The study area is located in Linzhi City, Tibet Autonomous Region, and includes three sample sites: Bayi District (BY), Chayu County (CY), and Mêdog County (MT). The sampling site in BY is the Xizang Agricultural and Animal Husbandry University, which has a plateau temperate semi-humid monsoon climate with an annual average temperature of 8.5°C and an average annual precipitation of 654 mm. This site represents an artificial ecosystem. CY has a subtropical climate, with an annual average temperature of 12°C and an average annual precipitation of 801.1 mm, featuring a natural ecosystem. MT has a subtropical humid climate, with an average annual temperature of 18.4°C and an annual precipitation of 2,358 mm, representing a natural ecosystem. This study designated these three sampling sites as BY, CY, and MT. The latitude, longitude, and average altitude of each site are presented in Table 1.

In September 2023, following the principle of random multi-point mixing, healthy C. tracyanum orchids with consistent growth were selected from each test plot. Orchids that had not been exposed to chemical fertilizers or pesticides were collected. The root-shaking method collected rhizosphere soil samples, with three samples taken from each plot, amounting to 500 g. The collected soil was stored in sterile bags at a low temperature, transported to the laboratory, and processed within 24 h.

The physicochemical properties of the soil were measured using conventional methods, following the procedures outlined by Bao (2000). The primary indicators analyzed included total phosphorus (TP), total potassium (TK), pH, TN, soil organic matter (SOM), available nitrogen (AN), available phosphorus (AP), and available potassium (AK). TP and AP were determined using the vanadium molybdate blue colorimetric method, while TK and AK were measured using a flame photometer. The pH value was determined using the electrode method. TN and AN were analyzed using the Kjeldahl method. SOM was determined using the potassium dichromate oxidation-external heating (volume) method.

Total DNA was extracted from the soil samples using a Soil Genomic DNA Extraction Kit (DP336, Tiangen Biotech, Beijing, Co., Ltd., China). The quality of the extracted DNA was assessed using 0.8% agarose gel electrophoresis, while its concentration and purity were measured using a NanoDrop ND-2000 Ultraviolet-Vis Spectrophotometer (Thermo Scientific, Wilmington, USA). The extracted DNA was further examined using 1% agarose gel electrophoresis to confirm its mass and integrity. PCR amplification was performed to target variable regions using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGG CTACCTTGTTACGACTT-3′). Additionally, the amplification of fungal internal transcribed spacer (ITS) sequences was conducted using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). The amplification process began with an initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min and 30 s. A final extension was performed at 72°C for 10 min, and the samples were subsequently stored at 4°C. The PCR products were analyzed using 1% agarose gel electrophoresis. The amplification conditions followed the methodology described by Jy and Liu (2024). PCR products that met the quality requirements were excised from the gel, purified, and sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for high-throughput sequencing.

Data organization and statistical analysis were conducted using Microsoft Excel 2010, while variance analysis was performed using Statistical Package for Social Sciences (version 26). One-way analysis of variance and Duncan’s multiple range test were used to determine significant differences in the data. The diversity and composition of soil microorganisms were analyzed using the i-Sanger platform¹ provided by Shanghai Majorbio Bio-Pharm Technology Co., Ltd.

As shown in Table 2, the pH of rhizosphere soil across the three sample sites ranged from 4.69 to 5.93, indicating acidic conditions. There was no significant difference in pH between BY and CY. The TP and TK contents were significantly higher in MT samples than in CY and BY samples, while CY samples had the lowest TP and TK contents. The TN content was highest in CY samples, significantly exceeding that in MT and BY samples, with BY samples having the lowest TN content. The SOM content in CY samples was significantly higher than in MT and BY samples, whereas there was no significant difference between MT and BY. The AN content did not differ significantly between MT and CY samples but was significantly lower in BY samples. The AP content was highest in MT samples, followed by BY, and lowest in CY. The AK content was significantly higher in CY samples than in MT and BY samples, with no significant difference between MT and BY. These findings highlight variations in the physicochemical properties of rhizosphere soil among the three sample sites.

As shown in Table 3, 154,325 valid bacterial sequences were obtained from the rhizosphere soil across the three sample sites, with an average sequence length of 1,446 bp and sequencing coverage ranging from 97.22 to 97.83%. For fungi, 143,146 valid sequences were obtained, with an average sequence length of 607 bp and sequencing coverage ranging from 98.8 to 99.06%. These results indicate that the sequencing data accurately represent the microbial community composition and diversity at the three sample sites.

At a similarity threshold of 97.00%, 7,467 bacterial operational taxonomic units (OTUs) were identified, including 2,766 in BY samples, 2,004 in CY samples, and 2,697 in MT samples. Two thousand eight hundred sixty-six fungal OTUs were detected in the rhizosphere soil, with 1,108 in BY, 1,077 in CY, and 681 in MT samples. CY samples had the lowest number of OTUs, while BY samples had the highest.

As illustrated in Figure 1A, 565 bacterial OTUs and 86 fungal OTUs were common across BY, CY, and MT samples, accounting for 7.56 and 3.00% of the total, respectively. The number of unique bacterial (Figure 1B) OTUs in BY, CY, and MT samples was 237, 86, and 230, respectively, while the number of unique fungal OTUs was 210, 251, and 117, respectively. These findings indicate variations in microbial community composition in the rhizosphere soil of C. tracyanum from different regions.

Figure 2A,B shows that CY had the lowest bacterial abundance index among the three sample sites, while MT had the lowest fungal abundance index. In terms of bacterial diversity (Figure 2B,D), BY had the highest bacterial diversity index, followed by MT and CY. For fungal diversity, BY also exhibited the highest index, followed by CY, whereas MT had the lowest. CY had the lowest bacterial abundance and diversity indices, whereas MT had the lowest fungal abundance and diversity indices. The rhizosphere soil of BY exhibited the highest bacterial and fungal diversity indices, with fungal diversity significantly exceeding bacterial diversity. Similarly, BY samples had the highest bacterial and fungal abundance indices, with fungal abundance significantly surpassing bacterial abundance. These results indicate distinct microbial community compositions in the rhizosphere soil of C. tracyanum from different regions.

Based on the principal component analysis results in Figure 3A, PC1 and PC2 accounted for 51.24 and 39.83% of the variation in rhizosphere soil bacterial communities, respectively, with a cumulative contribution of 91.07%. The samples from BY, CY, and MT were distributed in different quadrants and were relatively distant from each other, suggesting significant differences in bacterial community composition among the three sites. Similarly (Figure 3B), PC1 and PC2 explained 53.58 and 42.49% of the variation in rhizosphere soil fungal communities, respectively, with a cumulative contribution of 96.07%. The BY, CY, and MT samples were again located in different quadrants and were widely separated, indicating significant differences in fungal community composition among the three sites. These results highlight considerable differences in the microbial community structure of rhizosphere soil in C. tracyanum from different locations.

The genomic DNA sequencing results of rhizosphere soil microorganisms revealed that the rhizosphere soil from the three sample sites contained 1,535 bacterial OTUs, classified into 17 phyla, 50 classes, 93 orders, 162 families, 337 genera, and 462 species. Eight hundred seventy-one fungal OTUs belonging to 15 phyla, 49 classes, 107 orders, 215 families, 392 genera, and 545 species were identified.

At the phylum level, as shown in Figure 4A, bacterial samples from MT, CY, and BY with a relative abundance greater than 1% were annotated to five phyla, four of which were accurately classified: Proteobacteria, Acidobacteria, Planctomycetota, and Firmicutes. Specifically, bacterial samples from the rhizosphere soil of BY were classified into four phyla: Proteobacteria (41.65%), Acidobacteria (21.55%), Planctomycetota (8.70%), and Firmicutes (7.58%). Bacterial samples from CY were classified into Proteobacteria (51.00%), Acidobacteria (20.25%), Planctomycetota (9.80%), and Firmicutes (3.25%). Similarly, bacterial samples from MT were classified into Proteobacteria (37.32%), Acidobacteria (11.39%), Planctomycetota (14.71%), and Firmicutes (18.83%). These results indicate that Proteobacteria is the dominant bacterial phylum in the rhizosphere soil of C. tracyanum across all three sites. However, significant differences exist in their relative abundances and the composition and relative abundances of the secondary dominant phyla (Figure 4B).

The fungal samples from MT, CY, and BY were classified into three phyla: Basidiomycota, Ascomycota, and Mortierellomycota. Among them, the fungal samples from BY were classified into Basidiomycota (30.01%), Ascomycota (52.51%), and Mortierellomycota (11.62%). In CY, the fungal samples were classified into Basidiomycota (17.10%), Ascomycota (44.39%), and Mortierellomycota (30.01%). In MT, the fungal samples were classified into Basidiomycota (76.20%), Ascomycota (20.58%), and Mortierellomycota (1.71%). These findings suggest that Ascomycota is the dominant fungal phylum in the rhizosphere soil of C. tracyanum from BY and CY, whereas Basidiomycota is the dominant phylum in MT. Additionally, there are substantial variations in the relative abundances of fungal phyla among the three sites.

At the genus level, as shown in Figure 5A, in the rhizosphere soil of CY, unclassified_f__Acidobacteriaceae was the most dominant bacterial genus, with a relative abundance of 8.72%, followed by Paraburkholderia (7.85%) and Bradyrhizobium (7.05%). In MT, unclassified_f__Acidobacteriaceae was also the most dominant bacterial genus (4.62%), followed by Rhodoplanes (3.23%), unclassified_o__Pirellulales (3.11%), and Paenibacillus (3.08%). In BY, Vicinamibacter was the most dominant bacterial genus (10.21%), followed by unclassified_f__Acidobacteriaceae (4.10%), unclassified_c__Betaproteobacteria (3.99%), Pseudomonas (3.89%), and (Figure 5B) Bradyrhizobium (3.17%).

In the rhizosphere soil of MT, Psathyrella was the most dominant fungal genus (59.49%), followed by Apiotrichum (6.75%) and Burgoa (6.31%). In CY, Mortierella was the most dominant fungal genus (30.00%), followed by Archaeorhizomyces (4.66%), unclassified_f__Hyaloscyphaceae (4.61%), Geminibasidium (4.25%), and unclassified_o__GS11 (3.57%). In BY, Mortierella was again the most dominant fungal genus (11.59%), followed by Solicoccozyma (8.54%), Trichocladium (6.09%), Tausonia (6.05%), and unclassified_f__Stephanosporaceae (4.65%).

As shown in Figure 6A, in the rhizosphere soil of CY, unclassified_f__Acidobacteriaceae was the most dominant bacterial species (8.72%), followed by unclassified_g__Bradyrhizobium (7.05%), Paraburkholderia fungorum (5.44%), and unclassified_c__Acidobacteria (3.48%). In MT, unclassified_f__Acidobacteriaceae was also the most dominant bacterial species (4.62%), followed by unclassified_o__Pirellulales (3.14%), unclassified_g__Rhodoplanes (3.06%), Pseudolabrys taiwanensis (2.88%), and unclassified_f__Gemmatimonadaceae (2.85%). In BY, Vicinamibacter silvestris was the most dominant bacterial species (10.21%), followed by unclassified_f__Acidobacteriaceae (4.10%), unclassified_c__Betaproteobacteria (4.00%), Pseudolabrys taiwanensis (3.89%), and unclassified_g__Bradyrhizobium (3.17%).

As shown in Figure 6B, in the rhizosphere soil of MT, Psathyrella pygmaea was the most dominant fungal species (59.50%), followed by Burgoa anomala (6.31%) and unclassified_g__Apiotrichum (4.28%). In CY, Mortierella humilis was the most dominant fungal species (24.29%), followed by Hyaloscyphaceae sp. (4.53%), Geminibasidium sp. (4.25%), and GS11 sp. (3.67%). In BY, Trichoderma hamatum was the most dominant fungal species (6.09%), followed by Tausonia pullulans (6.05%), Solicoccozyma terricola (5.87%), and Stephanosporaceae sp. (4.65%).

These findings highlight significant differences in the dominant microbial communities and their relative abundances in the rhizosphere soil of C. tracyanum across different regions.

The core microbiome is considered a key component of the fundamental functions of the entire biological community. It is enriched, selected, and inherited through evolutionary processes (Zhang et al., 2020). Core microbial taxa can be analyzed from multiple perspectives, including consortium coexistence, OTU abundance, functional redundancy, and phylogeny. In this study, the coexisting microbial flora in the three plots was defined as the core microbial flora, following the method of Xu Guoqi et al. (Zhao et al., 2014). As shown in Figure 7A, 214 core bacterial species were identified in the rhizosphere soil samples from the three sample sites at the genus level, accounting for 2.86% of the total bacterial community. Among these, the 10 most abundant genera were unclassified_f__Acidobacteriaceae, Vicinamibacter, Bradyrhizobium, Paraburkholderia, unclassified_o__Hyphomicrobiales, Pseudomonas, unclassified_o__Pirellulales, Rhodoplanes, unclassified_f__Gemmataceae, and Caballeronia.

Similarly, Figure 7B illustrates that 79 core fungal species were identified at the genus level in the rhizosphere soil samples from the three sites, constituting 2.75% of the total fungal community. The 10 most abundant genera were Psathyrella, Mortierella, Solicoccozyma, Penicillium, Apiotrichum, Trichocladium, Tausonia, Saitozyma, unclassified_f__Hyaloscyphaceae, and Cladophialophora.

A correlation analysis based on the Pearson algorithm was conducted to examine the relationship between soil physicochemical factors and the 20 most abundant core microbial genera. As shown in Figure 8A, TP content was significantly positively correlated with the relative abundance of Rhodoplanes, unclassified_f__Gemmataceae, and Acinetobacter. TK content was positively correlated with the relative abundance of five genera, including unclassified_o__Pirellulales, Rhodoplanes, unclassified_f__Gemmataceae, Chthoniobacter, and Acinetobacter. Soil pH was positively correlated with the relative abundance of Bradyrhizobium, Paraburkholderia, Caballeronia, unclassified_f__Chitinophagaceae, and Mucilaginibacter but negatively correlated with unclassified_f__Gemmataceae, Niallia, and Paenibacillus. TN content was positively correlated with Vicinamibacter, Pseudomonas, and unclassified_c__Betaproteobacteria but negatively correlated with unclassified_f__Acidobacteriaceae, unclassified_o__Hyphomicrobiales, unclassified_c__Acidobacteria, Roseiarcus, and Chthoniobacter. SOM content was negatively correlated with unclassified_o__Pirellulales, Rhodoplanes, unclassified_f__Gemmataceae, and Acinetobacter. AN content was positively correlated with Vicinamibacter, Pseudomonas, unclassified_c__Betaproteobacteria, and Niallia but negatively correlated with unclassified_f__Acidobacteriaceae, unclassified_o__Hyphomicrobiales, unclassified_c__Acidobacteria, Roseiarcus, and Chthoniobacter. AP content showed a positive correlation with Rhodoplanes, unclassified_f__Gemmataceae, and Acinetobacter. AK content was positively correlated with Vicinamibacter, Pseudomonas, and unclassified_c__Betaproteobacteria but negatively correlated with unclassified_f__Acidobacteriaceae, unclassified_o__Hyphomicrobiales, unclassified_c__Acidobacteria, and Roseiarcus.

As illustrated in Figure 8B, TP content was significantly positively correlated with Psathyrella, Apiotrichum, Burgoa, and Trichoderma but negatively correlated with Penicillium, Tausonia, Saitozyma, and unclassified_f__Stephanosporaceae. Similarly, TK content was positively correlated with Psathyrella, Apiotrichum, and Burgoa but negatively correlated with Penicillium, Tausonia, Saitozyma, and unclassified_f__Stephanosporaceae. Soil pH was significantly positively correlated with Mortierella, unclassified_f__Hyaloscyphaceae, Cladophialophora, Archaeorhizomyces, Geminibasidium, unclassified_o__GS11, Cladosporium, Umbelopsis, Inocybe, and unclassified_o__Chaetothyriales but negatively correlated with Burgoa. TN content was positively correlated with Solicoccozyma, Trichocladium, and unclassified_f__Stephanosporaceae. SOM content was positively correlated with Penicillium, Tausonia, Saitozyma, and unclassified_f__Stephanosporaceae but negatively correlated with Psathyrella, Apiotrichum, and Trichoderma. AN content was positively correlated with Solicoccozyma, Trichocladium, and unclassified_f__Stephanosporaceae but negatively correlated with Inocybe. AP content was positively correlated with Psathyrella, Apiotrichum, and Trichoderma but negatively correlated with Penicillium, Tausonia, Saitozyma, and unclassified_f__Stephanosporaceae. AK content was positively correlated with Solicoccozyma, Trichocladium, and unclassified_f__Stephanosporaceae. These findings suggest that soil physicochemical properties influence the relative abundance of core microbial communities in rhizosphere soil.

As shown in Figure 9A, the composition of the bacterial community structure in rhizosphere soil was significantly positively correlated with TP, TK, pH, SOM, AP, and AK contents. Additionally, the α-diversity of rhizosphere soil bacteria exhibited a significant positive correlation with TP, pH, AN, and AP contents.

As depicted in Figure 9B, the fungal community structure in rhizosphere soil was positively correlated with TP, TK, TN, SOM, AN, AP, and AK contents but negatively correlated with pH. The α-diversity of rhizosphere soil fungi was significantly negatively correlated with pH and AN contents but positively correlated with other environmental factors. These results indicate that TP and AP contents influence microbial diversity in rhizosphere soil.