Habitat Characteristics and Root Mycobiome Diversity of Cypripedium shanxiense S. C. Chen in the Changbai Mountains

Abstract

Cypripedium shanxiense S. C. Chen has high ornamental value; it relies on specific habitats and fungi. Wild C. shanxiense populations need urgent conservation because they are declining rapidly. This study investigated three wild C. shanxiense populations under different canopy densities in the Changbai Mountains, analyzing habitat characteristics and plant morphology. Tissue isolation methods, molecular identification techniques, and metagenomic approaches were applied separately to purify root-colonizing fungi and to investigate the composition and functions of rhizosphere fungi, thereby revealing the diversity of root mycobiome in C. shanxiense. Results revealed that C. shanxiense achieved the best growth when the canopy density was 85.29%, and the lowest growth was under 96.13% canopy density. Soil phosphorus and potassium contents reached their highest levels under 69.33% canopy density, while soil nitrogen and organic matter contents peaked at 85.29%. Soil organic matter and available nitrogen constitute the core nutrient factors for the growth of C. shanxiense. A total of 16 fungal strains were mainly enriched in the roots, all belonging to Ascomycota. Including numerous growth-promoting fungi and pathogenic fungi. The rhizosphere fungi were mainly enriched with Basidiomycota and Ascomycota. Functional genes related to replication, recombination, and repair, and Glycoside Hydrolases. This study clarifies the optimal growth conditions of this species and the dominant rhizosphere and root fungi, providing a scientific basis for the ecological restoration and conservation of rare species.

1. Introduction

Orchidaceae is one of the most species-rich families of angiosperms [1]. It has a global distribution across diverse ecosystems, spanning tropical rainforests, temperate forests, and alpine meadows [2]. Orchid populations are strongly regulated by microenvironmental factors [3], including temperature, humidity, soil physicochemical properties, and canopy density [4]. As a key ecological group in Orchidaceae, terrestrial orchids are particularly close to microbial communities [5]. The composition and functional traits of fungi are especially pivotal to their survival and reproduction [6]. Fungi serve as essential energy sources for orchid seed germination [7]. Orchid seeds lack endosperm and cannot germinate independently under natural conditions. This unique biological characteristic inherently restricts the self-reproduction capacity of orchids [8]. Furthermore, habitat destruction and illegal harvesting of wild orchids often lead to accompanies these collection activities [9]. These anthropogenic disturbances have placed wild orchid populations in increasingly precarious survival conditions. Currently, wild orchid species with high ornamental and economic value are not fully protected in natural habitats. This has led to severe degradation and threats to wild orchid resources [10]. Notably, Orchidaceae accounts for over 73% of the species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), making it the most closely monitored and protected plant group [11].

Rhizosphere fungi exert key ecological functions by regulating plant fungal communities, thereby promoting host growth and enhancing nutrient acquisition [12]. Typical orchid rhizosphere fungi primarily belong to Basidiomycota, including taxa from Sebacinales, Ceratobasidiaceae, and Tulasnellaceae. These fungi form characteristic spiral mycelial structures on host roots [13]. Rhizosphere soil directly connects with plant root systems. Some rhizosphere fungi invade root cortex cells or colonize the rhizosphere environment surrounding roots, establishing mutualistic symbiotic relationships with host plants [14]. Consequently, the community composition and function of rhizosphere fungi influence the colonization efficiency and interaction stability of root fungi. These fungi also perform crucial ecological functions, such as modifying soil properties, enhancing plant tolerance to adverse environments, and inhibiting pathogen proliferation [15,16]. Dark septate fungi are commonly associated with plant roots, aiding survival and reproduction in harsh conditions, including drought, cold, nutrient-poor soils, alpine or subalpine ecosystems, high salinity, heavy metal pollution, and polar regions [17]. The habitat adaptability of Dendrobium officinale is regulated by beneficial fungi, whose colonization effectively enhances the plant’s tolerance to environmental fluctuations [18]. Key fungi identified from D. officinale roots included Epulorhiza, Tulasnella, and Sebacinales taxa. These fungi promoted seed germination, improved nitrogen uptake efficiency, and stimulated polysaccharide accumulation [19]. Two fungal strains isolated from Camellia oleifera significantly improved orchid rhizosphere soil fertility. They also promoted soil material cycling and energy flow. These strains increased beneficial microbe abundance while reducing pathogen levels, thereby establishing a microenvironment more conducive to orchid growth [20].

C. shanxiense is a typical terrestrial orchid species inhabiting temperate forest understories. It is light-sensitive and thrives in shaded environments [21]. With sparse populations and a restricted distribution range, this species is classified as rare and endangered, warranting high conservation priority. Changbai Mountain is widely recognized as one of the world’s biodiversity hotspots [22], where multiple wild C. shanxiense populations have been discovered during field surveys. This unique habitat provides valuable natural samples for further studies on the ecological habits of C. shanxiense and the assembly of its associated fungi. In addition to its ecological significance, C. shanxiense holds considerable ornamental value due to its distinctive floral morphology and coloration. However, its endangered status necessitates urgent conservation and research efforts. Therefore, this study conducted environmental surveys on wild C. shanxiense populations distributed across different canopy density levels in Jilin Province, China. The surveys focused on analyzing habitat characteristics, plant morphological traits, soil environmental factors, and associated plant species. Additionally, through metagenomic sequencing analysis of rhizosphere soils across a gradient of canopy densities, the community structure and functional traits were investigated. Through tissue isolation and molecular identification techniques, the fungal species associated with the roots of C. shanxiense were confirmed. This research aims to reveal the survival strategies and ecological adaptation mechanisms of C. shanxiense under specific environmental conditions, while also providing new insights and a theoretical basis for the conservation biology of rare and endangered plants. Exploring the fungal preference and functional characteristics of fungi associated with C. shanxiense will provide a basis for the effective conservation and sustainable utilization of this orchid species.

2. Materials and Methods

2.1. Sample Collection and Environmental Survey

A total of 12 natural populations of C. shanxiense were identified during field surveys conducted by our research team in the Changbai Mountains. To minimize the interference of other significant environmental variations on the survey results, three sampling sites in Linjiang City were selected for the formal experiment. Linjiang City (126°92′ E, 41°81′ N), where the average elevation is 776 m, the annual mean temperature is 6 °C, and the annual mean precipitation is 824 mm. The field survey was carried out in June, during which the average maximum temperature reached 26 °C, the average minimum temperature was 13 °C, and the average rainfall stood at 115 mm. Sampling plots were situated under broad-leaved forests and along forest edges. Figure 1A depicts C. shanxiense in its natural state, while Figure 1B presents its habitat conditions. Five sets of rhizosphere soil samples (10 g each) were collected from each plot. The soil layer was gently excavated until the fibrous roots of C. shanxiense were exposed, and the soil adhering to the roots was collected as rhizosphere soil. After removing sand and gravel particles, the rhizosphere soil samples from the same plots were thoroughly mixed and subjected to metagenomic sequencing. 5 cm root segments were collected from the root tips, with 2–3 roots sampled per plant, for the isolation and purification of endophytic fungi. All sampling and surveys conducted in this study were performed without affecting the normal growth conditions of C. shanxiense.

Five 5 m × 5 m plots were established, with each plot centered on a surveyed wild C. shanxiense population. Each plot was divided into 4 subplots of 2.5 m × 2.5 m, and the vertex of each subplot was set as a measurement point [23]. The average value of measurements from these points was used as the environmental indicator for the corresponding C. shanxiense population. Environmental factors, including altitude, canopy density, temperature, and humidity, were measured using a GPS receiver, a canopy analyzer, and a thermo-hygrometer (DELIXI ELECTRIC, China), respectively. Herbaceous and woody plants within the sampling plots were recorded as companion plants of C. shanxiense. After removing dead branches and fallen leaves around the rhizosphere of C. shanxiense, rhizosphere soil samples were collected using the quadrat sampling method. The soil samples were naturally air-dried, thoroughly mixed, and then sieved through 20-mesh and 100-mesh sieves sequentially for the determination of soil physicochemical properties. The determination methods for soil parameters were as follows [24,25]:

Available phosphorus (AP): 0.5 mol·L⁻¹ NaHCO₃ extraction method;

Available potassium (AK): NH₄OAc extraction followed by flame photometry;

Alkali-hydrolyzable nitrogen (AN): Alkali-hydrolyzable diffusion method;

Total phosphorus (TP): HClO₄-H₂SO₄ digestion method;

Total potassium (TK): NaOH fusion followed by flame photometry;

Total nitrogen (TN): Kjeldahl method;

Organic matter (OM): Potassium dichromate external heating method;

pH: Determined using a pH meter.

Eight soil physicochemical parameters were measured in total, with three technical replicates set for each parameter.

2.2. Isolation and Purification of Root-Isolated Fungi

The collected root samples were immersed in a 2% sodium hypochlorite solution for 3 min, rinsed repeatedly with sterile distilled water several times, and then immersed in 75% ethanol for 30 s. After thorough rinsing with sterile distilled water once more, the samples were dried using sterile filter paper. Sterilized roots were cut into 0.2 cm segments, which were then inoculated onto isolation medium supplemented with antibiotics (200 g/L potato, 20 g/L glucose, 20 g/L agar, kanamycin, and ampicillin). After 6–7 days of incubation, when fungal mycelia covered approximately 2/3 of the root segment area, edge portions of different colonies were picked and inoculated onto fresh potato dextrose agar (PDA) medium (200 g/L potato, 20 g/L glucose, 20 g/L agar). Inoculated PDA plates were incubated in a 25 °C constant-temperature incubator in the dark. This purification procedure was repeated continuously until pure fungal cultures were obtained. To verify the effectiveness of root surface sterilization, the final rinse water (sterile distilled water used for the last root rinse) was also inoculated onto the isolation medium. No fungal growth on these control plates confirmed successful surface disinfection.

2.3. Molecular Identification of Root-Isolated Fungi

Genomic DNA of purified fungal isolates was extracted using the Ezup Column-Based Fungal Genomic DNA Extraction Kit supplied by Sangon Biotech Co., Ltd., Shanghai, China. For molecular identification of the mycorrhizal fungi isolated from C. shanxiense, the universal primer pair ITS1/ITS4 was selected to amplify the fungal internal transcribed spacer (ITS) region. Polymerase chain reaction (PCR) amplification was performed under the following conditions: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min, and a final extension step at 72 °C for 7 min. A 3 μL aliquot of each PCR product was subjected to 1% agarose gel electrophoresis for fragment detection and verification. PCR products showing bright single bands were submitted to a commercial sequencing service provider for Sanger sequencing. Resultant sequences were subjected to BLAST analysis against the nucleotide database of the National Center for Biotechnology Information (NCBI). Mycorrhizal fungi were identified by combining the sequence alignment results with morphological observation data.

2.4. Metagenomic Data Processing

A total of 50 g of filtered rhizosphere soil was collected for metagenomic sequencing. Metagenomic sequencing was performed by Personalbio Technology Co., Ltd., Shanghai, China. The total DNA of rhizosphere soil microorganisms was extracted via the de novo sequencing method using the Mag-Bind Soil DNA Kit purchased from OMEGA Bio-tek, Inc. Subsequently, the concentration and purity of the extracted DNA were determined. Specifically, the DNA concentration was quantified with a Qubit 4 Fluorometer (Qubit™ 4 Fluorometer, with WiFi: Q33238; Qubit™ Assay Tubes: Q32856; Qubit™ 1X dsDNA HS Assay Kit: Q33231), and the genomic sequencing libraries were constructed following the standard experimental protocol described in the Illumina TruSeq DNA Sample Preparation Guide. The quality of the sequencing data before and after quality control was assessed using the fastp software, generating the Raw Data Statistics Table and Valid Data Statistics Table, respectively (

Table 1. Basic sequencing information.

Plot	Raw Reads	Raw Bases	Clean Reads	Clean Bases	Clean_Q20 (%)	Clean_Q30 (%)	Clean_GC (%)	Effective (%)
P1	111,283,900	16,692,585,000	105,833,602	15,627,316,557	100	98.44	62	93.62
P2	145,261,328	21,789,199,200	134,654,452	19,698,580,776	100	98.12	61.5	90.41
P3	123,541,232	18,531,184,800	113,150,864	16,452,009,235	100	97.51	62	88.78

). In total, 380,086,460 valid raw reads were obtained. For species annotation of the valid reads, Kaiju software was employed to align each read against the prokaryotic and eukaryotic microbial protein sequences in the NCBI-nr database, as well as the viral protein sequences in the RNDB-prot database. By counting the number of reads annotated to each species and collating the corresponding species annotation information, species abundance tables at different taxonomic levels were generated. Finally, the fungal genes were selected for subsequent visualization analysis.

2.5. Data Analysis

One-way analysis of variance (ANOVA) was performed in SPSS 27.0 to analyze soil nutrient contents, morphological indicators, and physiological parameters. Redundancy analysis (RDA) plots were generated using Wekemo Bioincloud (https://www.genescloud.cn (accessed on 3 February 2026)) [26]. Circos, Column, Veen, LDA Effect Size (LEFse), and Heatmap were all generated using R 4.5.2. Image integration was conducted with Adobe Illustrator 29.0. Phylogenetic trees were constructed using the neighbor-joining method in MEGA 11.0.13 software.

3. Results

3.1. Environmental Characteristics and Morphological Traits of C. shanxiense

In this study, environmental factors and plant growth indicators were measured and analyzed for 3 wild C. shanxiense plots in different canopy densities (

Table 2. Environmental and morphological characteristics of C. shanxiense.

Plot	Canopy Density (%)	Temperature (°C)	Humidity (%)	Elevatio (m)	Height (cm)	Width (cm)	Stem Diameter (cm)	Leaf Length (cm)	Leaf Width (cm)
P1	69.33	22.40	91.90	592	15.83 ± 1.76 ab	22.67 ± 2.52 a	0.30 ± 0.00 a	12.83 ± 0.28 c	5.87 ± 0.51 a
P2	85.29	23.70	90.70	609	19.17 ± 4.25 a	25.5 ± 3.91 a	0.27 ± 0.06 a	15.33 ± 1.76 b	7.67 ± 1.04 a
P3	96.13	22.60	95.90	608	11.17 ± 1.26 b	19.67 ± 2.2 a	0.23 ± 0.06 a	10.67 ± 0.58 a	5.83 ± 1.04 a

Note: Different letters after the values in the same column indicate that the difference between samples reaches a significant level of p < 0.05.

). P1 had the lowest canopy density of 69.33% and exhibited intermediate plant growth performance. P2 with a canopy density of 85.29% showed the optimal growth of C. shanxiense, while P3 with a canopy density as high as 96.13% displayed the most unfavorable growth.

Rhizosphere soil physicochemical properties of C. shanxiense were measured in three plots (

Table 3. Physical and chemical properties of the rhizosphere soil of C. shanxiense.

Plot	Organic Matter (g·kg−1)	Total Nitrogen (g·kg−1)	Total Phosphorus (g·kg−1)	Total Potassium (g·kg−1)	Alkali Hydrolyzed Nitrogen (mg·kg−1)	Available Phosphorus (mg·kg−1)	Available Potassium (mg·kg−1)	pH
P1	204.42 ± 10.09 b	3.03 ± 0.06 b	0.83 ± 0.017 a	24.69 ± 0.15 a	345.94 ± 12.40 b	7.30 ± 2.00 a	289.30 ± 14.00 a	6.49 ± 0.04 c
P2	281.36 ± 14.92 a	3.88 ± 0.04 a	0.80 ± 0.05 a	20.63 ± 0.61 c	365.17 ± 2.19 a	6.56 ± 0.53 a	253.63 ± 5.51 b	6.56 ± 0.02 b
P3	177.83 ± 6.18 c	2.93 ± 0.10 b	0.77 ± 0.07 a	22.55 ± 0.39 b	220.16 ± 9.96 c	8.10 ± 0.57 a	211.63 ± 2.52 c	6.77 ± 0.03 a

Note: Different letters after the values in the same column indicate that the difference between samples reaches a significant level of p < 0.05.

). Significant differences were observed in all eight measured soil physicochemical indicators in three plots. Specifically, P1 had the highest contents of TP, TK, and AK. P2 exhibited significantly higher contents of OM, TN, and AN compared with the other two plots. P3 had higher AP content than P1 and P2, along with the highest soil pH.

The habitat of C. shanxiense primarily consists of forest understories or grassy slopes, where it associates with a diverse array of plant species. Statistical analysis of woody and herbaceous plants in three plots revealed a total of six woody plant species and seven herbaceous plant species, summarized in

Table 4. Main associated plants of C. shanxiense.

Main Associated Plants
Woody Plants	Tilia tuan Szyszyl.
	Quercus mongolica Fisch. ex Ledeb.
	Salix raddeana Lacksch. ex Nasarow
	Acer pseudosieboldianum (Pax) Kom.
	Juglans mandshurica Maxim.
	Acer pictum subsp. Mono (Maxim.) Ohashi
Herbaceous Plants	Vicia unijuga A. Br.
	Phedimus aizoon (L.) ‘t Hart.
	Clematis florida Thunb.
	Brachybotrys paridiformis Maxim. ex Oliv.
	Arisaema amurense Maxim.
	Hylomecon japonica (Thunb.) Prantl and Kündig
	Convallaria keiskei Miq.

.

3.2. Correlation Analysis of Morphology and Environment

The results of the RDA were presented in Figure 2. Red dots represented plant growth indicators, whereas arrows denoted environmental factors. RDA1 explained 73.85% of the total variance, and RDA2 accounted for 19.03%, resulting in an overall explanatory rate of 92.88%. The selected environmental factors effectively explained their impacts on the measured plant growth indicators. AN, TN, and OM exhibited small vector angles with plant height, width, and leaf length and width; additionally, the angles among these three factors were narrow, indicating that they synergistically regulated the aboveground vegetative growth of plants. AK and TP showed small vector angles with stem diameter, suggesting a positive correlation with stem diameter. In contrast, TK formed large vector angles with most growth indicators, implying a weak regulatory effect on the plant growth indicators investigated in this study.

3.3. Morphological Description of C. shanxiense Root-Isolated Fungi

A total of 16 strains were isolated from C. shanxiense, and their morphological characteristics were described (

Table 5. Preliminary morphological identification of root-isolated strains.

Strain Number	Colony Texture	Colony Color	Colony Margin
SX1	Fluffy	Center: Light yellow Outer: Brown	Rules
SX2	Velvety	Center: Olive green Outer: White	Rules
SX3	Fluffy	White	Irregular
SX4	Fluffy	White	Rules
SX5	Fluffy	Central: Yellowish–green Outer: Olive green	Rules
SX6	Fluffy	Central: Grayish–white Outer: Dark gray	Rules
SX7	Cotton-like	White	Rules
SX8	Thin and waxy	Pale yellow	Irregular
SX9	Powdered	Pale yellow	Rules
SX10	Powdered	Pale yellow	Rules
SX11	Fluffy	Grayish–white	Rules
SX12	Thin and waxy	Center: Orange Outer: White	Irregular
SX13	Fluffy	Dark gray	Rules
SX14	Powdered	Olive green	Irregular
SX15	Velvety	Dark brown with white mycelium growing on top	Rules
SX16	Velvety	Black	Rules

). The colony textures were predominantly characterized as velvety, felt-like, powdery, waxy, or cottony. The felt-like texture was the most common colony type, with tightly packed and non-dispersive hyphae. Waxy mycelia were nearly transparent, and their morphology could only be observed within the culture medium. Colony colors ranged from white, pale yellow, and olive green to brown, black, and orange. Most colonies were circular in shape, while a few displayed irregular and scattered growth patterns.

3.4. Molecular Identification of C. shanxiense Root-Isolated Fungi

DNA was extracted from the mycelium of 16 fungal strains isolated from the roots of C. shanxiense (Figure A1). All sequences satisfied the quality criteria for Sanger sequencing. The sequencing results were uploaded to the NCBI database for BLAST alignment. The identification results of the 16 root-isolated fungal strains are presented in

Table A1. BLAST alignment results of 16 root-isolated fungal strains.

Strain Number	Similar Strains	Sequence Length	Login ID	Coverage	E-Value	Confidence Probability
SX1	Leptodontidium sp. BESC803f	1136	KC007189.1	96.00%	0	99.83%
SX2	Talaromyces sp.	561	MN845951.1	95.00%	0	99.63%
SX3	Xylaria longipes	573	MW492550.1	98.00%	0	99.63%
SX4	Trichoderma sp.	516	PQ219369.1	97.00%	0	97.01%
SX5	uncultured Leptodontidium	634	KU837930.1	96.00%	0	99.14%
SX6	uncultured Mycochaetophora	893	HG936157.1	98.00%	0	99.66%
SX7	Ilyonectria robusta	518	PP702654.1	95.00%	0	100.00%
SX8	Sordariomycetes sp. 120 XC-2016	510	KT719202.1	92.00%	0	100.00%
SX9	Clonostachys parasporodochialis	551	PP385740.1	98.00%	0	99.44%
SX10	Leptodophora orchidicola	630	PQ517212.1	96.00%	0	99.66%
SX11	uncultured fungus	885	MW215362.1	14%	2 × 10−32	91.15%
SX12	Nemania sp.	579	PP494010.1	97.00%	0	99.65%
SX13	Leptodontidium sp.	635	OP895755.1	97.00%	0	99.66%
SX14	Talaromyces variabilis	579	MZ573039.1	98.00%	0	99.64%
SX15	Didymella sp. QSY-51.4-5	569	PP446742.1	98.00%	0	99.22%
SX16	Colletotrichum spaethianum	566	OR083294.1	98.00%	0	100.00%

. All strains belonged to the Ascomycota (

Table A2. BLAST comparison of strain classification.

Strain Number	Similar Strains	Phylum	Class	Order	Family
SX1	Leptodontidium sp. BESC803f	Ascomycota	Leotiomycetes	Helotiales	unclassified
SX2	Talaromyces sp.	Ascomycota	Eurotiomycetes	Eurotiales	Trichocomaceae
SX3	Xylaria longipes	Ascomycota	Sordariomycetes	Xylariales	Xylariaceae
SX4	Trichoderma sp.	Ascomycota	Sordariomycetes	Hypocreales	Hypocreaceae
SX5	uncultured Leptodontidium	Ascomycota	Leotiomycetes	Helotiales	unclassified
SX6	uncultured Mycochaetophora	Ascomycota	Leotiomycetes	Helotiales	unclassified
SX7	Ilyonectria robusta	Ascomycota	Leotiomycetes	Helotiales	unclassified
SX8	Sordariomycetes sp. 120 XC-2016	Ascomycota	Sordariomycetes	unclassified	unclassified
SX9	Clonostachys parasporodochialis	Ascomycota	Sordariomycetes	Hypocreales	Bionectriaceae
SX10	Leptodophora orchidicola	Ascomycota	unclassified	unclassified	unclassified
SX11	uncultured fungus	Ascomycota	unclassified	unclassified	unclassified
SX12	Nemania sp.	Ascomycota	Sordariomycetes	Xylariales	Xylariaceae
SX13	Leptodontidium sp.	Ascomycota	Leotiomycetes	Helotiales	unclassified
SX14	Talaromyces variabilis	Ascomycota	Eurotiomycetes	Eurotiales	Trichocomaceae
SX15	Didymella sp. QSY-51.4-5	Ascomycota	Dothideomycete	Pleosporale	Didymellaceae
SX16	Colletotrichum spaethianum	Ascomycota	Sordariomycetes	Glomerellale	Glomerellacea

), with two strains unidentifiable at the class level. The remaining 14 strains were classified into four classes (Leotiomycetes, Eurotiomycetes, Sordariomycetes, and Dothideomycetes), encompassing six orders and seven families. Among these, seven strains could not be identified to the species level. Three additional specimens were identified as fungi not previously cultured. To clarify the phylogenetic positions of the isolated strains, a phylogenetic tree was constructed based on the ITS sequences using the neighbor-joining method (Figure 3). The results showed that Sordariomycetes sp. 120 XC-2016, Nemania sp., and Xylaria longipes clustered together with bootstrap support values of 59%, 100%, and 38%, respectively. Colletotrichum spaethianum, Ilyonectria robusta, and Clonostachys parasporodochialis formed a subclade with bootstrap support values of 42% and 68% for the respective nodes. Leptodophora orchidicola, Leptodontidium sp., Leptodontidium sp. BESC803f, uncultured Leptodontidium, and uncultured Mycochaetophora clustered into a clade with bootstrap support values of 62%, 100%, 96%, and 80% at the corresponding nodes. Talaromyces sp. and Talaromyces variabilis formed a well-supported clade (99% bootstrap value) and further clustered with Trichoderma sp. and Didymella sp. QSY-51.4-5 into a major clade with 49% bootstrap support. The uncultured fungus formed an independent outgroup. The evolutionary distance scale of the phylogenetic tree was 0.050. The support rates of each branch reflect the reliability of clustering, providing molecular biological evidence for strain classification and phylogenetic relationship analysis.

3.5. Different Classes Root-Isolated Fungi in C. shanxiense

After comprehensive identification based on morphological and molecular characteristics, the colony and mycelial morphologies of the strains were observed and described. Nine strains representing four different classes with distinct morphological variations, along with their mycelial structures (SX1, SX2, SX3, SX7, SX9, SX12, SX14, SX16), were selected for presentation.

Figure 4 shows two strains (SX1 and SX7) belonging to Leotiomycetes. Figure 4A,C illustrate the colony morphology and mycelium structure of SX1. Colonies were regular and circular, with dense mycelium exhibiting a velvety appearance. The central portion of the colony was pale yellow, while the outer region was brown. ITS sequence alignment revealed 99.83% similarity to Leptodontidium sp. BESC803f (KC007189.1).

Figure 4B,D illustrate the colony morphology and mycelial structure of strain SX7, respectively. The colony was regular and circular, with a cottony texture and an overall white coloration. ITS sequence alignment showed that SX7 shared 100.00% similarity with Ilyonectria robusta (PP702654.1).

Figure 5 shows two strains (SX2 and SX14) belonging to Eurotiomycetes. Figure 5A,C illustrate the colony morphology and mycelial structure of SX2, respectively. The colonies were regular and circular, with dense mycelia expanding gradually outward and exhibiting a felt-like texture. The central portion of the colonies was olive green, while the outer edges were white. ITS sequence alignment revealed that SX2 shared 99.63% similarity with Talaromyces sp. (MN845951.1).

Figure 5B,D illustrate the colony morphology and mycelial structure of SX14, respectively. SX14 formed multiple irregular circular olive green colonies on the medium, with a powdery and relatively flat surface. The grayish–white mycelia were slender and filamentous, with distinct septa and a smooth surface. Small conidia were distributed along the hyphae. ITS sequence alignment showed that SX14 shared 99.64% similarity with Talaromyces variabilis (MZ573039.1).

Figure 6 shows four strains (SX3, SX9, SX12, and SX16) belonging to Sordariomycetes. Figure 6A,E illustrate the colony morphology and mycelial structure of strain SX3, respectively. The colonies were irregularly circular with a velvety texture and maintained a uniform white coloration throughout cultivation. ITS sequence alignment revealed that SX3 shared 99.63% similarity with Xylaria longipes (MW492550.1).

Figure 6B,F illustrate the colony morphology and mycelial structure of SX9, respectively. The colony was generally circular, with mycelia spreading outward in irregular circular patterns within the medium. A pale yellow powdery substance formed on the colony surface. The mycelia were robust, filamentous, moderately curved, transparent, smooth, septate, and branched. ITS sequence alignment showed that SX9 shared 99.44% similarity with Clonostachys parasporodochialis (PP385740.1).

Figure 6C,G illustrate the colony morphology and mycelial structure of SX12, respectively. The colony had a waxy texture, with a distinct orange center and white periphery, expanding in a concentric ring pattern to form a petal-like appearance. The mycelia were slender and filamentous, smooth, with clearly visible septa, and pale green in color. Green spores, spherical or irregularly oval in shape, were distributed along the mycelia. ITS sequence alignment revealed that SX12 shared 99.65% similarity with Nemania sp. (PP494010.1).

Figure 6D,H illustrate the morphological characteristics of SX16, respectively. The colonies were felt-like, regularly circular, with an uneven surface exhibiting a granular texture, and displayed a uniform black coloration. The brownish-gray mycelia were rod-like, with distinct septa and a smooth surface. ITS sequence alignment revealed that SX16 shared 100.00% similarity with Colletotrichum spaethianum (OR083294.1).

Figure 7 shows SX15, which is classified into Dothideomycetes. Figure 7A,B illustrate the colony morphology and mycelial structure of SX15, respectively. The colony was regularly circular with a felt-like texture, displaying an overall deep brown color and producing white mycelia. ITS sequence alignment revealed that SX15 shared 99.22% similarity with Didymella sp. QSY-51.4-5 (PP446742.1).

3.6. Analysis of the Composition and Function of Rhizosphere Fungi

Circos diagram of the rhizosphere fungal community in C. shanxiense showed composition and distribution characteristics at different taxonomic levels (Figure 8). At the Phylum level (Figure 8A), rhizosphere fungi primarily comprised Basidiomycota, Ascomycota, and other low-abundance groups, including Mucoromycota and Olpidiomycota, with distinct association patterns in fungal composition between groups. At the family level (Figure 8B), the dominant families included Russulaceae, Cortinariaceae, and Gloniaceae. The composition of rhizosphere fungi at the family level was more complex in 3 plots, reflecting specific distribution patterns of fungal communities at this taxonomic level within the rhizosphere environment.

The Unigene set obtained from metagenomic sequencing was aligned against the eggNOG database, yielding 29,569 annotated genes. These genes were screened for Clusters of Orthologous Groups of proteins (COG) functions, and 5125 genes were selected for subsequent analysis. Analysis of the top 20 functional genes by abundance and nine dominant functional categories (Figure 9) revealed that rhizosphere fungi were primarily involved in the following functional categories (Figure 9A): L (replication, recombination, and repair), C (energy production and conversion), and G (carbohydrate transport and metabolism). Specifically (Figure 9B), rhizosphere fungi were enriched in the COG functional genes COG2801, COG2319, COG0507, COG0666, COG0843, and COG3866. Among these, COG2801 and COG0507 belonged to L, which were primarily involved in transposition and the preparation of double-strand DNA breaks for recombinational DNA repair, COG2319 was mainly associated with anaphase-promoting complex binding, COG0666 and COG3866 belonged to G, which were primarily involved in the response to abiotic stimuli and pectate lyase function, respectively, while COG0843 was associated with C, primarily involved in heme-copper terminal oxidase activity.

Analysis of carbohydrate-active enzymes (CAZy) functional genes was performed on the rhizosphere fungi of C. shanxiense (Figure 10). CAZy annotation results in 3 plots revealed significant differences in the abundance distribution of different CAZy enzyme classes. Among all CAZy enzyme classes, Glycoside Hydrolases (GHs) exhibited the highest abundance, which was far greater than that of other enzyme types, while Carbohydrate Esterases (CEs) showed the lowest abundance. No significant differences in the abundance of carbohydrate-active enzyme genes were observed in 3 plots for the rhizosphere fungi associated with C. shanxiense.

3.7. Differential Analysis

The species and functional gene composition characteristics of rhizosphere fungi in three plots of C. shanxiense were analyzed (Figure 11A,B). At the species level, the numbers of endemic species in P1, P2, and P3 were 34, 30, and 42, respectively. The number of shared species between P1 and P2, P1 and P3, and P2 and P3 was 17, 49, and 44, respectively, and the total number of shared species across the three plots reached 333. At the functional gene level, the number of unique functional genes in P1, P2, and P3 was 63, 40, and 110, respectively. The number of shared functional genes between P1 and P2, P1 and P3, and P2 and P3 was 45, 66, and 57, respectively, with the total number of shared functional genes in the three plots reaching 280. Overall, P1 exhibited intermediate levels of endemic species and functional genes, while P3 showed the highest abundances of both endemic species and functional genes in the three plots. In contrast, P2 recorded the lowest numbers of endemic species and functional genes.

LEfSe analysis (LDA score > 4) identified significantly different fungal taxa in rhizosphere communities across different plots of C. shanxiense (Figure 12). Specifically, Serendipitaceae was a statistically enriched taxon in P1, whereas Gloniaceae and Russulaceae were significantly enriched taxa in P2. These taxa served as biomarkers distinguishing rhizosphere fungal community structures across plots, reflecting the selective preferences of distinct rhizosphere microenvironments for specific fungal taxa.

3.8. Correlation Analysis of Rhizosphere Fungi and Environment

RDA results (Figure 13) indicated that RDA1 explained 64.86% of the variance, while RDA2 explained 35.14%. Significant associations existed between the rhizosphere fungal communities of C. shanxiense and environmental factors. Specifically, Geoglossaceae, Serendipitaceae, and Russulaceae showed significant positive correlations with AK and TK; Strophariaceae and Amanitaceae exhibited positive correlations with pH and canopy density; Atheliaceae and other taxa were closely associated with AN; while Cortinariaceae, Rhizopogonaceae, and other groups showed strong associations with OM.

Heatmap analysis of the correlation between eggNOG annotation functions and environmental factors (Figure 14) revealed strong associations between different COG functional groups and environmental factors in the rhizosphere of C. shanxiense. Functional groups represented by COG1020 and COG2072 showed significant positive correlations with AP; functional groups such as COG0841 and COG4197 exhibited significant positive correlations with factors including TN, OM, and TP. Conversely, groups such as COG2114 and COG2124 displayed significant negative correlations with TN and OM. Additionally, COG1452 demonstrated clear associations with pH and canopy density. These results indicated that soil physicochemical factors (N, OM, and pH) and the habitat factor (canopy density) significantly regulated the distribution of core functional gene clusters in the rhizosphere of C. shanxiense.

4. Discussion

C. shanxiense survival and reproduction are highly dependent on specific habitat conditions and its symbiotic relationship with fungi [27]. In this study, canopy density significantly influenced the growth and development of C. shanxiense. Its growth under 85.29% canopy density exhibited optimal morphological traits, including plant height, crown spread, and leaf length. By contrast, growth was unfavorable under a high canopy density of 96.13%. This indicated that moderate shading promoted photosynthesis and energy accumulation in C. shanxiense, whereas excessive shading might lead to insufficient light intensity, limiting its carbon assimilation capacity [28] and consequently affecting biomass accumulation [29]. This phenomenon aligned with the light response mechanisms of most orchids [30]. This study further quantified the optimal canopy density range for C. shanxiense under natural conditions, providing actionable thresholds for habitat management.

Soil physicochemical properties serve as crucial indicators for evaluating habitat suitability for orchids, with nutrient content and pH directly influencing root absorption efficiency and symbiotic microbial activity [31]. In this study, the rhizosphere soil of C. shanxiense exhibited weak acidity, with a pH ranging from 6.49 to 6.77, which was consistent with the preference for acidic soils among most terrestrial orchids [32]. Plants grown under 85.29% canopy density exhibited the most favorable morphological traits and higher contents of soil organic matter, total nitrogen, and alkali-hydrolyzable nitrogen compared with those in other plots. Their available potassium content remained at a moderate level, indicating that soil organic matter and nitrogen supply were key nutrient-limiting factors for C. shanxiense growth. Meanwhile, organic matter accumulation promoted the proliferation of microorganisms and nutrient transformation [33,34]. Under high canopy density conditions, organic matter accumulation and alkaline-hydrolyzable nitrogen content were relatively low. This might be attributed to insufficient available nitrogen supply caused by enhanced microbial nitrogen immobilization [35]. Furthermore, no significant differences in soil phosphorus content were observed in 3 plots. This suggested that microbial activation of phosphorus might have compensated for natural variations in soil phosphorus pools, providing a starting point for further investigations into microbial nutrient transformation functions.

This study isolated and identified 16 fungal species from the roots of C. shanxiense. However, at the phylum level, they all belonged to the Ascomycota. This composition differed from previously reported fungal assemblages associated with temperate terrestrial orchids. Most studies indicated that Basidiomycota were the dominant symbiotic fungi of orchids [36]; however, no Basidiomycota were isolated in this study. This discrepancy might be related to the sampling season, altitude, and vegetation. The dominant status of Ascomycota in C. shanxiense roots might result from their superior acid tolerance and organic matter decomposition capacity [37]. These traits were highly compatible with the soil characteristics of the study area. Notably, Leptodontidium sp. and Clonostachys parasporodochialis have been shown to enhance nutrient uptake in orchids and inhibit pathogen colonization [38,39]. Leptodophora orchidicola is an orchid-specific symbiotic fungus. Its presence in the roots of C. shanxiense further confirms the specialized symbiotic association between orchids and fungi [40]. These fungi also exhibited lignin-degrading capabilities [41] and thus might participate in rhizosphere litter degradation and carbon cycling processes in the C. shanxiense rhizosphere. Talaromyces, which belongs to Eurotiomycetes, produces stress-resistant conidia that aid plants in coping with adverse environments [42]. This resembled the functional characteristics of endophytic fungal communities in orchid species such as Dendrobium and Bletilla striata [43,44], confirming the functional conservation of orchid–fungal symbiotic systems. This discovery enriched the diversity database of fungi associated with orchids. However, this study also isolated the pathogenic fungus Ilyonectria robusta and Colletotrichum spaethianum, which cause root rot in various plant species [45,46]. Their presence in C. shanxiense roots suggests a potential “symbiotic-pathogenic” dynamic equilibrium within natural populations. This finding emphasized that conservation and artificial introduction efforts must not only screen for beneficial strains but also monitor the structure of root microbial communities to mitigate potential pathogen threats.

The functional specificity of fungi colonizing roots is central to understanding the mechanisms underlying orchid endangerment [47]. In this study, velvety and fluffy colonies accounted for the highest proportion at 75%. This colony structure facilitated hyphal attachment and nutrient exchange, while the transparent hyphae of waxy colonies might enhance adaptation to low-light environments. Such strains gained a competitive advantage in nutrient acquisition, while other strains might exert defensive functions by producing secondary metabolites [48]. The diversity of colony colors, including white, pale yellow, and black, correlates with pigment synthesis. Dark-colored colonies might produce pigments to resist UV damage [49], which represented a morphological adaptation suited to the light conditions under low canopy density. These findings indicated that the root fungal communities of C. shanxiense had developed synergistic adaptation mechanisms to heterogeneous habitats through differentiation in morphology and growth strategies.

The community composition and function of dominant rhizosphere fungi in C. shanxiense remained unchanged under varying canopy density. At the phylum level, Ascomycota and Basidiomycota dominated, while Russulaceae predominated at the family level. From the perspective of carbohydrate-active enzymes, rhizosphere fungi primarily participated in Glycoside Hydrolase processes. Studies on autotrophic-heterotrophic nutritional mode switching in orchids revealed that trehalosidase genes were highly enriched in fully heterotrophic orchids, indicating carbon acquisition from fungi [50]. This suggested that rhizosphere fungi associated with C. shanxiense might supply trehalose for its growth and participate in carbohydrate transport. Comparative studies of root sections from Cypripedium macranthos [51] under varying canopy density levels revealed that increased canopy density also heightened fungal infection rates on roots. This indicated that high canopy density enhanced fungal activity both in the rhizosphere and within the roots of Cypripedium.

This study has certain limitations. The tissue block isolation method failed to obtain dominant rhizosphere fungi, possibly because some fungi were strongly endophytic and difficult to culture in vitro [52]. Additionally, functional validation experiments for the isolated fungi were not performed, meaning their growth-promoting effects on C. shanxiense remained to be confirmed through systematic inoculation trials. Therefore, the fungi isolated from the roots cannot be identified as the symbiotic fungi of C. shanxiense. Moreover, sampling was only carried out during one growing season, resulting in unclear seasonal dynamics of the fungal community. Future research should focus on screening highly effective growth-promoting fungal strains through inoculation trials for application in artificial propagation, investigating the regulatory mechanisms of canopy density and soil nutrients on the fungal community structure, and conducting long-term, fixed-site monitoring to reveal the association patterns between C. shanxiense population dynamics and symbiotic systems.

5. Conclusions

This study systematically clarified the optimal habitat conditions of the endangered species C. shanxiense in the Changbai Mountain, as well as the composition, structure, and functional characteristics of its root mycobiome. Canopy density and soil nutrients were identified as the core regulating factors for its growth, under 85.29% canopy density being the optimal growth condition. Both excessive and insufficient shading were found to inhibit the morphological development of C. shanxiense. Soil organic matter and nitrogen are key factors influencing not only plant growth but also the dynamics of the fungal community. 16 fungal strains isolated from C. shanxiense roots all belong to Ascomycota, covering 3 classes, 6 orders, and 7 families. The dominant genera include Leptodontidium, Talaromyces, and Xylaria. These fungi exhibited functional diversity, with some strains promoting nutrient uptake, suppressing pathogens, or enhancing plant stress resistance. Pathogenic fungi such as Ilyonectria robusta and Colletotrichum spaethianum were also present, forming a “symbiotic-pathogenic” dynamic equilibrium. Basidiomycota and Ascomycota were the dominant phyla in the rhizosphere fungal community, and these fungi were involved in carbohydrate transport processes of C. shanxiense, providing carbon sources essential for plant growth. This study elucidated the survival strategy of C. shanxiense from a plant–microbe–environment interaction perspective, enriching the fungal diversity database of the subterranean parts of temperate orchids. It provided critical theoretical foundations and fungal strain resources for the conservation, artificial propagation, and ecological restoration of this endangered species, while offering new insights into the microecological adaptation mechanisms of endangered orchids.