Characteristics of the Rhizosphere Fungal Community and Isolation and Identification of Root Endophytes from Wild Cymbidium goeringii and Cymbidium faberi in the Qinling Mountains of China

Characteristics of the Rhizosphere Fungal Community and Isolation and Identification of Root Endophytes from Wild Cymbidium goeringii and Cymbidium faberi in the Qinling Mountains of China | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 January 2025 V1 Latest version Share on Characteristics of the Rhizosphere Fungal Community and Isolation and Identification of Root Endophytes from Wild Cymbidium goeringii and Cymbidium faberi in the Qinling Mountains of China Authors : Siyu Wen , Xinying Hao , and Junyang Song 0000-0002-3529-8113 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173676459.96504541/v1 313 views 123 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Wild orchids hold significant ecological value, yet current research mainly focuses on germplasm resources, lacking systematic studies on the native habitats and root endophytic fungi of species like Cymbidium goeringii and Cymbidium faberi. This study used fungal sequencing to explore the fungal community in the native habitats of these orchids in the Qinling Mountains. We compared rhizosphere and non-rhizosphere soil fungi and assessed the ecological functions of dominant fungal groups. The impact of soil chemical indicators on fungal communities was also analyzed. Endophytic fungi were isolated from orchid roots to evaluate their growth-promoting potential. Results showed that Ascomycota and Basidiomycota were dominant in the rhizosphere of both species. Symbiotic nutrient and pathology-saprophytic-symbiotic mixed fungal types were predominant, accounting for 56.76% and 79.17% of the fungal community, respectively. Soil factors like total nitrogen and ammonium nitrogen correlated strongly with these communities. A total of 17 fungal strains were isolated, with strains HL01, HL02, HL06, and HL17 exhibiting indole-3-acetic acid (IAA) production, nitrogen fixation, and phosphorus solubilization. Strain HL08 showed IAA production, nitrogen fixation, and potassium solubilization. These strains contribute to enriching growth-promoting fungal resources for both species. This research provides a theoretical and scientific basis for the conservation, artificial cultivation, and breeding of Cymbidium goeringii and Cymbidium faberi. Characteristics of the Rhizosphere Fungal Community and Isolation and Identification of Root Endophytes from Wild Cymbidium goeringii and Cymbidium faberi in the Qinling Mountains of China Siyu Wen 1 , Xinying Hao 1 , Junyang Song 1* 1 College of Landscape Architecture and Art, Northwest A&F University(NWAFU), 712100, Yangling, China; [email protected] (SW); [email protected] (XH); [email protected] (JS) First author: Siyu Wen 1 [email protected] (SW) *Corresponding author: Junyang Song1* [email protected] (JS); ORCID：0000-0002-3529-8113 Abstract: Wild orchids hold significant ecological value, yet current research mainly focuses on germplasm resources, lacking systematic studies on the native habitats and root endophytic fungi of species like Cymbidium goeringii and Cymbidium faberi . This study used fungal sequencing to explore the fungal community in the native habitats of these orchids in the Qinling Mountains. We compared rhizosphere and non-rhizosphere soil fungi and assessed the ecological functions of dominant fungal groups. The impact of soil chemical indicators on fungal communities was also analyzed. Endophytic fungi were isolated from orchid roots to evaluate their growth-promoting potential. Results showed that Ascomycota and Basidiomycota were dominant in the rhizosphere of both species. Symbiotic nutrient and pathology-saprophytic-symbiotic mixed fungal types were predominant, accounting for 56.76% and 79.17% of the fungal community, respectively. Soil factors like total nitrogen and ammonium nitrogen correlated strongly with these communities. A total of 17 fungal strains were isolated, with strains HL01, HL02, HL06, and HL17 exhibiting indole-3-acetic acid (IAA) production, nitrogen fixation, and phosphorus solubilization. Strain HL08 showed IAA production, nitrogen fixation, and potassium solubilization. These strains contribute to enriching growth-promoting fungal resources for both species. This research provides a theoretical and scientific basis for the conservation, artificial cultivation, and breeding of Cymbidium goeringii and Cymbidium faberi . Keywords: Cymbidium goeringii , Cymbidium faberi , Native habitat, Rhizosphere fungal community, Root endophytes, Qinling Mountains 1 Introduction The Orchidaceae, one of the two major families of angiosperms, comprises over 880 genera and 25,000 species. Orchids hold diverse values, including medicinal, nutritional, and ornamental significance(Freudenstein, 2003, Amy et al., 2018). China is home to one of the richest and most diverse floras in the Northern Hemisphere, with over 1,300 species of orchids(Du et al., 2020). New species and varieties of orchids continue to be discovered and documented(Du et al., 2020). The Qinling Mountains, situated in central China, mark the boundary between the warm temperate zone and the subtropical zone. This region serves as an ecological transition zone in China, characterized by a rich diversity of orchid species and a high level of endemism. The soil types in the Qinling Mountains are primarily dominated by yellow-brown, brown, and dark brown soils(He, 2018). The diverse geography and complex climate of the Qinling Mountains have created a unique habitat for orchids, with approximately 45 genera and 101 species. Of these, 50 species are endemic to China, and 4 species are endemic to the Qinling Mountains, including wild spring orchids and cymbidium orchids(Tsun-shen, 1994). The interaction between the roots and rhizosphere soil of wild orchids involves numerous complex biological and abiotic factors. The relationship between the roots and rhizosphere microorganisms is essential for the survival, growth, and reproduction of orchids(Chaudhary et al., 2015, Singh et al., 2004). Wild orchids can establish symbiotic relationships with specific mycorrhizal fungi, which invade root cells and form a complex symbiotic system with the orchids. This system facilitates the absorption of water and nutrients, particularly in nutrient-poor soil environments. By secreting chemical signals, orchids attract beneficial mycorrhizal fungi(Toal et al., 2000) and help regulate soil pH(Kim and Silk, 1999). Information transfer and material exchange occur between the orchid root system and the rhizosphere soil, thereby creating a distinct soil ecological niche(L, 1904). The influence of orchids on rhizosphere soil extends to affect the surrounding non-rhizosphere soil through various mechanisms, thereby forming an ecological chain that radiates outward. The results indicate that the diversity and richness of the rhizosphere fungal community are higher than those in the non-rhizosphere soil, with distinct differences observed in community structure(Kozdrój and Elsas, 2000). The growth and development of orchids are highly reliant on the nutrients supplied by rhizosphere microorganisms, necessitating the evolution of beneficial interactions with soil microbes. Throughout nearly their entire life cycle, orchids depend on mycorrhizal fungi for nutritional support(Yue et al., 2019). The symbiotic relationship between orchids and mycorrhizal fungi can be directly or indirectly influenced by the physicochemical properties of the soil and the structure of the plant community in their natural environment(Xu-Ge et al., 2014). Endophytic fungi play a crucial role in the long-term co-evolution with host plants, promoting plant growth through mechanisms such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and IAA production(Allen et al., 2007). Orchidaceae exhibit an exceptionally high diversity of species and ecotypes(Hans, 2014). However, an increasing number of orchid species are experiencing a sharp decline(Gao, 2015). In addition to the factors of illegal digging and trade(MF., 2015), habitat destruction (such as logging, fires, road construction, and agricultural expansion) and environmental degradation (including climate change, soil erosion, and droughts) have resulted in a significant decline in orchid populations(Pereira et al., 2010). Cymbidium goeringii and Cymbidium faberi are two significant genera within the Orchidaceae family, prized for their high ornamental, cultural, and economic value. However, their wild populations are on the brink of depletion due to overcollection, reproductive challenges, and limited habitat availability. In response to the global surge in demand for plants with distinct ornamental traits, many countries have begun to develop and trade orchid resources, including germplasm with unique colors, morphologies, fragrances, and resistance characteristics(Archana et al., 2020). The expansion of production scale and the exchange of germplasm have contributed to the protection and development of orchids to some extent. It has been demonstrated that conservation technologies and scientific research on wild orchids play a crucial role in promoting the rapid and sustainable growth of the orchid industry, yielding significant economic, ecological, and social benefits. Currently, research on orchids primarily focuses on germplasm resources(Xingchen et al., 2020), rapid breeding through histoculture(Jinao et al., 2021), molecular breeding(Fei and Liu, 2019), genetic diversity analysis(Xiao-yu et al., 2022), and the analysis of root-associated endophytic microbial diversity(Jianxin et al., 2023). The wild populations of Cymbidium goeringii and Cymbidium faberi , along with the characteristics of their native habitats, have not been thoroughly and systematically studied. Soil fungal communities in these native habitats are crucial environmental factors influencing the growth and development of orchids; however, research in this area remains limited. Therefore, studying the rhizosphere and non-rhizosphere soil microbial communities, as well as culturable endophytic fungi in the wild populations of Cymbidium goeringii and Cymbidium faberi , is of great significance. The wild populations of Cymbidium goeringii and Cymbidium faberi from the same forest area in the Qinling Mountains were selected as the research subjects. We isolated and identified endophytic fungi from the roots of these two orchids and analyzed the diversity of culturable fungi in their roots in the Qinling Mountains. Using high-throughput sequencing technology, we examined rhizosphere and non-rhizosphere soil microorganisms within their habitat, identifying the characteristics of the soil fungal community structure and functional groups. Additionally, we explored the impact of soil chemical factors on the structure of the soil fungal community. This study provides both a theoretical and scientific foundation for the protection and restoration of orchid populations, the development of efficient artificial cultivation and propagation systems, and the sustainable growth of the orchid industry. 2 Materials and Methods 2.1 Plot setting In mid-October 2020, a natural native distribution site of Cymbidium goeringii and Cymbidium faberi (species commonly found together) was identified in Zhenan County, Shangluo City, Shaanxi Province, within the Qinling Mountains (33° 43’N, 109° 15’E). Based on the natural distribution, three sampling plots were randomly selected. In each plot, seedlings with the same age, optimal growth, and good health were chosen, with a minimum of six plants of Cymbidium goeringii and Cymbidium faberi selected. The roots of these orchids were carefully excavated, and the soil loosely adhering to the roots was shaken off and classified as non-rhizosphere soil after removing animal residues and dead leaves. The soil adhering to the root surface within a 2-mm radius was gently brushed off using a sterile small brush and categorized as rhizosphere soil. Both soil types were then placed into sterile bags, temporarily stored in an ice box, and transported to the laboratory for further processing within 24 hours. Additionally, five composite soil samples were randomly collected from each plot. Rhizosphere and non-rhizosphere soil samples were sieved through a 2-mm mesh, and each sample was divided into two parts. One part was naturally dried for the determination of soil physicochemical properties, while the other was stored at -80°C for the extraction of total fungal DNA from both rhizosphere and non-rhizosphere soils for high-throughput sequencing analysis. Table 1: Material and Origin 1-1 C. faberi 34 East 1170 Open forest, mid-slope position, Crustacea trees, Lacertidae shrubs associated 1-2 C. faberi 30 East 1170 Open forest, mid-slope position, Crustacea trees, Lacertidae shrubs associated 1-3 C. faberi 39 East 1170 Open forest, mid-slope position, Crustacea trees, Lacertidae shrubs associated 2-1 C. goeringii 30 Northwest 1170 Open forest, mid-slope position, Crustacea trees, Lacertidae shrubs associated 2-2 C. goeringii 29 Northwest 1160 Open forest, mid-slope position, Crustacea trees, Lacertidae shrubs associated 2-3 C. goeringii 29 Northwest 1170 Open forest, mid-slope position, Crustacea trees, Lacertidae shrubs associated Note: In order to protect the wild populations of Cymbidium goeringii and Cymbidium faberi in the Qinling Mountains, the latitude and longitude coordinates of the collection sites have been withheld. 2.2 Determination of soil chemical properties We refer to Soil Sampling and Methods of Analysis for the determination of soil chemical properties(Carter and Gregorich, 2007). Soil pH was measured using a pH meter with a soil-to-deionized water ratio of 1:2. The organic matter content was determined using the potassium dichromate volumetric method. Soil nitrate nitrogen and ammonium nitrogen contents were measured through 1 mol/L KCl leaching (flow assay). Total soil nitrogen content was determined by the Kjeldahl method. Soil available phosphorus content was assessed using sodium bicarbonate leaching, followed by molybdenum-antimony colorimetry. Available potassium was quantified through ammonium acetate leaching using the flame photometric method(Shidan, 2000). 2.3 ITS sequencing of soils Soil DNA was extracted using the E.Z.N.A™ Mag-Bind Soil DNA Kit (Omega, USA), and DNA concentration and purity were assessed using the Qubit 3.0 DNA Assay Kit. High-quality DNA was then amplified using ITS universal primers, ITS1 and ITS4, to analyze the fungal microbial communities in the soil. The first round of PCR amplification followed these conditions: 94°C for 3 minutes, then 94°C for 30 seconds, 45°C for 20 seconds, and 65°C for 30 seconds for 5 cycles, followed by 94°C for 20 seconds, 55°C for 20 seconds, and 72°C for 30 seconds for 20 cycles, with a final extension at 72°C for 5 minutes and a hold at 10°C. The second round of amplification was performed under the same conditions as the first PCR round. Detection was carried out by 2% agarose gel electrophoresis (150 V for 2 minutes), and DNA sample concentrations were quantified using a Qubit 3.0 fluorescence quantifier. Sequencing was performed by Bioengineering. 2.4 Isolation and identification of root endophytic fungi Root segments that were observed to be infested with mycelium under a microscope were selected for surface sterilization and homogenization in an ultra-clean bench. A 100 µL aliquot of tissue homogenate was transferred onto PDA medium, with 5 replicates per group, and incubated at 28°C in a light-protected constant temperature incubator. To sterilize the root segments, they were treated with 75% ethanol followed by a 5% sodium hypochlorite solution. The sterilized root segments were then cut into approximately 6 mm pieces and ground in a low-temperature environment. Root segments showing mycelial infection, as observed microscopically, were cultured on PDA medium and subcultured 2-3 times until a single fungal colony appeared. The fresh mycelium was incubated in the dark for 5-7 days, after which DNA was extracted from both rhizosphere and non-rhizosphere soils. DNA concentration and purity were subsequently assessed. The fungal universal primers ITS-1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’) were employed to amplify high-quality DNA strains(Shimiao et al., 2023). The PCR amplification program consisted of an initial denaturation at 98°C for 3 minutes, followed by 35 cycles of denaturation at 98°C for 10 seconds, annealing at 53°C for 10 seconds, and extension at 72°C for 1 minute. The PCR products, exhibiting clear, distinct, and sharp electrophoretic bands, were sent for sequencing to Beijing Kengke Biotechnology Co., Ltd. The sequencing results were then analyzed using BLAST on NCBI to determine the taxonomic classification of each strain. 2.5 Assay of growth-promoting ability (GPA) The purified fungi were inoculated onto Ashby solid medium and incubated at 28°C for 7 days, with three replicates for each fungal strain. Subculturing was performed three times. If a strain exhibited normal growth on Ashby solid medium, it was considered to possess nitrogen-fixing ability. For potassium solubilization testing, the strains were inoculated onto potassium solubilization medium, with three replicates per strain. These cultures were incubated at 28°C for 7 days. The presence of hyaline rings around the colony edges was observed, as this indicates the strain’s ability to solubilize potassium. The strength of this ability was quantified by calculating the D/d ratio. The secretion of IAA by the strain was assessed using the method described by Glickmann E. (Glickmann and Dessaux, 1995). The assay method for determining the dissolved phosphorus of the strain was based on the procedure described by Yang Gang(Gang et al., 2020). 2.6 data analytics Data collection and processing were performed using Excel 2020, while plotting was conducted with Origin 2022. Statistical analysis for differences was carried out using SPSS 26 software (p<0.05). Diversity and abundance indices were calculated using QIIME software. Box plots, heat maps, and PCoA diagrams were generated using R software. Redundancy analysis (RDA) of dominant bacteria and soil physicochemical factors was performed using Canoco 5.0 software. Fungal community functions were annotated using the FunGuild database. 3 Results and analysis 3.1 Analysis of rhizosphere and non-rhizosphere soil fungal communities of C. goeringii and C. faberi To gain a deeper understanding of plant-microbial interactions and plant adaptability, it is essential to analyze the differences in the root-associated soil fungal community structure. We investigated the fungal community structure of C. goeringii and C. faberi as well as the distinction between rhizosphere and non-rhizosphere soils at the OTU level. The results from principal coordinate analysis (PCoA) revealed that the first principal axis (PCoA1) and the second principal axis (PCoA2) contributed 19.46% and 13.76%, respectively, with a cumulative contribution rate of 33.22%. This indicated significant differences in the microbial community structure between the rhizosphere and non-rhizosphere soils of both C. goeringii and C. faberi (Fig. 1b). A comparison of the α diversity index showed that fungal diversity was higher in the rhizosphere soil of both orchids compared to the non-rhizosphere soil. The number of OTUs in the rhizosphere soil fungi of C. goeringii and C. faberi was 318, with 218 and 247 endemic OTUs, respectively, accounting for 40.67% and 43.72% of the total OTUs. This suggests that although the two orchids share a significant proportion of fungal communities, they each harbor a considerable number of unique fungal species. The number of rhizosphere OTUs in C. faberi was higher than in C. goeringii , indicating that C. faberi may establish a closer symbiotic relationship with a more specific set of fungal communities. Fig 1: Soil Fungal Community Structure in the Rhizosphere of C. goeringii and C. faberi Note: a. The Venn diagram illustrates the shared and unique fungal OTUs between C. goeringii and C. faberi in rhizosphere (R) and non-rhizosphere (NR) soils. Four colored circles represent distinct soil samples: C. goeringii non-rhizosphere (C-NR), C. goeringii rhizosphere (C-R), C. faberi non-rhizosphere (H-NR), and C. faberi rhizosphere (H-R). The diagram presents the number of OTUs in each sample and their intersections. b. Principal coordinate analysis (PCoA) reveals the differences in fungal community structure between C. goeringii and C. faberi across rhizosphere and non-rhizosphere soil samples, with each point representing an individual sample. c. The heatmap displays the relative abundance of each fungal group across various soil samples, with color intensity corresponding to abundance levels. The horizontal axis represents different samples, while the vertical axis lists distinct fungal groups. d. The stacked histogram shows the relative abundance of Ascomycetes and Basidiomycetes in the soil samples of C. goeringii and C. faberi . e. Stacked bar charts illustrate the proportions of the ten most abundant fungal groups in rhizosphere versus non-rhizosphere soils of C. goeringii and C. faberi . Each bar corresponds to a specific soil sample, with different colored segments representing different fungal groups. The structural analysis of fungal communities in the rhizosphere and non-rhizosphere soils of C. goeringii and C. faberi revealed that Ascomycota and Basidiomycota were the dominant fungal phyla common to both orchids’ soils at the phylum level (Fig. 1c). The relative abundance of Ascomycota was ranked as follows: C. faberi non-rhizosphere > C. goeringii non-rhizosphere > C. goeringii rhizosphere > C. faberi rhizosphere, with percentages of 58.54%, 55.10%, 40.97%, and 33.76%, respectively. The relative abundance of Basidiomycota was ranked as C. faberi rhizosphere > C. goeringii rhizosphere > C. goeringii non-rhizosphere > C. faberi non-rhizosphere, with values of 58.54%, 55.10%, 40.97%, and 33.76%, respectively (Fig. 1d). Ascomycota was most abundant in the non-rhizosphere soil of C. faberi , followed by C. goeringii non-rhizosphere, C. goeringii rhizosphere, and C. faberi rhizosphere. This suggests that Ascomycota may be better suited to performing ecological functions in non-rhizosphere environments, potentially related to organic matter decomposition processes in these soils. In contrast, Basidiomycota was more abundant in rhizosphere soils than in non-rhizosphere soils, particularly in C. faberi . This supports the hypothesis that Basidiomycota play a crucial role in establishing symbiotic relationships with plant roots. At the genus level, the top 10 genera in the soils of C. goeringii and C. faberi included Filamentum, Rubra, Dendrospora, Crassicorpora, and Trichoderma, among others. The relatively high abundance of Trichoderma in both rhizosphere and non-rhizosphere soils (4.51% and 4.03%, respectively) suggests that Trichoderma plays a significant role in the soil environment of C. goeringii , likely contributing to enhanced plant stress resistance and promoting healthy growth. Filamentum and Russulaceae were more prevalent in the rhizosphere soil of C. faberi (33.50% and 20.62%, respectively), indicating that these fungi may have stronger interactions or form symbiotic or mutualistic relationships with the root system of C. faberi . In contrast, C. goeringii exhibited lower abundances of these genera, which may reflect distinct adaptation strategies of the two orchids to their respective soil environments and microbial resources. Additionally, Mycosymbioces accounted for 4.89% of the microbial community in the C. faberi rhizosphere, while its abundance was less than 0.10% in other samples. This marked increase in the rhizosphere suggests that Mycosymbioces may play a role in the root-associated microbiota of C. faberi . 3.2 Analysis of ecofunctional groups of soil fungi in the rhizosphere of C. goeringii and C. faberi The ecological functions of rhizosphere soil fungi, whether directly or indirectly, influence the growth and development of plants. Using the FunGuild database, we identified the nutrient types of rhizosphere and non-rhizosphere soil symbiotic fungal communities in C. goeringii and C. faberi and analyzed the known functional fungal OTUs. The predominant soil symbiotic fungi types in the rhizosphere of both orchids were Symbiotroph, Pathotroph-Saprotroph, Saprotroph, Pathotroph-Saprotroph-Symbiotroph, and Saprotroph-Symbiotroph hybrids. In the rhizosphere soil ecological niches of the spring orchids, Symbiotroph and Pathotroph-Saprotroph-Symbiotroph dominated, accounting for 56.76% and 22.44% of the fungal community structure, respectively. The primary fungal genera, Rubescens and Cenococcum , were relatively abundant in the rhizosphere soil. The Saprotroph-Symbiotroph group, accounting for 7.83% of the fungal community structure, consisted of Myxotrichaceae and Helotiaceae . There were relatively few Saprotroph and Pathotroph fungi in the rhizosphere soil of C. goeringii , suggesting that the orchid may possess a higher pathogen defense capacity or that its rhizosphere environment is less conducive to the growth of such fungi. The dominant fungal type in the rhizosphere soil niche of C. faberi was Pathotroph-Saprotroph-Symbiotroph, comprising 79.17% of the community structure, with Agaricales being the primary fungal group. This indicates that C. faberi may have established a relatively strong symbiotic relationship with its soil symbiotic fungi, which plays a crucial role in nutrient absorption and plant growth. In non-rhizosphere soil, significant differences in fungal community structure were observed between the two species. The functional groups of rhizosphere soil fungi in both C. goeringii and C. faberi were primarily Ectomycorrhizal and other functional groups, including Wood Saprotroph and Undefined Saprotroph, among others. This further highlights the diversity of rhizosphere soil fungi. Fig 2: Nutrient Strategies of Rhizosphere Soil Fungal Communities in C. goeringii and C. faberi Note: Using the FunGuild database, we explored the distribution of nutrient strategies within the rhizosphere (R) and non-rhizosphere (NR) soil fungal communities of Cymbidium spp. and Habenaria spp. 3.3 Effects of soil chemical factors on dominant fungal communities Different soil chemical factors, such as pH, organic matter content, and nutrient status, can influence nutrient uptake, disease resistance, and the adaptability of plants by shaping their associated fungal communities. Redundancy analysis (RDA) was employed to investigate the relationship between dominant fungal communities and soil chemical factors in orchid soil samples. The results revealed that the content of total nitrogen and ammonia nitrogen had a significant positive impact on fungal community composition, suggesting that nitrogen availability may be a key factor influencing the structure of soil fungal communities. Additionally, pH was found to play a crucial role in shaping fungal diversity and community structure, with its effects being opposite to those of nitrogen-related factors. The distribution of fungal communities along the RDA1 axis indicated that different fungal phyla exhibit varying adaptability to soil chemical conditions. For instance, Basidiomycota and Glomeromycota showed strong associations with organic matter (OM) and exchangeable potassium (K), which may relate to the role of these fungi in soil nutrient cycling, such as decomposing organic matter and influencing plant mineral nutrient uptake.The distribution of rhizosphere and non-rhizosphere soil samples from C. goeringii and C. faberi revealed that their respective sample points were dispersed in the RDA space, indicating significant differences in the effects of the rhizosphere and non-rhizosphere environments on fungal community structure. The sample points of C. goeringii were more strongly associated with nitrate nitrogen and available phosphorus, while C. faberi exhibited a stronger correlation with total phosphorus (TP) and organic matter (OM). In conclusion, soil chemistry significantly influenced the composition of fungal communities in both rhizosphere and non-rhizosphere soils of the two orchids, which may further impact the composition of mycorrhizal fungi in these orchids. Fig 3: Correlation Analysis of Soil Chemical Factors and Dominant Fungal Communities in Two Orchids Note: Redundancy analysis (RDA) plots illustrate the relationship between dominant fungal communities and soil chemical factors in both rhizosphere (R) and non-rhizosphere (NR) soil samples of Cymbidium sp. and Habenaria sp. Different colored points represent distinct soil sample types: C. goeringii rhizosphere (CR, purple), C. goeringii non-rhizosphere (CNR, blue), C. faberi rhizosphere (HR, pink), and C. faberi non-rhizosphere (HNR, orange). Arrows on the plots indicate the direction and strength of the influence exerted by various physicochemical factors on the composition of fungal communities, including total nitrogen (TN), ammonia nitrogen (NH3-N), nitrate nitrogen (NH4-N), exchangeable potassium (K), total phosphorus (TP), available phosphorus (AP), organic matter (OM), pH, and total carbon (TC). 3.4 Isolation and identification of endophytic fungi from the roots of C. goeringii and C. faberi The isolation and characterization of endophytic fungi from orchid roots offer valuable insights into the symbiotic relationships between these fungi and their orchid hosts. A total of 17 fungal strains were isolated from the roots of C. goeringii and C. faberi in the Qinling Mountains using the dilution plating method. Among these, 2 strains were isolated from C. goeringii , while 15 strains were isolated from C. faberi (Fig. 4). The colonies were primarily white and grayish-brown in color, with a texture predominantly fluffy and felt-like. Notable morphological features included circular concentric circles, radial patterns, and folds. The morphological characteristics of the 17 strains are summarized in Table 2. The two endophytic fungi isolated from the roots of C. goeringii were classified into the genera Aspergillus (family Eurotiaceae) and Tubercularia (family Tuberculariaceae) within the Deuteromycota phylum. Of the 15 strains isolated from C. faberi , they were classified into 3 phyla, 5 classes, 7 orders, 8 families, and 8 genera. Among these, Fusarium was the dominant genus, with an isolation frequency of 43.48%. The highest isolation frequencies were observed for Hemicycetes (47.83%) and Ascomycetes (34.80%). Seven genera of common endophytic fungi were identified in the roots of C. faberi , with Beauveria , Irpex , Trametes , and Acrocalymma exhibiting isolation frequencies of 8.70%. Additionally, Trichoderma , Cladosporium , and Chaetomium were isolated at frequencies of 4.35%. Fig 4: 17 strains isolated from C. goeringii and C. faberi Table 2: Colony Morphological Characteristics 1 HL01 dark green subcircular tabular tapetal, radiant growth 2 HL02 white subcircular high in the center, thin at the edges tapetal, concentric rings 3 HL03 white subcircular high in the center, thin at the edges villiform、aerial mycelium 4 HL04 white circle high in the center, thin at the edges villiform 5 HL05 white circle high villiform 6 HL06 white circle high tapetal, radiant growth 7 CL07 turquoise,white rim subcircular tabular tapetal, wrinkled 8 HL08 taupe subcircular high in the center, thin at the edges tapetal, wrinkled 9 HL09 brown subcircular tabular tapetal, wrinkled 10 HL10 white subcircular tabular villiform 11 HL11 white circle tabular in the center, thin at the edges villiform、radiant growth 12 HL14 white subcircular tabular apetal, concentric rings 13 HL15 taupe、white subcircular high in the center, thin at the edges Villiform, wrinkled 14 HL16 white circle high villiform 15 HL17 taupe、White circle high Villiform, concentric rings 16 CL21 pale yellow circle high villiform 17 HL23 white subcircular tabular tapetal, radiant growth Note: The initial letters of the strain numbers correspond to the isolated plant species: ”CL” represents Cymbidium goeringii , and ”HL” represents Cymbidium faberi . 3.5 Determination of endophytic fungal growth-promoting ability The growth-promoting mechanisms of plant endophytic fungi are complex. These fungi can directly stimulate plant growth by synthesizing plant hormones such as indole-3-acetic acid (IAA) and gibberellins. Additionally, they can indirectly influence plant growth and development through processes such as nitrogen fixation, phosphorus solubilization, and potassium solubilization(Júlia et al., 2004). The growth-promoting abilities of 17 strains of root endophytic fungi from Cymbidium goeringii and Cymbidium faberi were assessed, including their IAA secretion, nitrogen fixation, phosphorus solubilization, and potassium solubilization capabilities. Using the Salkowski colorimetric method, 16 fungal strains were identified as capable of secreting IAA, namely HL01, HL02, HL03, HL04, HL05, HL06, CL07, HL08, HL09, HL10, HL11, HL14, HL15, HL16, HL17, and CL21. The IAA production ranged from 0.08 to 32.68 μg/mL. In the IAA qualitative assay, strain HL03 from the no tryptophan test group produced the highest amount of IAA, at 12.47 μg/mL, while strain CL07 from the tryptophan-supplemented group secreted the highest IAA level, reaching 32.68 μg/mL. These results align with previous studies demonstrating that IAA synthesis is enhanced under tryptophan mediation(Ignatov, 1999, Mehmood et al., 2019, Aly et al., 2021). Using Ashby medium, 11 fungal strains were screened for normal growth, indicating their nitrogen fixation ability. These strains included HL01, HL02, HL04, HL06, CL07, HL08, HL15, HL16, HL17, HL20, and HL21. Four strains, namely HL01, HL02, HL06, and HL17, were initially screened using NBPIR solid medium for their phospholysis capacity, exhibiting D/d ratios ranging from 1.12 to 2.76. The available phosphorus content in the culture medium was determined using the molybdenum-antimony resistance colorimetric method. The results indicated that the phosphorus solubilizing capacity followed the order HL01 > HL02 > HL06 > HL17, with available phosphorus contents of 210.32, 185.57, 50.20, and 2.26 μg/mL, respectively. Although the available phosphorus content in strain HL01 was higher, its D/d ratio was lower than that of HL06 (D/d = 2.76). This suggests that the phosphorus dissolution zone can serve as a primary screening indicator, but the molybdenum-antimony resistance colorimetric method is necessary for quantitative analysis. Additionally, two fungal strains, HL08 and HL23, formed transparent zones on potassium solubilizing medium, indicating their potassium solubilization ability, with D/d ratios of 1.11 and 1.14, respectively. In summary, strains HL01, HL02, HL06, and HL17 demonstrated IAA production, nitrogen fixation, and phosphorus solubilization capacities, while strain HL08 exhibited IAA production, nitrogen fixation, and potassium solubilization abilities. Strains HL04, CL07, HL15, HL16, and CL21 displayed two growth-promoting abilities, namely IAA production and nitrogen fixation. Table 3: Summary of Fungal Growth-promoting Ability HL01 + + + - HL02 + + + - HL03 + - - - HL04 + + - - HL05 + - - - HL06 + + + - CL07 + + - - HL08 + + - + HL09 + - - - HL10 + - - - HL11 + - - - HL14 + - - - HL15 + + - - HL16 + + - - HL17 + + + - CL21 + + - - HL23 - - - + 4 Conclusion and discussion 4.1 Differences exist in the structure of fungal communities in the root systems of wild C. goeringii and C. faberi in the Qinling Mountains In this study, we compared the fungal community’s diversity in the rhizosphere and non-rhizosphere soils of wild C. goeringii and C. faberi from the same forested area in the Qinling Mountains. The results revealed that fungal diversity in the rhizosphere soil of both orchid species was generally higher than that in the non-rhizosphere soil. This finding is consistent with the research by Cao Yongchang et al.(Yongchang et al., 2017) on the autumn rhizosphere microbial diversity of Pinus tabulatus , Pinus huashanensis , and spruce in the Qinling Mountains. Root exudates, leaching, and other plant-root activities can contribute to variations in microbial community structures between rhizosphere and non-rhizosphere soils(LipariniPereira et al., 2005), further highlighting the pivotal role of the rhizosphere microenvironment in shaping soil microbial diversity. By analyzing the OTU-level data, we found that although both orchid species share a portion of the fungal community, each also harbors a substantial number of unique fungal species. Notably, the proportion of endemic fungi in the rhizosphere soil of C. faberi was higher than that of C. goeringii . This suggests that C. faberi may harbor a more diverse fungal community, potentially due to the secretion of distinct chemical compounds by its root system, which could be more effective in attracting and sustaining a wide variety of microbial communities. Phylum-level analysis revealed that Ascomycota and Basidiomycota were the dominant fungal phyla in the orchid root soils. The relative abundance of Ascomycota was highest in the non-rhizosphere soil of C. faberi , while Basidiomycota predominated in its rhizosphere soil. It has been established that Ascomycota and Basidiomycota typically dominate soil ecosystems, playing key roles in organic matter decomposition and forming mycorrhizal symbioses with plants(Manici et al., 2024). Studies have demonstrated that Trichoderma and Mycosymbioces play significant roles in C. goeringii and C. faberi , respectively, at the genus level. In terms of nutrient types, C. goeringii was primarily dominated by Symbiotroph and Pathotroph-Saprotroph-Symbiotroph, while C. faberi exhibited dominance by Pathotroph-Saprotroph-Symbiotroph. The symbiotic relationships between the two orchids have important implications for plant nutrient acquisition and overall growth. Redundancy analysis further revealed that different fungal communities exhibit varying degrees of adaptability to soil chemical conditions. The fungal community associated with C. goeringii showed greater sensitivity to nitrate nitrogen and available phosphorus, while the fungal community associated with C. faberi demonstrated a stronger correlation with total phosphorus (TP) and organic matter (OM). 4.2 Functional diversity characterizes the endophytic fungi of wild C. goeringii and C. faberi in Qinling Mountains According to Huang Min et al.(Min et al., 2022), at least 24 genera (12.8%) of fungi are shared between endophytic and soil fungal communities, suggesting that some endophytic fungi may originate from the rhizosphere soil. This transfer from the rhizosphere to the internal plant tissues may have significant implications for nutrient absorption, disease defense, and the plant’s environmental adaptation. Some soil fungi are capable of penetrating the root epidermis and colonizing internal tissues after prolonged contact with plant roots. This process not only fosters the symbiotic relationship between orchidaceae plants and fungi but may also influence plant growth performance and ecological adaptation strategies(Jie et al., 2019). Mycorrhizal fungi of the Orchidaceae family are a group of fungi that establish a symbiotic or unidirectional positive relationship with the roots or rhizoid structures of orchid species(Meina et al., 2021). Thus, root endophytic fungi are capable of colonizing and being reisolated as orchid mycorrhizal fungi within plant roots following isolation, culture, and subsequent symbiotic reestablishment (Man-Yun, 2017). Through isolation and characterization, two and fifteen endophytic fungal strains belonging to nine genera were obtained from the root systems of wild C. goeringii and C. faberi in the Qinling Mountains, respectively. Functional assays revealed that these fungi exhibit beneficial properties, including IAA production, nitrogen fixation, phosphorus solubilization, and potassium solubilization. We isolated Aspergillus(Jian-bing et al., 2011) and Fusarium(Xiao-guo et al., 2021) from the roots of C. goeringii , and isolated Beauveria bassiana, Harodon, Embolus(Fan et al., 2017), Acrocalymma, Trichoderma(Zhou et al., 2021), cladospora(Ling-Ling et al., 2019) and Trichochium(JIN-TANG and -XING, 1989) from the roots of C. faberi . These bacterial genera were further demonstrated to be symbiotic with orchids and may play a crucial role in orchid growth. The primary mycorrhizal fungal groups commonly reported in orchids, such as Coleomycete s , were not isolated in this experiment. This may be due to their strong host specificity, as they likely form orchid mycorrhizae (OM) with Cypripedium and Paphiopedilum(Dressler and Rasmussen, 1996, Xijun and Fengrong, 2018). Factors such as the isolation method, sterilization conditions, and the type of medium can also influence the isolation efficiency and stability to some extent(Fang and Chun-ying, 2013). Some Aspergillus phosphorous-solubilizing fungi are capable of converting insoluble phosphorus in the soil into bioavailable phosphate components, thereby providing an enriched source of phosphate fertilizer for plants(KHAN et al., 2007, Chi, 2013). from ancient acacia leaves solubilized the highest concentrations of Ca₃(PO₄)₂ phosphorus, ranging from 941.22 to 1238.28 mg/L(Jiaojiao et al., 2016). Zhan Shoufa et al. isolated Aspergillus awamori and Aspergillus niger from the endophytic fungi of the dominant fern Manzanita in a potassium-mining area. Both species demonstrated efficient capabilities in phosphorus solubilization, potassium solubilization, and IAA secretion, consistent with the results of this experiment(Shou-fa et al., 2017). n the experiment, a trend was observed where the pH decreased as the effective phosphorus content increased. Similarly, Ren Qingxin et al. identified a positive correlation between available phosphorus and organic acid content through Pearson analysis, noting that the pH of the bacterial solution decreased with the growth of strain WR1-4(Qing-Xin et al., 2022). In summary, the growth-promoting capabilities of the 17 strains indicate that they hold potential as biofertilizers or bioaugmenters. These strains could be applied to enhance soil quality and promote orchid growth, thereby improving both the survival rate and quality of cultured orchids. The results of this study offer new insights into the interactions between C. goeringii and C. faberi and soil microorganisms, providing valuable information for the cultivation and conservation of orchids. Author Contributions Siyu Wen: conceptualization, investigation, sample collection, experiment, data curation and visualization, formal analysis, and writing-original draft preparation. Xinying Hao: Writing -original draft preparation, review, and editing Junyang Song: conceptualization, methodology, investigation, supervision, project administration, resources, and initial funding. All authors have read and agreed to the published version of the manuscript. This research was funded by the Xi’an Municipal Science and Technology Bureau, grant number 21NYYF0012; the Department of Science and Technology of Shaanxi Province, grant number 2024NC-YBXM-072. Acknowledgements We would like to express our gratitude to Northwest A&F University for providing the research infrastructure and various other support towards this inter-university collaborative research activity. We would like to thank professor Jing Zhang (Northwest A&F University) for providing us with the technical guidance, experimental site, and equipment. The authors would like to express gratitude to professor Jean Wan Hong Yong (Swedish University of Agricultural Sciences) for his helpful suggestions for this manuscript. Declaration of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Reference Allen H E, C M L, A K D, et al. 2007. Ecological implications of anti-pathogen effects of tropical fungal endophytes and mycorrhizae[J]. Ecology, 88(3): 550-558.Aly K a M, Eldin H S, M. A S, et al. 2021. 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Characteristics of microbial community in forest soil between rhizosphere and non-rhizosphere in summer and autumn in Qinling Mountains, China[J]. Acta Ecologica Sinica, 37(05): 1667-1676.Yue G, Shun-Xing G, Xiao-Ke X 2019. Fungal diversity and mechanisms of symbiotic germination of orchid seeds: a review[J]. Mycosystema, 38(11): 1808-1825.Zhou J, Xie T, Liu J, et al. 2021. Community structure and biological function of the root symbiotic fungi of wild Cymbidium ensifolium[J]. Acta Microbiologica Sinica, 61(07): 2136-2153. Data Accessibility Statement: In order to ensure the transparency, reproducibility, and broad utilization of our research findings, we hereby provide a comprehensive data accessibility statement for the dataset supporting our study. The dataset utilized in this research encompasses a wide range of variables pertinent to Microbial gene sequence, and has been meticulously curated to ensure its accuracy and completeness. The data have been collected through Sequencing company, and have undergone rigorous quality control procedures to address potential issues such as missing values, outliers, and measurement errors. To facilitate data accessibility, the dataset has been deposited in a reputable data repository The dataset is publicly available which allows for reuse, distribution, and adaptation of the data, provided that appropriate credit is given to the original authors and source. We encourage researchers, scholars, and practitioners in related fields to utilize this dataset for further analysis, validation of our findings, and to address new research questions. We are committed to responding promptly to any inquiries or requests for additional information or clarification regarding the dataset. In conclusion, by making our dataset publicly accessible, we aim to contribute to the advancement of knowledge in microorganism and to foster an open and collaborative research environment. Benefit-Sharing Statement: Benefits Generated: Benefits from this research accrue from the sharing of our data and results on public databases as described above. Information & Authors Information Version history V1 Version 1 13 January 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords agriculture bioinfomatics/phyloinfomatics fungi host parasite interactions microbial biology Authors Affiliations Siyu Wen Northwest A&F University View all articles by this author Xinying Hao Northwest A&F University View all articles by this author Junyang Song 0000-0002-3529-8113 [email protected] Northwest A&F University View all articles by this author Metrics & Citations Metrics Article Usage 313 views 123 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Siyu Wen, Xinying Hao, Junyang Song. Characteristics of the Rhizosphere Fungal Community and Isolation and Identification of Root Endophytes from Wild Cymbidium goeringii and Cymbidium faberi in the Qinling Mountains of China. Authorea . 13 January 2025. DOI: https://doi.org/10.22541/au.173676459.96504541/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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