1. Introduction
The Orchidaceae family is recognized as one of the most diverse and widely distributed families among angiosperms, noted for its unique floral morphology, growth habits, and ecological adaptability. Globally, the family comprises approximately 880 genera and 25,000 species, found across tropical, subtropical, and temperate regions [1]. The Orchidaceae family fulfills crucial ecological roles within ecosystems [2]. Additionally, they possess high economic value in ornamental [3], medicinal, and cultural domains and represent a vital component of the horticultural industry [4,5]. These orchids, which employ the ‘opportunistic/generalist’ strategy, form symbiotic and synergistic relationships with local soil bacteria. However, most orchid species are very demanding in terms of their habitats.
The rhizosphere—the narrow soil zone directly influenced by root exudates—undergoes profound microbial and physicochemical transformations, including altered nutrient cycling, pH regulation, and pathogen suppression, which critically shape plant health and ecosystem functioning. Despite this, the rhizosphere effect in Orchidaceae remains underexplored, particularly regarding bacterial community assembly and functional differentiation. Rhizosphere bacterial diversity is closely related to the growth, development, and stress resistance of Orchidaceae [6,7]. Recent studies have demonstrated that the rhizosphere and endophytic bacterial communities associated with Orchidaceae can enhance plant growth through various mechanisms, including nitrogen (N) fixation, organic matter decomposition, secretion of plant growth hormones, and increased resistance to pathogens [8]. Furthermore, the symbiotic association between mycorrhizal bacteria and Orchidaceae significantly contributes to nutrient uptake and plant adaptation to environmental stress [9,10]. Therefore, comprehensive research on the bacterial diversity of Orchidaceae is vital for understanding their ecological adaptation mechanisms, conserving endangered species, and optimizing cultivation management [11].
Numerous studies have investigated the bacterial diversity of Orchidaceae, including the effects of soil bacteria on the germination of Orchidaceae seeds [12] and the bacterial communities present in the rhizosphere soil of Orchidaceae, as well as potential orchid mycorrhizal bacteria [13]. However, from an ecological perspective, the influence of soil bacterial composition, environmental factors, and soil bacterial functions on the structure of Orchidaceae soil bacterial communities remains inadequately understood [14].
China is abundant in Orchidaceae resources and harbors many species with unique ornamental and medicinal value [15]. For example, C. amoena and C. faberi are widely cultivated for their ornamental appeal, whereas G. elata is highly prized for its medicinal properties [16]. C. macranthos and C. appendiculata also possess significant ornamental value. All five of these Orchidaceae species are protected plants in China.
Based on a comprehensive survey of Orchidaceae in the north subtropical region of China [17], this study selected five common Orchidaceae species [18]. Utilizing high-throughput sequencing technology, we systematically analyzed the characteristics of rhizosphere soil bacterial diversity in Orchidaceae [19]. We explored the dominant bacterial groups, variations in diversity indices, and functional differences in soil bacterial communities among different Orchidaceae species, thereby providing a theoretical foundation for the conservation and sustainable use of Orchidaceae [20,21].
2. Materials and Methods
2.1. Overview of the Study Area
This study was conducted at the Funiu Mountain Nature Reserve in Henan Province, China [22]. The geographic coordinates of the reserve are 111°17′–112°17′ E longitude and 32°50′–33°54′ N latitude, covering a total area of 56,024 hectares (Attachment S2). The reserve occupies a transitional zone between the north subtropical and warm-temperate regions, exhibiting a continental monsoon climate characterized by four distinct seasons. Spring is dry and windy, summer is cool and humid, autumn is rainy, and winter is cold and dry. Most of the area has an elevation exceeding 1500 m.
The five Orchidaceae species selected for this study—C. amoena, C. macranthos, C. appendiculata, C. faberi, and G. elata—are typical and relatively common in Henan Province and are all nationally protected species [23].
2.2. Sampling Design
Following a review of the relevant literature and preliminary surveys of sampling sites (Table 1), we selected 10 representative Orchidaceae plants from a homogeneous population area in the Funiu Mountain Nature Reserve as study subjects in July 2023 [24]. The rhizosphere soil was defined as the soil within the 0–3 cm range surrounding the surface of the plant’s fibrous roots, excluding the surface humus layer, as well as the 5–15 cm soil layer. We employed a five-point composite sampling method. All soil samples were passed through a 2 mm sieve, thoroughly mixed in equal portions, and transported to the laboratory on the same day [3]. A portion of the soil samples was preserved at −80 °C for subsequent DNA analysis, while the remaining samples were stored at −4 °C for assessment of rhizosphere soil physicochemical properties [25].
2.3. Determination of Soil Bacterial Communities
Total DNA was extracted from 0.5 g of fresh soil sample using the Fast DNA SPIN extraction kit (Mobio Laboratories, Carlsbad, CA, USA) for bacterial analysis [26]. The DNA concentration was quantified using a spectrophotometer (Thermo Scientific, Wilmington, DE, USA); the samples were then stored at −80 °C. Subsequently, the V3–V4 region of the 16S rDNA was amplified and sequenced using primers 515F and 806R [27]. Sequencing was conducted on the Illumina HiSeq platform (Illumina Inc., San Diego, CA, USA), employing a paired-end sequencing strategy (2 × 150 bp). The sequencing and subsequent bioinformatics analysis were performed by Guangzhou Kidio Biotechnology Co., Ltd. (Guangzhou, China).
2.4. Determination of Soil Physicochemical Properties
Soil samples were air-dried, sieved through a 60-mesh sieve (0.25 mm aperture) to remove coarse fragments, and stored for analysis. The soil pH was measured potentiometrically using a 1 M KCl solution at a soil-to-solution ratio of 1:2.5. Soil Organic Matter (SOM) was quantified using the dichromate oxidation method. Available N was assessed using the alkaline hydrolysis diffusion method. Available phosphorus (P) was determined with the molybdenum blue method. Soil Moisture Content (SMC) was evaluated by drying the soil at 105 °C overnight. Available potassium (AK) was quantified using flame photometry [28].
2.5. Statistical Analysis
The VennDiagram package in R was utilized to generate Venn diagrams illustrating the number of unique and shared operational taxonomic units (OTUs) among the soils of different Orchidaceae species. Visualization was achieved through boxplots [29].
Stacked bar charts were employed to analyze the relative abundance of dominant bacterial communities in the rhizosphere soil of various Orchidaceae species [16]. Statistical analysis was conducted using the ggplot2 package in R. Redundancy analysis was employed to investigate the associations between environmental factors and soil bacterial communities across different Orchidaceae species [30]. Calculations were performed using the vegan package, and the envfit function was utilized to evaluate the effect of environmental factors on bacterial community distribution [31,32,33].
Null model methods were implemented to examine the relative significance of deterministic and stochastic processes in bacterial community assembly. The β nearest taxon index (βNTI) and the taxonomic diversity index (RCBray) were calculated to quantify variations in community phylogenetic and taxonomic diversity. A |βNTI| value greater than 2 indicates that deterministic processes, specifically environmental filtering, predominantly influence community turnover. A βNTI value exceeding 2 suggests heterogeneous selection (where differing environmental conditions lead to variations in communities), whereas a βNTI < 2 points to homogeneous selection (indicating similar environmental conditions foster community convergence). Conversely, a |βNTI| < 2 implies that stochastic processes, such as migration and dispersal, are the primary drivers of community assembly. For RCBray, values below 0.95 indicate homogeneous dispersal processes, whereas values exceeding 0.95 suggest dispersal-limited processes. A |βNTI| < 0.95 indicates the absence of a dominant process.
The neutral community model was employed to predict the association between the occurrence frequency of species across all samples and their relative abundance within the community [34]. The vegan package in R facilitated the fitting of the neutral model, the assessment of the association between species relative abundance and occurrence frequency, and the calculation of the R value (correlation coefficient) and N value (number of samples) to evaluate model fit and sample adequacy [35].
Bacterial functional prediction methods were utilized to reveal functional differences among the bacterial communities in the soils of the five Orchidaceae species. Using 16S rDNA gene sequence data, the Tax4Fun2 tool on the Biocloud platform was employed to perform OTU taxonomic annotation by comparing it with the SILVA database [36]. Functional annotations were integrated with information from the KEGG database (
3. Results
3.1. Species Composition and Diversity of Soil Bacterial Communities
The Venn diagram results revealed that among all species, C. amoena exhibited the highest number of soil bacterial OTUs. In contrast, C. appendiculata displayed a lower number of soil bacterial OTUs and shared fewer OTUs with other plants (Figure 1). Among generalized and specialized species, C. faberi recorded the greatest number of soil bacterial OTUs.
Boxplot analysis indicated significant differences in the species richness of soil bacteria among the five different Orchidaceae species. C. amoena and G. elata demonstrated higher species richness of soil bacteria, whereas C. appendiculata exhibited lower species richness (Figure 1).
At the order level, Bacteroidales and Burkholderiales exhibited higher abundance in the rhizosphere soil of the five Orchidaceae species, with these order-level groups being particularly prevalent in samples of C. amoena and G. elata. At the family level, Chitinophagaceae and Sphingomonadaceae were dominant in the rhizosphere soil of the five Orchidaceae species. Notably, Pyrinomonadaceae was more prevalent in other soils but demonstrated lower abundance in the rhizosphere soil of C. appendiculata. At the genus level, Udaeobacter and Solibacter were predominant in the rhizosphere soil of the five Orchidaceae species. In addition, the RB41 and Pir4 lineages were more enriched in the rhizosphere soil of G. elata, while being relatively scarce in the rhizosphere soil of other Orchidaceae species (Figure 2).
3.2. Response of Bacterial Communities to Environmental Factors
The results of the RDA analysis indicated that the first and second axes accounted for 43.02% and 28.22% of the variation in bacterial communities, respectively, yielding a combined explanation rate of 61.24% (Figure 3). Soil pH significantly influenced the soil bacterial community of C. amoena. Total N was identified as the primary influencing factor for the soil bacterial community of C. macranthos. Available P and SMC were shown to significantly affect the soil bacterial communities of C. faberi and G. elata. The selected environmental factors had a relatively minor effect on the soil bacterial community of C. appendiculata.
For the generalized species, the RDA analysis revealed a cumulative explanation rate of 58.31%, with SMC identified as the primary environmental factor affecting the soil bacterial community of C. amoena. Total N significantly affected the soil bacterial communities of C. macranthos and C. appendiculata. In the case of specialized species, the RDA analysis indicated a cumulative explanation rate of 71.05%, with Available P, SOM, and AK significantly influencing the soil bacterial communities of C. amoena, G. elata, and C. faberi. Total N emerged as the principal environmental factor affecting the soil bacterial communities of C. macranthos and C. appendiculata.
3.3. Differences in Rhizosphere Soil Bacterial Community Assembly
The results of the NCM analysis indicated (Figure 4) that the fit of different bacterial communities to the neutral community model was as follows: all species (R2 = 0.804), neutral taxa (R2 = 0.792), specialized species (R2 = 0.759), and generalized species (R2 = 0.349). In the soil bacterial OTU data of the five different Orchidaceae species, the number of OTUs for all species, neutral taxa, specialized species, and generalized species were Nm = 8236, Nm = 3740, Nm = 1905, and Nm = 253, respectively.
During the assembly of rhizosphere soil bacterial communities, different species of Orchidaceae exhibited notable differences in their ecological strategies. The rhizosphere soils of C. amoena, C. macranthos, and C. faberi were primarily influenced by heterogeneous selection within deterministic processes. In contrast, in the rhizosphere soils of C. appendiculata and G. elata, non-dominant processes within stochastic frameworks were predominant. In the assembly of rhizosphere soil bacterial communities of generalized species, heterogeneous selection within deterministic processes was dominant across all five Orchidaceae species. In contrast, in the rhizosphere soil of C. macranthos for specialized species, homogeneous selection within stochastic processes was prevalent (Figure 5).
3.4. Functional Diversity of Soil Microorganisms in Different Orchidaceae
The pie charts representing metabolic functions indicate that fermentation predominates among the metabolic functional bacteria within the soils of the five Orchidaceae species. In the soil bacterial community of C. appendiculata, fermentation comprises as much as 81.15%. In the soil of G. elata, fermentation and chitinolysis are notably prevalent, accounting for 64.20% and 16.76% of the metabolic functional bacteria, respectively. In the soils of C. amoena and C. faberi, nitrate respiration is more pronounced (Figure 6A).
Nutritional bacteria are categorized into 12 types based on their nutritional strategies, which include interactions with hosts through phototrophy, parasitism, or symbiosis. Among these, aerobic chemoheterotrophy is the dominant nutritional strategy in the soil bacterial communities of the five Orchidaceae species. In C. amoena, nitrate respiration is particularly abundant. In contrast, the soil bacteria of C. macranthos and C. appendiculata exhibit a higher relative abundance of animal parasites or symbionts (Figure 6B).
4. Discussion
The structure of bacterial communities exhibits variation among different species of Orchidaceae, which may correlate with the specific plant species. The soil bacterial communities associated with G. elata and C. amoena demonstrate considerable richness, suggesting that these plants can sustain more diverse bacterial communities [39]. The higher number of shared OTUs in the rhizosphere soil of C. amoena and G. elata indicates that these two species may inhabit similar ecological environments or possess analogous requirements for bacterial community composition [40,41].
At the order level, Bacteroidales and Burkholderiales exhibit a higher relative abundance in the rhizosphere soil of the five different Orchidaceae species [28]. At the family level, Chitinophagaceae and Sphingomonadaceae are dominant, whereas at the genus level, Udaeobacter and Solibacter are the most prevalent. This highlights the universality and significance of these bacterial groups in the rhizosphere soil associated with the five species of Orchidaceae. However, certain bacteria, including Pyrinomonadaceae, RB41, and the Pir4 lineage, show a lower relative abundance in the rhizosphere soil of C. appendiculata. Overall, the structure of rhizosphere soil bacterial communities among different Orchidaceae species reveals significant variations, which may be attributed to plant species, growth environments, and the selective preferences of plants for bacterial communities.
The structure of rhizosphere soil bacterial communities across various Orchidaceae species is influenced by multiple physicochemical soil factors [42]. The bacterial community associated with C. amoena is primarily regulated by soil pH, whereas that of C. macranthos is mainly affected by total N content. The rhizosphere soil bacterial communities of C. faberi and G. elata are significantly driven by Available P and SMC. These findings indicate that the structure of rhizosphere soil bacterial communities in different Orchidaceae species is shaped by various physicochemical factors, with distinct plant species demonstrating varied responses to these influences. This variation may be closely associated with the ecological differentiation of Orchidaceae species, their strategies for resource utilization, and their symbiotic interactions with bacteria.
The results of the NCM analysis indicate that the fit of different bacterial communities to the neutral community model varies significantly. For all species, neutral taxa demonstrate a superior fit, indicating that stochastic processes have a substantial influence on community assembly. In contrast, specialized and generalized species exhibit lower fit values, indicating that deterministic processes play a more prominent role in shaping their communities.
In the assembly of the rhizosphere soil bacterial community associated with various Orchidaceae species, notable differences in ecological strategies are evident [43]. For the species studied, the rhizosphere soil of C. amoena, C. macranthos, and C. faberi is primarily shaped by heterogeneous selection within deterministic processes. This suggests that these plants selectively enrich specific bacterial taxa, enabling them to better adapt to their growth environments. Conversely, in the rhizosphere soil of C. appendiculata and G. elata, non-dominant processes within stochastic frameworks are more prevalent. This observation may be related to the adaptive evolution of these species in response to their environment. For generalized species, heterogeneous selection within deterministic processes predominates in the assembly of rhizosphere soil bacterial communities across all five Orchidaceae species, indicating a strong selectivity in bacterial community assembly. Overall, the assembly of rhizosphere bacterial communities in Orchidaceae species is influenced by both stochastic and deterministic processes. The varied assembly strategies observed among different Orchidaceae species are likely closely associated with their ecological niche differentiation and environmental adaptability [44].
Fermentation functional bacteria predominate in the rhizosphere soil bacterial communities associated with all five species of Orchidaceae, representing the primary metabolic type of bacteria [45]. This observation indicates that fermentation functions are likely vital to the rhizosphere soil bacterial communities of Orchidaceae [46]. These bacteria not only participate in the decomposition and transformation of organic matter but may also significantly affect soil nutrient cycling, plant health, and ecosystem stability. Notable differences in the relative abundance of metabolic and nutritional bacteria exist within the rhizosphere soil bacterial communities of various Orchidaceae species. For example, the rhizosphere soil of C. appendiculata exhibits a comparatively high abundance of bacteria associated with the degradation of aromatic compounds. In contrast, the rhizosphere soils of C. amoena and G. elata are characterized by a higher abundance of bacteria that contribute to chitin degradation. These variations likely correlate closely with the physiological characteristics of the respective plants and their rhizosphere environments. The rhizosphere soil of C. amoena and C. faberi supports a relatively high bacterial diversity, whereas C. macranthos, C. appendiculata, and G. elata exhibit lower bacterial numbers. Such differences may reflect varying strategies employed by different Orchidaceae species in constructing and maintaining their rhizosphere bacterial communities. These findings highlight the pivotal role of fermentation functional bacteria in the rhizosphere soil bacterial communities of Orchidaceae [32,47]. The observed differences in metabolic functions and bacterial abundance among the rhizosphere bacterial communities of different Orchidaceae species highlight their diverse ecological adaptation strategies [48].
5. Conclusions
This study provides a comprehensive analysis of the structure, function, and associations with environmental factors of the rhizosphere soil bacterial communities associated with five species of Orchidaceae [49]. The results indicate significant structural differences in the rhizosphere soil bacterial communities among the five Orchidaceae species. The primary environmental factors influencing these communities vary across different species. Fermentation functional bacteria dominate these communities and are crucial to the ecological functions of Orchidaceae rhizosphere ecosystems. Additionally, variations in the abundance of metabolic and nutritional bacteria are evident among the rhizosphere bacterial communities of different plants. The community assembly processes of specialized and generalized species are governed by deterministic and stochastic processes, respectively.
The five Orchidaceae species are not only protected plants in China but also possess significant ornamental and medicinal values. This study examines the structural characteristics, functional differences, and response mechanisms of the rhizosphere bacterial communities associated with various Orchidaceae species from the perspective of soil microbiology. This research offers a crucial theoretical foundation for the conservation and sustainable utilization of Orchidaceae.
Conceptualization, J.D.; data curation, J.D. and S.G.; formal analysis, J.D., Z.Y. and X.L.; funding acquisition, Z.Y.; investigation, X.S.; methodology, J.D. and Z.G.; project administration, Z.Y.; resources, J.D.; software, Z.G.; supervision, S.G. and Z.Y.; validation, Z.Y. and S.G.; visualization, J.D.; writing—original draft, X.L.; writing—review and editing, X.S. and S.G. All authors have read and agreed to the published version of the manuscript.
Not applicable.
The raw data presented in this study are available on request from the corresponding author. The data are not publicly available due to the risk of potential threats to some endangered plant species.
We thank the staff members of the Urban Ecosystem Biodiversity Group at Henan Agricultural University for their technical guidance. We are grateful to Shengqian Guo, Zhonghu Geng, and Xiaohang Li from the College of Life Sciences of Henan Agricultural University for their contributions to data collection.
The authors declare no conflicts of interest.
Footnotes
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Figure 1 (a): Venn diagram of OTUs of rhizosphere soil bacteria of five orchid species, (b): boxplot of species richness of rhizosphere soil bacteria of five orchid species. DHL represents C. amoena, DHSL represents C. macranthos, DJL represents C. appendiculata, HL represents C. faberi, and TM represents G. elata.
Figure 2 (a): Relative abundance of rhizosphere soil bacteria among all species for different orchids, (b): relative abundance of rhizosphere soil bacteria among generalist species for different orchids, (c): relative abundance of rhizosphere soil bacteria among specialist species for different orchids. DHL represents C. amoena, DHSL represents C. macranthos, DJL represents C. appendiculata, HL represents C. faberi, and TM represents G. elata.
Figure 3 Redundancy analysis of rhizosphere soil bacterial community structure and environmental factors for different orchid species. (a): All species, (b): general species, (c): special species, SMC represents Soil Moisture Content, PH represents Potential of Hydrogen, AK represents Available Potassium (
Figure 4 Relative abundance distribution of rhizosphere soil microorganisms across different orchid species. (a) Total microbial community; (b) generalist species; (c) neutral taxa; (d) specialist species. Non-species represents the concept of neutral taxa. Cl represents Confidence Interval, Rsqr represents R-squared, and “Nm” represents Number of Measurements.
Figure 5 Ecological processes of rhizosphere soil bacteria for five orchid species. The inner circle indicates whether deterministic or stochastic processes dominate community assembly. The outer circle illustrates the proportion of specific ecological processes within stochastic and deterministic frameworks. DHL represents C. amoena, DHSL represents C. macranthos, DJL denotes C. appendiculata, HL signifies C. faberi, and TM refers to G. elata.
Figure 6 Bacterial biological function in the soil of five orchid species. (A): Proportional circular diagram illustrating the different organic compound metabolism functions of soil bacteria in five orchid species, arranged from the inside out as follows: C. amoena (DHL), C. macranthos (DHSL), C. appendiculata (DJL), C. faberi (HL), and G. elata (TM). (B): Stacked bar chart depicting the relative abundance of phototrophic, parasitic, and symbiotic bacteria in the soil of the five orchid species in Funiu Mountain Nature Reserve.
Sampling sites of five orchid species.
| Species Names | Elevation/m | Longitude | Latitude |
|---|---|---|---|
| Cremastra appendiculata | 1258 | 112.005364 | 33.693947 |
| Cremastra appendiculata | 1388 | 112.002483 | 33.697272 |
| Changnienia amoena | 944 | 111.914447 | 33.574636 |
| Changnienia amoena | 953 | 111.91445 | 33.5746 |
| Cypripedium macranthos | 1205 | 111.905097 | 33.585867 |
| Cypripedium macranthos | 1278 | 111.905042 | 33.585114 |
| Gastrodia elata | 1248 | 112.067697 | 33.789036 |
| Gastrodia elata | 1094 | 111.9133333 | 33.57277778 |
| Cymbidium faberi | 1009 | 112.0002778 | 33.68416667 |
| Cymbidium faberi | 1009 | 112.00029 | 33.68412 |
Supplementary Materials
The following supporting information can be downloaded at:
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1 College of Tropical Agriculture and Forestry, Hainan University, No. 58 Renmin Avenue, Meilan District, Haikou 570228, China; [email protected]
2 College of Life Sciences, Henan Agricultural University, No. 63 Agricultural Road, Zhengzhou 450002, China; [email protected] (S.G.); [email protected] (X.L.); [email protected] (Z.G.); [email protected] (Z.Y.)