heterogama, Racocetra coralloidea, Scutellospora sp., S. calospora, S. rubra¸ Claroideoglomus sp., C. claroideum, C. etunicatum, C. geosporum, Funneliformis sp., F. geosporum, F. mosseae, Glomus sp., G. multicaule, Rhizophagus irregularis, and Sclerocystis coremioides (

Table 3. Frequency of occurrence (FOi) of spores from AM fungi and ecological indices observed in monospecific stands of F. orientalis as influenced by thinning intensities.

AMF Species	FOi 1 (Classification 2)				Species Classification 3
	Control	MT	MIT	IT
Order Diversisporales
Family Acaulosporaceae
Acaulospora dilatata	4.3 (R)	-	-	-	Exclusive
A. bireticulata	-	8.3 (R)	-	-	Exclusive
Acaulospora sp.	17.4 (C)	-	-	-	Exclusive
Order Gigasporales
Family Dentiscutataceae
Dentiscutata heterogama	-	15.7 (C)	-	-	Exclusive
Family Gigasporaceae
Racocetra coralloidea	-	9.7 (R)	-	-	Exclusive
Scutellospora calospora	4.8 (R)	-	-	-	Exclusive
S. rubra	4.5 (R)	8.5 (R)	8.7 (R)	17.5 (C)	Generalists
Scutellospora sp.	-	-	-	9.1 (R)	Exclusive
Order Glomerales
Family Entrophosporaceae
Claroideoglomus claroideum	13.0 (C)	-	-	-	Exclusive
C. etunicatum	8.7 (R)	-	18.7 (C)	-	Intermediate
C. geosporum	5.6 (R)	-	9.3 (R)	9.1 (R)	Intermediate
Claroideoglomus sp.	-	-	8.3 (R)	8.5 (R)	Intermediate
Family Glomeraceae
Funneliformis geosporum	4.9 (R)	16.3 (C)	-	-	Intermediate
F. mosseae	9.1 (R)	-	-	-	Exclusive
Funneliformis sp.	-	-	-	16.2 (C)	Exclusive
Glomus multicaule	-	-	-	9.7 (R)	Exclusive
Glomus sp.	19.5 (C)	24.8 (C)	23.7 (C)	-	Intermediate
Rhizophagus irregularis	7.5 (R)	16.7 (C)	21.9 (C)	17.6 (C)	Generalists
R. intraradices	-	-	-	12.3 (C)	Exclusive
Sclerocystis coremioides	-	-	9.4 (R)	-	Exclusive
Ecological indices
Species richness (S)	11.00 a	7.00 b	7.00 b	8.00 b
Simpson index (D)	0.93 a	0.94 a	0.91 a	0.94 a
Shannon index (H′)	2.31 a	1.89 c	1.83 c	2.02 b
Hurlbert’s PIE	0.93 b	0.94 b	1.07 a	0.95 b
Pielou index (J)	0.93 a	0.97 a	0.94 a	0.97 a

¹ FOi = n/N, where N is the total of AMF spores observed and n is the number of times an AMF species were encountered from each research area; ² Classification of AMF frequency of occurrence was analyzed according to [30]: D–dominant (FO > 50%), MC—most common (31% ≤ FO ≤ 50%), C—common (10% ≤ FO ≤ 30%), and R—rare (FO < 10%); and ³ Species classification suggested by [31] most common (present in all fields), intermediate (present in 2 fields) or exclusive (present in only one field). The same lowercase letters represent no significant differences among thinning intensities by Duncan’s test (p < 0.05). MT = moderate thinning; MIT = moderately intense thinning; and IT = intense thinning. Bold cells represent the highest significant observed values.

). We identified 11 AM fungi species in the control, 7 with moderate thinning and moderately intense thinning, and 8 with intense thinning. The most common identified AMF species were R. irregularis, S. rubra, Glomus sp., and C. geosporum, whereas the exclusive AM fungi species identified were A. dilatata, A. Bireticulata, D. heterogama, R. coralloidea, S. calospora, C. claroideum, F. mosseae, G. multicaule, R. intraradices, and S. coremioides. For the ecological indices, the highest significant values of species richness and Shannon’s index were found in the control, while the highest values of Hurlbert’s PIE were found with moderately intense thinning. For the Simpson and Pielou indices, we did not find any significant differences among the different thinning intensities (Table 3).

We observed significant differences among the thinning intensities regarding root colonization by AMF (p < 0.001). The highest values of root colonization were found in the roots of Z. mays that were inoculated with rhizospheric soil from intense thinning (Figure 2).

3.3. Multivariate Analysis: A Deeper View of Thinning İntensities’ İnfluence on Soil Properties and AM Fungi Community Composition

The PCA showed that species richness, Shannon’s diversity, K₂O, P₂O₅, total nitrogen, soil organic carbon, electrical conductivity, infiltration, and permeability were the main factors contributing to the variance of the samples (Figure 3). The thinning intensities were dissimilar to each other. Additionally, the analysis provided the following relationships: (i) a negative correlation among P₂O₅, species richness, and Shannon’s diversity; (ii) a positive relationship between the control and AMF richness and diversity; (iii) a positive relationship between moderate thinning and electrical conductivity; (iv) a positive relationship between moderately intense thinning and soil P₂O₅ content; and (v) a positive relationship between intense thinning and soil K₂O content (Figure 3).

The NMDS grouped the thinning intensities into four groups considering the dissimilarities in AMF community composition (the frequency of occurrence of spores). The NMDS had a good fit with stress values equal to 0.04. The soil samples from the control were characterized by a high frequency of occurrence of C. claroideum (R² = 0.80, p < 0.01) and Acaulospora sp. (R² = 0.91, p < 0.01). For moderate thinning, this was characterized by a high frequency of occurrence of C. claroideum (R² = 0.79, p < 0.01), A. bireticulata (R² = 0.71, p < 0.01), and Glomus sp. (R² = 0.65, p < 0.05). Next, moderately intense thinning was characterized by a high frequency of occurrence of R. irregularis (R² = 0.65, p < 0.05) and S. coremioides (R² = 0.90, p < 0.001). Finally, intense thinning was characterized by a high frequency of occurrence of R. intraradices (R² = 0.93, p < 0.001) and Funneliformis sp. (R² = 0.90, p < 0.001) (Figure 4).

4. Discussion

4.1. Relationship among Soil Properties, AM Fungi, and Thinning Intensities in F. orientalis Stands

Our results demonstrate the influences of different thinning intensities on bulk density, grain density, pore volume, permeability, infiltration, electrical conductivity, P₂O₅, field capacity, permanent wilting point, available water, soil pH, soil organic carbon, total nitrogen, and K₂O₂, and how these factors may affect the AM fungi community composition and root colonization by AMF. Previous studies have shown that Ultisols are recognized by their acidic nature, high degree of weathering, clay-rich composition, low natural fertility, well-developed soil horizons, moderate to high aluminum content, and susceptibility to erosion [32,33]. These soils exhibit a history of extensive chemical weathering, resulting in the leaching of soluble minerals, which makes forestry highly dependent on appropriate management practices such as thinning and symbiotic relationships with AM fungi [8]. AM fungi species were found in the rhizosphere of F. orientalis, but additionally, we found evidence supporting our first and second hypotheses about thinning intensities directly changing the soil physical–chemical properties and indirectly changing the AM fungi community composition [34]. Our main objective was to understand how different thinning intensities, a management practice that aims to reduce plant density and competition, could alter both the soil properties and AM fungi community, as hypothesized by Barbosa et al. [11], who described plant densities influencing the AM fungi community structure through changes in the soil physical–chemical properties. Our results show how different thinning intensities affect the soil properties to enhance AM fungi symbiosis [35,36]. We found that root colonization and the frequency of occurrence of Scutellospora rubra, Funneliformis sp., and Rhizophagus intraradices with intense thinning were higher than in the control. These results align with previous studies by Masebo et al. [37] and Mohamed et al. [38], which reported high root colonization and the dominance of specific AM fungi in soil samples under management practices [39].

In such conditions, it is important to consider that different thinning intensities directly influence plant density, which can alter the soil physical and chemical properties by affecting root density, nutrient and water uptake rates, light acquisition, and plant competition [40]. Typically, changes in the soil physical and chemical properties occur within the tree’s rhizosphere or rhizobiome, supporting the ‘island of fertility’ hypothesis described by Laurindo et al. [41]. The changes in the rhizobiome influenced by different thinning intensities include decreases in bulk density, grain density, pore volume, permeability, infiltration, and electrical conductivity, with increases in field capacity, permanent wilting point, available water, soil pH, soil organic carbon, total nitrogen, P₂O₅, and K₂O, when comparing the results from the control to those from intense thinning. These changes promoted the AM fungi community by increasing the frequency of occurrence of specific AM fungi and root colonization [22,41]. In subtropical ecosystems, other studies have reported that thinning, as a management practice, may modulate the soil’s physical properties in the rhizosphere of adult trees over time [42,43,44]. This modulation occurs through improvements in habitat provision, an increase in the soil organic carbon content, and bioturbation [45,46]. When comparing the results from the control to those from intense thinning, positive relationships have been found between bulk weight and field capacity, wilting point, and clay content, while a negative relationship has been observed between bulk density and sand content [47,48].

Over time, these changes in the rhizosphere of F. orientalis may affect the structure of the AM fungi community (species richness, diversity, dominance, and evenness) by selecting AM fungi species that are adapted to specific ranges of soil pH, nutrient content, organic matter, and moisture [11,49]. Overall, spores from Acauslospora, Claroideoglomus, Dentiscutata, Funneliformis, Glomus, Racocetra, Rhizophagus, Sclerocystis, and Scutellospora were found in the rhizosphere of F. orientalis, as influenced by different thinning intensities. In soil ecosystems with a soil pH ranging from 4.0 to 7.0 and an average soil organic carbon content, these genera are commonly found, as described by Souza et al. (2024) [1]. For example, high values of soil pH, soil organic carbon, total N, and K₂O can be attributed to high root colonization and a high frequency of occurrence of Scutellospora rubra, Funneliformis sp., and Rhizophagus intraradices [11,41]. It is widely reported that thinning improves plant performance (growth rate, plant biomass, and root density), and such improvements may promote AM fungi symbiosis and root colonization [48]. We observed that high root colonization by AMF does mean a soil ecosystem with average values of soil physical–chemical properties.

4.2. The İnfluence of Different Thinning İntensities on AM Fungi Community Composition: Evidence of AM Fungi–Host Pairing Specificity

Our results indicated that the different thinning intensities altered the AM fungi community (frequency of occurrence of AM fungi species, species richness, and diversity) by establishing a changed rhizobiome, as described in the “enhanced mutualism” hypothesis [50]. This shift in the AM fungi community enabled root colonization by specific species [51]. We found a high frequency of occurrence of (i) Acaulospora sp. and Glomus sp. in the control; (ii) Dentiscutata heterogama, Funneliformis geosporum, Glomus sp., and Rhizophagus irregularis with moderate thinning; (iii) Claroideoglomus etunicatum, Glomus sp., and Rhizophagus irregularis with moderately intense thinning; and (iv) Funneliformis sp., Rhizophagus irregularis, Rhizophagua intraradices, and Scutellopora rubra with intense thinning (ESM_1). We found strong evidence that these AM fungi species have improved the root colonization by AM fungi (ESM_2) in maize roots [50,52]. An improvement in the root colonization by AM fungi can enhance plant growth, biomass, nutrition, and yield [53]. The root colonization results offered insights into the symbiosis functionality in the F. orientalis rhizobiome. For instance, the high root colonization with intense thinning indicated nutritional benefits for F. orientalis, while the frequency of occurrence of some AM fungi taxa suggested that thinning promotes host–AM fungi pairing specificity [54,55].

The ecological relationship between AM fungi and host plants is controlled by several external stressors, such as root activity, root exudates, plant competition, moisture, temperature, nutrient content, and soil pH. For example, we found an increase in the total nitrogen content with intense thinning. It enhances biological activity and soil organic matter decomposition [56]. Thus, it depolarizes AM fungi membranes, enhances hyphal growth, and activates lipid catabolism and protein synthesis genes [57]. Additionally, changes in soil pH and electrical conductivity can regulate electrochemical capacity, which, in turn, enhances the uptake of nutrients, carbohydrates, lipds, and especially the uptake of inorganic P [11].

Arbuscular mycorrhizal (AM) fungi play a crucial role in forest sustainability by enhancing nutrient uptake, promoting plant growth, and improving the soil structure [35]. The diversity of AM fungi in forest ecosystems is directly linked to the health and resilience of the forest [1]. A high AM fungi diversity increases the range of beneficial interactions between plants and fungi, allowing for better nutrient cycling and distribution among different plant species. This symbiotic relationship is particularly important in nutrient-poor soils, where AM fungi can significantly enhance the availability of essential nutrients like phosphorus, thus supporting plant growth and maintaining the productivity of the forest ecosystem [56].

Changes in AM fungi diversity can have profound effects on forest sustainability [41]. A reduction in AM fungi diversity, often caused by environmental disturbances or changes in land use, can lead to a decreased nutrient availability and weakened plant–fungal interactions, resulting in a reduced forest resilience [30]. Conversely, maintaining or increasing AM fungi diversity can improve forest sustainability by enhancing the ecosystem’s ability to recover from disturbances, resist pathogens, and adapt to changing environmental conditions. This highlights the importance of conserving AM fungi diversity as a key component of forest management practices aimed at promoting long-term ecological stability and sustainability [8,9].

4.3. Thinning Intensities Changing F. orientalis Rhizosphere: Evidence for Changes in Soil Physical–Chemical Properties

Our results subsection on changes in the soil physical–chemical properties demonstrated that intense thinning could decrease bulk density, grain density, pore volume, permeability, infiltration, and electrical conductivity. On the other hand, intense thinning increased field capacity, permanent wilting point, available water, soil pH, soil organic carbon, total nitrogen, P₂O₅, and K₂O. It is important to emphasize that the experimental plots had the soil type classified as Ultisols. Monospecific stands of F. orientalis received the same management practices and fertilization over the years. Studies focused on soil physical–chemical properties have demonstrated that thinning can improve root density and root activity by releasing organic acids and H⁺ into the rhizosphere [30,58]. AM fungi symbiosis presents three distinct phases (asymbiotic, presymbiotic, and symbiotic) that are controlled by the root system, root exudation, and H⁺ extrusion. Consequently, a management practice that can improve the root system will directly enhance root colonization by selected AM fungi species [59].

With intense thinning, we observed the highest values for field capacity, available water, soil pH, soil organic carbon, total nitrogen, and K₂O, which can, in various pathways, promote the aymbiotic phase and a higher frequency of occurrence of the Funneliformis, Rhizophagus, and Scutellospora genera [41]. Changes in the soil physical–chemical properties in the F. orientalis rhizobiome caused a decrease in the H⁺ flux, since we observed an increase in the soil pH in plots with intense thinning. High values of soil pH can change the total nitrogen and K₂O contents [60]. Other studies in Ultisols have shown that the root system may improve its own rhizosphere through exudation, which helps the plant to create a favorable habitat for symbionts and other key microorganisms [61,62].

The NMDS groups support our hypothesis that different thinning intensities create distinct rhizobiomes with unique soil properties and AM fungi–F. orientalis pairings. Studies on AM fungi and F. orientalis in Turkey’s natural beech forests are rare. This is the first study to assess the impact of different thinning intensities on soil AM fungi communities. Specific AM fungi and F. orientalis pairings can (i) reduce species richness and Shannon’s diversity, as observed in the PCA score plot; (ii) enhance root colonization by Scutellospora rubra, Funneliformis sp., and Rhizophagus intraradices; and (iii) improve rhizobiome dynamics by altering root activity [63]. These results align with the NMDS analyses reported by Laurindo et al. (2022) [41] and Souza et al. (2024) [1]. In fact, our results highlighted that different thinning intensities can promote the rhizobiome of F. orientalis in different pathways, thus, it is important to consider management practices with the ability to enhance the root system as a promoter of the symbiotic relationship between tree species and AM fungi [64].

5. Conclusions

The different thinning intensities influenced the soil physical–chemical properties and the community structure of the AM fungi, which, in turn, influenced the root colonization by the AM fungi, supporting the AM fungi–host pairing hypothesis, as evidenced in the rhizosphere of F. orientalis. Specific pairings of AM fungi and F. orientalis can (i) reduce species richness and Shannon’s diversity, as observed in the PCA score plot; (ii) enhance root colonization by Scutellospora rubra, Funneliformis sp., and Rhizophagus intraradices; and (iii) improve rhizobiome dynamics by altering root activity. We observed a high frequency of occurrence of (i) Acaulospora sp. and Glomus sp. in the control; (ii) Dentiscutata heterogama, Funneliformis geosporum, Glomus sp., and Rhizophagus irregularis with moderate thinning; (iii) Claroideoglomus etunicatum, Glomus sp., and Rhizophagus irregularis with moderately intense thinning; and (iv) Funneliformis sp., Rhizophagus irregularis, Rhizophagus intraradices, and Scutellospora rubra with intense thinning. Our results indicated decreases in bulk density, grain density, pore volume, permeability, infiltration, and electrical conductivity, while we observed increases in field capacity, permanent wilting point, available water, soil pH, soil organic carbon, total nitrogen, P₂O₅, and K₂O when comparing the results from the control to those from intense thinning. This study emphasizes the importance of forest management practices that sustainably enhance root systems and improve plant performance, soil fertility, and AM fungi symbiosis.