Variation in Culturable Rhizosphere Fungal Communities of Abies koreana in Mt. Jiri, Korea, in Relation to Topographic and Physicochemical Soil Properties

Abstract

This study was aimed at characterizing the culturable rhizosphere fungal community of Abies koreana on Mt. Jiri, Korea and to investigate the influence of topographic and physicochemical soil properties on them. Samples were collected from two contrasting sites: the Seseok Plateau (gentle-slope basin with poor drainage) and Oesamsinbong (steep slope with excellent drainage). A total of 620 isolates spanning four phyla, 11 classes, 21 orders, 34 families, and 51 genera were identified. Penicillium was the dominant genus, comprising 367 isolates. Shannon’s diversity index increased as Penicillium dominance decreased, indicating that reduced exclusive dominance enhanced community evenness. Although both sites had loamy soils, their chemical properties varied significantly with topography. The Seseok Plateau showed extreme acidification (pH 3.80–4.20) and severe cation leaching with a very low average base saturation (BS) of 4.9%. In contrast, Oesamsinbong maintained higher BS (avg. 12.3%) because the steep terrain inhibited acid accumulation. Penicillium dominance was significantly higher in Oesamsinbong (72.7–76.0%) than in the Seseok Plateau (31.9–48.9%). Pearson’s correlation revealed a strong positive relationship between the BS and Penicillium dominance (r = 0.957, p = 0.0028). Although available phosphate was higher in the Seseok Plateau, the high Penicillium dominance in Oesamsinbong supports a potential hypothesis regarding microbial contribution to phosphorus availability for the host. Overall, the reduced vigor of A. koreana in the Seseok Plateau was likely associated with impaired microbe-mediated nutrient uptake under extreme acidic stress. Enhancing the rhizosphere fungal ecosystem is crucial for ensuring the sustainability of endangered subalpine conifer forests.

1. Introduction

Abies koreana Wilson is an endemic species found only in high-altitude regions of the Korean Peninsula. It is recognized for its high ecological value and is classified as an endangered species by the International Union for Conservation of Nature (IUCN) [1]. Recently, high-temperature, humid environments, and moisture stress resulting from climate change have been identified as the primary causes for the rapid decline of A. koreana forests, particularly on Mt. Jiri and Mt. Halla [2,3]. To address this crisis, research on meteorological factors and physiological characteristics is being conducted actively [3–5]. However, studies on the soil environment, foundation of A. koreana survival, and preservation and utilization of symbiotic microbial resources remain relatively insufficient.

Soil microorganisms, particularly rhizosphere fungi, play vital roles in maintaining host plant health by solubilizing insoluble nutrients and suppressing pathogens [6–8]. Therefore, investigating rhizosphere microbial communities in A. koreana habitats and securing beneficial strains is crucial, not only for habitat restoration, but also for the systematic preservation of biological resources as unique national assets [9]. Fungi adapted to the extreme conditions of high-altitude environments possess unique physiological traits [10–12], representing significant value as biological resources.

This study was aimed at analyzing the relationship between soil physicochemical properties and cultural rhizosphere fungi at two distinct sites on Mt. Jiri with contrasting topographical features: the Seseok Plateau (Seseok-pyeongjeon) and Oesamsinbong. Culturable rhizosphere fungi from each site were isolated and identified to evaluate their potential for preservation as a national biological resource. Furthermore, diversity indices (Shannon, Simpson, and Margalef) were calculated to quantitatively evaluate the health of the microbial ecosystem in relation to topographic and soil conditions.

This study sought to clarify how soil acidification and base saturation (BS) influence the dominance of beneficial fungi, such as Penicillium, and the overall fungal diversity, so as to provide insights into the mechanism for the decline in A. koreana abundance from a soil–microbe perspective. The strains and ecological data obtained in this study will serve as fundamental data for establishing sustainable conservation strategies for A. koreana forests and for the national management of microbial resources.

2. Materials and methods

2.1. Sampling site description

Rhizosphere fungi were sampled from the Seseok Plateau and Oesamsinbong, where A. koreana communities are relatively preserved [13] (Table 1). Because the ecology of rhizosphere fungi is influenced by the host plant, samples were collected from three sufficiently spaced locations within each site. The Seseok Plateau is a high-altitude basin on Mt. Jiri, where cold air at elevations of 1500–1700 m tends to become trapped and stagnant. It is a high-level undulating plateau covering approximately 1 km² with a gentle slope of 15°–20°. Because of this topography, poor drainage has been reported to adversely affect the survival of A. koreana [2] (Table 1).

Table 1.

Sampling sites and taxa distribution.

Geographical name	Sampling site no.	GPS coordinates	Total isolates	Phylum	Class	Order	Family	Genus
Seseok Plateau (Seseok-pyeongjeon)	1	35°19′06.51″N 127°41′41.23″E	93	4	7	9	13	18
	2	35°19′06.50″N 127°41′42.15″E	92	4	7	7	10	16
	3	35°19′06.50″N 127°41′43.50″E	97	3	7	13	19	27
Oesamsinbong (mountain peak)	4	35°15′40.74″N 127°42′50.61″E	96	2	5	8	13	15
	5	35°15′41.53″N 127°42′49.05″E	88	4	6	7	9	12
	6	35°15′42.57″N 127°42′46.01″E	154	4	10	17	26	29

Oesamsinbong, one of the major ridges on Mt. Jiri, is located at a lower elevation (1350–1450 m) than the Seseok Plateau, and has steep slopes ranging from 25° to 35°. As it forms part of the southern ridge of Mt. Jiri, it receives abundant solar radiation. However, high solar exposure combined with steep terrain leads to rapid evaporation of soil moisture and excellent drainage [14,15].

2.2. Soil sampling

Rhizosphere soil represents the area of soil around plant roots where highly active interactions among soil, host plant, and microbiome takes place [16,17]. Samples were collected from each site within 15–20 cm of tree trunks. Rhizosphere soil samples were collected at a depth of 25–30 cm from the surface, where a large proportion of the active root zone existed. Samples were collected from three live A. koreana trees at each site. The samples were immediately transferred to the laboratory under refrigerated conditions (4 °C), and samples from each site were pooled to generate composite samples representing site-level conditions for exploratory analysis.

2.3. Culture and isolation

The specimens were transferred to sterile bottles, maintained at 4 °C, and quickly transferred to the laboratory. Each soil sample was mixed with sterile 0.85% NaCl solution at a volumetric ratio of 9:1 and agitated at 40 rpm for 30 min. The supernatant (100 µL) was serially diluted 10⁴ to 10⁶-times and smeared on 1/2-strength potato dextrose agar (Difco, Detroit, MI) plates. Thereafter, fungal isolates from the agar were streaked onto non-diluted PDA plates to exclude morphologically identical isolates.

2.4. Genomic DNA extraction and PCR amplification

Each isolate was inoculated into sterile potato dextrose broth (PDB) and cultured at 25 °C and 120 rpm for seven days. The mycelia were then filtered and lyophilized for two days. Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS) region was amplified using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [18]. The PCR conditions were as follows: initial denaturation at 94 °C for 4 min; 35 cycles of denaturation (94 °C, 1 min), annealing (55–58 °C, 1 min), and extension (72 °C, 2 min); and a final extension at 72 °C for 10 min. The amplified PCR products were purified using an AccuPrep PCR Purification & Gel Extraction Kit (Bioneer, Daejeon, South Korea) and sequenced using an ABI 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA).

2.5. Identification and diversity analysis

Fungal isolates were preliminarily identified based on BLASTn search results of their ITS region sequences. Given the limitations of ITS barcode resolution for species-level differentiation within specific lineages (e.g., Penicillium), these assignments should be interpreted as the closest matching taxonomic units rather than definitive species placements. All isolated fungal sequences (n = 620) were deposited in GenBank under the accession numbers PX628495–PX629114 (Supplemental Table 1). To evaluate fungal diversity, species richness was assessed using Margalef’s (Dmg) and Menhinick’s (Dmn) indices, whereas species diversity was measured using the Shannon (H′) and Simpson (D) indices [19–22]. All diversity indices were calculated strictly at the genus level based on the total raw abundance data for the recovered isolates without rarefaction or downsampling, ensuring a full reflection of the secured cultural biological resources.

2.6. Chemical properties analysis

The chemical properties of the collected rhizosphere soils were analyzed according to standard soil analysis methods of the National Institute of Agricultural Science and Technology [23,24].

• pH and electrical conductivity (EC): Soil pH and EC were measured using a pH meter and an EC meter, respectively, after mixing the soil with distilled water at a 1:5 (w/v) ratio and stirring for 30 min.

• Organic matter (OM) and total nitrogen (T-N): Organic matter content (%) was determined using the Walkley–Black redox titration method, and T-N (%) was analyzed using the micro-Kjeldahl method.

• Available phosphate (P₂O₅): After extraction using the Lancaster method, available phosphate was quantified using a colorimetric method and expressed in mg/kg.

• Exchangeable cations (Ca, Mg, K, and Na): Exchangeable cations were extracted with a 1 N NH₄OAc (pH 7.0) solution and measured using inductively coupled plasma optical emission spectrometry (ICP-OES), with concentrations calculated in mg/kg.

• Cation exchange capacity (CEC): The total nutrient retention capacity of the soil was measured using the 1 N NH₄OAc extraction method and expressed in cmol⁺/kg.

To investigate the effects of soil acidification and mineral distribution on the dominance of rhizosphere fungi, Pearson’s correlation coefficients (r) were calculated to evaluate the relationships between major soil variables (pH, OM, exchangeable cations, etc.) and the dominance of Penicillium. Statistical significance was assessed using these coefficients.

2.7. Particle size analysis

Sieve analysis was performed in accordance with the ASTM D422 standard test methods [25] to determine the particle size distribution of the soil samples used in this study. As a pretreatment, the collected soil was air-dried at room temperature and the aggregated particles were crushed to prepare the samples for analysis. The total dry weight of each sample was measured to establish a baseline value for analysis.

For the mechanical sieve analysis, a series of standard sieves including 4.75 mm (No. 4), 2.0 mm (No. 10), 0.85 mm (No. 20), 0.425 mm (No. 40), 0.25 mm (No. 60), 0.11 mm (No. 140), and 0.075 mm (No. 200) were arranged vertically. The samples were agitated for 10–15 min on a mechanical sieve shaker.

Data were calculated by determining the retained weight (g) of each sieve based on the difference between the initial and final sieve weights. These values were used to calculate the cumulative retained weight (g) and percentage (%). Finally, the proportion of the sample passing through each sieve was expressed as the cumulative passing percentage, which served as the fundamental data for the particle size distribution curve. To determine the fine-grained soil (silt and clay) content, the weight of the particles collected in the bottom pan (residual) after passing through a 0.075 mm (No. 200) sieve was measured.

2.8. Base saturation analysis

Base saturation was calculated using soil physicochemical data [26,27]. The concentrations of the exchangeable cations, measured in mg/kg, were converted into cmol⁺/kg. The sum of these exchangeable cations was then divided by CEC to determine the percentage of BS. The conversion factors used in the calculations were Ca/200, Mg/121.5, K/391, and Na/230.

3. Results and discussion

3.1. Taxonomic distribution

In the present study, 620 fungal isolates were identified based on their morphological characteristics. These isolates were classified into four phyla: 11 classes, 21 orders, 34 families, and 51 genera (Table 1). The phyla identified included Ascomycota (550 isolates), Mortierellomycota (53), Mucoromycota (10), and Basidiomycota (7). The dominant genus was Penicillium, represented by 367 morphologically distinct isolates. All sequences obtained in this study were assigned GenBank accession numbers (Table 1 and Supplemental Table 1).

In addition, 18 isolates belonging to the genus Oidiodendron from several sites (Supplemental Table 2) were reported to play a crucial role in the conservation of A. koreana. These isolates showed high sequence similarity with O. chlamydosporicum, O. citrinum, O. echinulatum, O. flavum, O. maius, and O. tenuissimum. Notably, some isolates exhibited less than 98% similarity, suggesting their potential as new biological resources. O. maius, recently discovered in A. koreana communities, has been reported to yield significantly positive results in the restoration and survival of this endangered tree species [28,29].

3.2. Diversity indices

The genus-level diversity indices of the culturable fungal community isolated from the rhizosphere of A. koreana were calculated as follows (Table 2): Shannon’s index (H′) 2.00, Simpson’s index (D) 0.36, Margalef’s richness 7.78, and Menhinick’s index (Dmn) 2.05. A Shannon index >2.0 generally indicates a balanced distribution of species. Although a high concentration of Penicillium was observed in Oesamsinbong, its overall statistical dominance was moderated by the appearance of other fungal genera across the study sites. Furthermore, a Menhinick’s index of approximately 2.05 confirms a high level of species richness in the surveyed areas.

Table 2.

Fungal diversity in the rhizosphere of Abies koreana.

Geographical name	Sampling site no.	Number of isolates	Genera richness	Margalef’s richness	Menhinick’s index (Dmn)	Shannon’s index (H′)	Simpson’s index (D)	Ratio (%) of Penicillium	No. of Penicillium
Seseok Plateau (Seseok-pyeongjeon)	1	93	19	3.971	1.970	2.093	0.204	40.8	38
	2	92	16	3.317	1.668	1.916	0.262	48.9	45
	3	97	27	5.683	2.741	2.652	0.123	31.9	31
	Subtotal	282	35	6.026	2.084	2.446	0.185	40.57	114
Oesamsinbong (mountain peak)	4	96	15	3.067	1.531	1.157	0.566	75.0	72
	5	88	12	2.457	1.279	1.143	0.537	72.7	64
	6	154	29	5.559	2.337	1.331	0.577	75.97	117
	Subtotal	338	40	6.698	2.176	1.394	0.562	74.57	253
Total		620	51	7.776	2.048	2.001	0.359	57.57	367

Diversity indices were calculated at the genus level using the raw data on the number of isolates, without standardization or rarefaction, to accurately reflect the total scope of the secured cultural resources.

In terms of the topographical distribution, the rhizosphere of the Seseok Plateau exhibited a stable taxonomic structure. In total, 282 isolates were obtained from this area, with 3–4 phyla and 16–27 genera consistently distributed across each sampling site. All three sites in this region maintained steady distributions at the class (7) and order (7–13) levels, forming a well-balanced community structure that was not biased toward any specific species.

In contrast, Oesamsinbong showed significant variation between sites and high diversity at specific locations. A total of 338 isolates were obtained from Oesamsinbong, site 6 possessed 154 isolates belonging to 10 classes, 17 orders, and 29 genera, representing the highest taxonomic richness among all the surveyed sites. Conversely, sites 4 and 5 had relatively fewer taxonomic groups, suggesting that fungal community density and composition varied significantly depending on microenvironmental factors, even within the same geographical area.

Simpson’s index for Oesamsinbong (0.562) was approximately three times higher than that for the Seseok Plateau (0.185), primarily because of the overwhelmingly high Penicillium ratio (74.57%) in Oesamsinbong. While a high Simpson’s index indicates strong dominance by specific species, it reveals that despite having healthy soil with good drainage and high BS, Oesamsinbong maintains an ecologically simpler community structure dominated by the beneficial fungus Penicillium [30]. Analysis showed that Shannon’s index tended to decrease as Penicillium dominance increased, suggesting that the fungal community in the A. koreana rhizosphere is governed by the establishment of Penicillium, which positively influences host plant growth and disease resistance [31–33]. However, it must be noted that the exclusive use of 1/2-strength PDA medium introduces a strong culture bias toward fast-growing opportunists and saprophytes (e.g., Penicillium and Trichoderma). This culture-dependent approach captures only the culturable fraction of the rhizosphere microbiome, potentially underrepresenting slow-growing, obligate, and unculturable taxa including many crucial ectomycorrhizal (ECM) fungi.

We hypothesized that these differences in community structure arise from topographic characteristics and their subsequent effects on soil properties, even though the Seseok Plateau and Oesamsinbong are located in the same region. To clarify the relationship between soil chemical properties and microbial dominance, Pearson’s correlation analysis was conducted to link community structure data with the soil analysis results.

3.3. pH variation of rhizosphere soil

The rhizosphere soils at both sites exhibited strong acidity (Table 3), and the pH of the Seseok Plateau soil was extremely low, ranging from 3.80 to 4.20 (mean 3.97). A key observation was the lower relative standard deviation (%RSD) of pH in Oesamsinbong than in Seseok Plateau. This indicates that the hydrogen ion concentration in Oesamsinbong was maintained more stably, whereas the Seseok Plateau suffered from both severe acidification and high environmental variability.

Table 3.

Soil chemical properties of the rhizosphere of Abies koreana.

Geographical name	Sampling site no.	pH	Exchangeable cation (mg/kg)				Electrical conductivity (dS/m)	Cation exchange capacity (cmol/kg)	P2O5 (mg/kg)	T-N (%)	Organic matter content (%)	Sum of exchangeable cations (cmol+/kg)	Base saturation (%)
			Mg2+	Ca2+	Na+	K+
Seseok Plateau (Seseok-pyeongjeon)	1	3.80	51.58	198.50	10.67	89.99	1.63	41.41	179.93	0.84	2.22	1.70	4.10
	2	4.20	88.70	342.00	14.79	136.16	1.98	39.35	190.08	1.09	2.70	2.85	7.24
	3	3.90	38.99	112.75	9.13	91.68	1.57	35.81	198.88	0.74	1.84	1.16	3.24
	Average	3.97	59.76	227.38	11.53	105.94	1.73	38.86	189.63	0.89	2.25	1.43	4.9
	(%) RSD	5.25	43.25	71.29	25.38	24.71	12.82	7.29	5.00	20.44	19.13	26.70	43.3
Oesamsinbong (mountain peak)	4	4.60	123.24	709.74	12.62	102.52	0.74	37.63	125.01	0.93	2.77	4.90	13.03
	5	4.40	93.37	450.82	26.84	115.08	0.84	35.37	130.91	0.89	2.47	3.43	9.69
	6	4.30	121.08	865.98	24.26	164.60	1.67	41.56	210.43	1.04	2.82	5.85	14.07
	Average	4.43	112.56	675.51	21.24	127.40	1.08	38.47	155.45	0.95	2.69	4.73	12.3
	(%) RSD	3.45	14.80	31.04	35.67	25.76	47.13	11.38	30.69	8.30	7.05	25.80	18.7

The extreme acidification of the Seseok Plateau can be attributed to its topographical features. The high-level plateau with gentle slopes inhibits effective drainage, leading to the continuous accumulation of humic acids and terpenes, including resin, from the needles. Furthermore, the low-temperature characteristics of Mt. Jiri delay the decomposition of OM, accelerating the accumulation of organic acids. The resulting oxygen depletion due to poor drainage likely suppresses nitrification and induces anaerobic decay. The metabolic byproducts of this process, such as methane and hydrogen sulfide, are considered detrimental to the growth of A. koreana.

In acidic environments, phosphorus (P) predominantly binds with iron (Fe) and aluminum (Al) to form insoluble phosphates. Although this strong fixation reduces the risk of phosphorus leaching, it simultaneously decreases the bioavailability of P in plants [34–36]. Chemical stress affects the microbial community. Although fungi generally exhibit higher acid tolerance than bacteria, and the genus Penicillium is known for its resilience to acidity, the drop in pH below the physiological threshold of 4.0 at the Seseok Plateau appears to have severely restricted mycelial growth and sporulation. Consequently, the lower dominance of Penicillium in the Seseok Plateau (31.9–48.9%) than in Oesamsinbong (72.7–76.0%) is interpreted as a direct result of extreme acidic stress.

3.4. Cation exchange capacity and electrical conductivity

While the optimal pH for A. koreana ranges from 5.5 to 6.5, the species can tolerate and grow in more acidic conditions [37]. However, from a soil chemical perspective, a pH <4.0 can trigger the leaching of essential base cations such as Ca²⁺ and Mg²⁺, as well as associated P loss [38]. The severe depletion of base cations observed in the Seseok Plateau requires careful interpretation of its hydrological and chemical characteristics. Although the site is characterized by poor drainage, which typically limits solute transport, the accumulation of organic acids under such conditions may create a chemically unstable environment. During periods of intense rainfall, these accumulated acidic compounds can facilitate episodic leaching events in which exchangeable base cations such as Ca²⁺ and Mg²⁺ are rapidly displaced and removed from the soil system. This mechanism provides a plausible explanation for the paradoxical coexistence of poor drainage and severe base depletion, ultimately resulting in the extremely low BS values observed, and establishes a clear link between soil acidification and the depletion of exchangeable base cations in the Seseok Plateau.

In addition to base cation loss, strongly acidic conditions (pH < 4.0) suggest a high likelihood of Al³⁺ mobilization. Although Al concentrations were not directly measured in this study, it is well established that such conditions promote Al solubilization, which can exert toxic effects on plant roots and inhibit microbial activity. Therefore, the decline in A. koreana abundance in the Seseok Plateau is hypothesized to be influenced not only by nutrient depletion but also by combined chemical stress, including a possible mechanism involving aluminum toxicity; however, this hypothesis needs to be empirically validated.

In this study (Table 3), extremely low levels of exchangeable K, Na, and Ca were observed at sites 1 and 3 (pH < 4.0), providing empirical support for the proposed episodic leaching mechanism. This suggests that the base cations previously adsorbed onto soil colloids were displaced by H⁺, further lowering soil pH [38]. Consequently, although the acidity was within the survival range of A. koreana, the resulting mineral deficiency acted as a significant growth-limiting factor.

In Oesamsinbong, the %RSD of monovalent cations was higher than that of divalent cations. This aligns with the typical soil behavior, where monovalent cations, which have weaker adsorption affinities, exhibit higher mobility and sensitivity to environmental changes. Conversely, the Seseok Plateau showed a reverse trend, with divalent cations exhibiting higher variability than monovalent cations. This unusual phenomenon suggests that severe localized soil acidification on the Seseok Plateau causes severe chemical stress, leading to the displacement of strongly adsorbed divalent cations from the soil exchange complex.

The EC values at all sites remained below 2.0 dS/m, which is considered stable for plant growth [38]. The average CEC was approximately 38 cmol⁺/kg with minimal spatial variation, indicating high soil fertility, a robust buffering capacity, and abundant organic carbon. The relatively higher EC observed in the acidified areas of the Seseok Plateau was attributed to the release of exchangeable cations from soil colloids into the soil solution during acidification.

3.5. Distribution of available phosphate and microbial-mediated solubilization mechanism

The analysis revealed that the available phosphate levels (Table 3) were higher in the Seseok Plateau than in Oesamsinbong. In subalpine regions, phosphate is typically strongly bound to soil particles, making them susceptible to physical scouring and subsequent losses during heavy rainfall events associated with climate change [39–44]. Therefore, the lower phosphate levels observed in Oesamsinbong were attributed to enhanced physical removal driven by steep slopes.

However, the relationship between phosphate concentration and plant nutrient status appeared to be decoupled in this system. Despite the relatively high levels of extractable phosphate in the Seseok Plateau, the reduced dominance of phosphate-solubilizing fungi (PSF), particularly Penicillium, suggests that the bioavailability of phosphorus may be limited under strongly acidic conditions. Although direct measurements of bioavailable phosphorus or microbial solubilization activity were not conducted, the observed patterns indicate that microbial-mediated processes play a critical role in regulating nutrient availability.

Under strongly acidic conditions (pH < 4.0), phosphate is prone to fixation through interactions with Fe and Al, forming insoluble compounds that are not readily accessible to plants [34–36]. In the Seseok Plateau, this chemical limitation is exacerbated by the severe depletion of exchangeable base cations (Ca²⁺ and Mg²⁺), which are essential for maintaining nutrient balance and facilitating biological activity. These findings indicate that chemical processes driven by acidification play a more dominant role than physical scouring in determining the nutrient availability in this region.

In contrast, although Oesamsinbong experiences substantial physical phosphate loss due to its steep terrain, relatively stable exchangeable cation levels and milder acidification conditions support the dominance of functional microbial groups such as Penicillium. This microbial community is hypothesized to potentially contribute to phosphorus solubilization and nutrient supply, despite lower total phosphate levels.

These findings are consistent with previous studies reporting that the Seseok Plateau suffers from poor drainage [28], and that such conditions can limit the height growth of A. koreana [45]. Although the population density and survival rate of A. koreana on the Seseok Plateau remained relatively high, growth performance appeared to be constrained, as evidenced by reduced height development (Figures 1 and 2).

Figure 1.

Condition of healthy Abies koreana individuals at each sampling site: Seseok Plateau (sites 1–3) and Oesamsinbong (sites 4–6).

Figure 2.

Topographical characteristics of the Seseok Plateau and restricted growth height of Abies koreana forests. (A) Representative landscape of the Seseok Plateau, characterized by low mortality but suppressed growth of A. koreana owing to environmental stressors; (B) visual representation of the A. koreana community on the Seseok Plateau showing a dense distribution of healthy individuals with restricted vertical growth.

In conclusion, this study suggests that the nutritional status of A. koreana may be associated with microbe-mediated phosphate solubilization processes rather than bulk phosphate concentration. The high dominance of Penicillium in Oesamsinbong represents a key mechanism that enables efficient nutrient acquisition under conditions of physical nutrient loss. Because, in this study, we focused on establishing baseline data and securing cultural resources, direct functional validation remains a limitation. Therefore, follow-up studies are recommended to address this issue. In contrast, the decline of A. koreana in the Seseok Plateau is interpreted as a consequence of the disruption of microbial-mediated nutrient acquisition systems [46–49] under extreme acidification. Although these secured cultural isolates represent a significant pool of potential biological resources, claims regarding novel taxa or specific regional endemicity must be evaluated cautiously. Further multilocus phylogenetic analyses incorporating the BenA and CaM loci are currently underway to confirm their exact taxonomic status.

3.6. Soil texture

Particle size analysis showed that the soils of both Seseok Plateau and Oesamsinbong were classified as loam and exhibited identical textures (Table 4). Although this texture generally provides a favorable physical environment for plant growth, subtle differences in particle distribution were observed. The clay content of the Seseok Plateau is approximately 5–7% higher than that of Oesamsinbong. This characteristic endows the Seseok Plateau with higher nutrient retention and water-holding capacity, which may be advantageous during drought. However, during heavy rainfall, this higher clay content, combined with gentle topography, exacerbates poor drainage and the formation of stagnant water.

Table 4.

Soil classification.

Geographical name	Sampling site no.	Sand (%)	Silt (%)	Clay (%)	Soil texture
Seseok Plateau (Seseok-pyeongjeon)	1	40.36	38.52	21.12	Loam
	2	46.24	33.32	20.44	Loam
	3	42.48	35.40	22.12	Loam
Oesamsinbong (mountain peak)	4	45.24	39.00	15.76	Loam
	5	51.60	34.76	13.64	Loam
	6	48.92	35.40	15.68	Loam

In contrast, Oesamsinbong exhibited higher sand content and lower clay content than the Seseok Plateau. Specifically, site 5 approached a sandy loam texture, with sand content exceeding 50%. This higher sand fraction increases soil permeability and facilitates drainage; however, it also appears to accelerate the physicochemical leaching of available phosphate and exchangeable cations, particularly given the steep terrain of the area.

3.7. Base saturation

The calculated BS (Table 3) at all study sites was significantly lower than the typical range for healthy forest soils (20–50%) [27,50]. Despite the high CEC, ranging from 35 to 41 cmol⁺/kg across all sites, the extremely low BS suggests that while the soil has a large nutrient retention capacity, the exchange complex is predominantly occupied by H⁺ rather than essential mineral nutrients.

In the Seseok Plateau, the BS was exceptionally low (3.24–7.24%), indicating that more than 90% of the exchange sites were occupied by H⁺. This extreme base-depleted state is consistent with the soil characteristics of declining A. koreana habitats on Mt. Jiri and suggests that the nutritional imbalance may have intensified due to cation leaching under severe acidification. In particular, site 3 (3.24%) on the Seseok Plateau was predicted to experience the most severe nutrient deficiency stress. Nutritional imbalance, driven by the displacement and leaching of essential cations by H⁺, may be a key factor contributing to the reduced growth of A. koreana. Although dissolved Al concentrations were not directly measured in this study, extreme acidity (pH < 4.0) and low BS suggest a potential risk of Al mobilization and associated toxicity under strongly acidic conditions, as has been widely reported in acidic forest soils [50]. However, because aluminum concentrations were not directly quantified in this baseline study, this interpretation remains strictly inferential and serves as a hypothesis that requires further validation. To overcome this limitation, follow-up soil mineral analyses are currently underway to empirically confirm the actual exchangeable Al³⁺ levels.

Consequently, although the observed soil acidification and base leaching were clearly associated with shifts in the culturable fungal assemblage, direct physiological evidence linking these factors to nutrient deficiency in A. koreana needles or actual root damage remains limited in this baseline study. To establish a definitive causal link, follow-up studies involving direct plant tissue profiling and root health assays are required.

3.8. Correlation between soil properties and dominance of Penicillium in the rhizosphere

Pearson’s correlation analysis revealed that the ratio of Penicillium was highly correlated with several soil chemical properties (Table 5; Figure 3). The BS showed the strongest positive correlation (r = 0.957, p < 0.01), followed by exchangeable magnesium (r = 0.935, p < 0.01), calcium (r = 0.896, p < 0.05), and pH (r = 0.891, p < 0.05) (Table 5).

Table 5.

Correlation between soil properties and the dominance ratio of Penicillium (n = 6, degrees of freedom = 4).

Independent variables (soil properties)	Correlation coefficient (r)	Coefficient of determination (R2)	p Value	Statistical significance
Base saturation (BS)	0.957	0.917	0.0028	Highly significant (**)
Exchangeable magnesium	0.935	0.875	0.0062	Highly significant (**)
Exchangeable calcium	0.896	0.835	0.0157	Significant (*)
Soil pH	0.891	0.794	0.0171	Significant (*)
Organic matter content	0.812	0.659	0.0496	Significant (*)
Exchangeable sodium	0.743	0.552	0.0906	Not significant (ns)
Exchangeable potassium	0.543	0.295	0.2655	Not significant (ns)
Available phosphate	−0.490	0.240	0.3239	Not significant (ns)

(*) Significant at the (p < 0.05) level.

(**) Highly significant at the (p < 0.01) level.

Figure 3.

Coefficient of determination (R²) for the relationship between the dominance ratio (%) of Penicillium and rhizosphere soil properties. Linear regression analysis showed a high coefficient of determination (R² = 0.917), indicating that soil base saturation may be an important factor associated with the dominance ratio (%) of Penicillium.

In contrast, P₂O₅ showed a weak negative correlation with Penicillium dominance (r = −0.490), which was not statistically significant. These results suggest that the depletion of exchangeable bases, rather than the absolute concentration of phosphate, is the primary environmental factor suppressing the dominance of beneficial fungi in the rhizosphere of A. koreana.

Base saturation emerged as a strong indicator for the dominance of Penicillium (BS) (r = 0.957), which was the most critical indicator among the analysis results. Base saturation emerged as the strongest variable determining the dominance of Penicillium. This suggests that beyond simple pH adjustment, an abundance of exchangeable cations (such as Ca and Mg) is essential for Penicillium to achieve a dominant status within the community. The low dominance observed on the Seseok Plateau (31.9–48.9%) is interpreted as a direct consequence of low BS (average 4.9%) resulting from severe cation leaching.

Relationship with Mg (r = 0.935): Similar to exchangeable calcium (r = 0.896), Mg showed a very high correlation with the dominance of Penicillium. This indicated that the rhizosphere microbial ecosystem of A. koreana is highly sensitive to the presence of divalent cations (Ca²⁺ and Mg²⁺). This supports the hypothesis that the leaching of these ions is a key factor hindering the establishment of beneficial fungi.

Lack of correlation with available phosphate (r = −0.490): The lack of correlation between phosphate levels and the proportion of Penicillium was a distinctive finding of this study. This supports the mechanism where environmental pressures (topography and acidity) show a strong trend of influencing the survival of Penicillium, which, in turn, facilitates phosphate absorption in A. koreana, rather than Penicillium abundance being driven by high soil phosphate levels.

Relationship with the diversity index (Shannon’s H′): Data show that as pH increases and Penicillium becomes more dominant (as seen in Oesamsinbong), the Shannon index (H′) tends to decrease. This can be explained as an ecological stabilization phenomenon, in which a specific beneficial fungus, Penicillium, establishes monopolistic dominance in the healthy rhizosphere of A. koreana.

The statistical independence of the replicates was limited because of the use of composite samples; thus, the results of these correlation analyses must be interpreted cautiously as indicative of general exploratory trends rather than for drawing strict or definitive causal inference.

4. Conclusions

This study provides evidence that the decline in A. koreana at specific high-altitude topographical sites is closely associated with the collapse of the rhizosphere fungal ecosystem. Our findings establish a critical environmental chain: in high-altitude basins, such as the Seseok Plateau, stagnant topography triggers extreme soil acidification (pH < 4.0), which in turn induces severe cation leaching and base depletion. This chemical stress may contribute to the suppression of the dominance of functional fungal networks, particularly in the phosphate-solubilizing genus Penicillium, thereby impairing the microbial-mediated nutrient uptake system that is essential for host vigor.

In contrast, well-drained steep terrains, such as Oesamsinbong, maintain a resilient symbiotic environment where Penicillium thrives, facilitating efficient phosphorus acquisition despite physical nutrient scouring. These results have shifted the conservation paradigm for endangered subalpine conifers from a purely climatic perspective to one that integrates soil chemical stability and microbial ecology. In conclusion, this study demonstrates that soil acidification and low BS significantly shape the culturable rhizosphere fungal assemblages of the endangered A. koreana. Although these environmental shifts are hypothesized as potential predisposing stressors for the decline in the abundance of this subalpine conifer, further comprehensive monitoring of plant nutritional status and root vitality is necessary to fully elucidate the exact causal mechanisms.

Funding Statement

This work was supported by grants from the National Institute of Biological Resources (NIBR) funded by the Ministry of Climate, Energy, Environment (MCEE) of the Republic of Korea (Nos. NIBR202505101, NIBR202502105, and NIBR202602105).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data are available within the manuscript and its supplementary materials.