Analysis of the Endophytic Fungi Community Structure and Function of Stephania tetrandra Based on High-throughput Sequencing

Analysis of the Endophytic Fungi Community Structure and Function of Stephania tetrandra Based on High-throughput Sequencing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of the Endophytic Fungi Community Structure and Function of Stephania tetrandra Based on High-throughput Sequencing Lijun Yang, Shaogang Li, Qian Cao, Han Gao, Wei Luo, Qiong Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6691995/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Analyzing the endophytic fungi in various Stephania tetrandra tissues is crucial for investigating strain resources, enhancing S. tetrandra cultivation, and carrying out further studies. In order to investigate the variety, community structure, and function of endophytic fungus in various tissues, the three distinct tissues of S. tetrandra 's roots, stem, and leaves were sequenced and analyzed using Illumina MiSeq high-throughput sequencing technology. Findings: 539 OTUs and 212,783 valid sequences throughout 11 phyla, 29 classes, 64 orders, 135 families, 222 genera, and 286 species were found. The major phylum and genus were Ascomycota and Cladosporium . Eight functional ecological groups were found in the endophytic fungi of S. tetrandra , according to the FUNGuild software analysis. The undefined functional groups made up a comparatively large percentage of the various tissues of S. tetrandra , which required more research. This work established a foundation for the development and use of S. tetrandra resources by revealing the diversity and structure of endophytic fungi in the roots, stems, and leaves of various S. tetrandra tissues. endophytic fungal diversity function prediction high-throughput sequencing organizational differences Stephania tetrandra Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The root of Stephania tetrandra is its therapeutic component; this information was initially documented in " Shennong's Herbal Classic ." It is well-known for its detumescence and diuresis, which help to expel wind and ease pain [ 1 ] .The investigation of S. tetrandra 's chemical makeup and potential usefulness in medicine are currently the primary areas of study. A class of fungus known as endophytic fungi inhabits plants for a specific amount of time or at all stages and does not clearly harm their host plants [ 2 ] .Research on its pharmacological effects and therapeutic components has long been popular. Endophytic fungi and host plants have a special and intimate symbiotic relationship in the intricate system of plant-environment interaction, which has a significant impact on the physiological and biochemical traits, growth and development process, and environmental adaptability of plants [ 3 ] . The detection range and resolution constraints of conventional microbial research methods can be overcome by high-throughput sequencing technology, which can thoroughly examine complex microbial communities with high precision and high sensitivity [ 4 ] . In addition to shedding light on the internal mechanism of plant-microbe interaction, a thorough study of the endophytic fungal community structure in S. tetrandra is crucial for comprehending the organism's growth, development, and adaptability mechanisms. The quality of Chinese medicinal materials may be impacted by these endophytic fungi's role in the synthesis and control of S. tetrandra 's therapeutic components. It might also be crucial for the development of plant tolerance to biotic and abiotic stressors, which offers fresh concepts for disease prevention and ecological planting. The growth of biotechnology and the pharmaceutical sector may benefit from the discovery of novel bioactive compounds and enzymes that endophytic fungi themselves may provide.To provide a new theoretical foundation for the thorough investigation, development, and use of S. tetrandra , this study employed high-throughput sequencing technology to examine the community structure of endophytic fungi in various tissues of the species and performed functional prediction based on this information. Materials and Methods Source of Sample In Xinyang City, Henan Province, China, fresh S. texandra was gathered (GPS coordinates: 114 ° 01'-114 ° 06'E, 31 °46'-31°52'N). The experiment included samples from three different plants, with three copies for each type of sample: root samples (FG): FG1, FG2, FG3; leaf samples (FY): FY1, FY2, FY3; and stem samples (FJ): FJ1, FJ2, FJ3. Sample Surface Sterilization and Treatment Rinse the new plant's roots, stems, and leaves. Then, take 5.0 g of each tissue material, wash it three times with sterile water in an ultra-clean bench, and use sterile filter paper to absorb the moisture from the surface. After soaking in 75% ethanol for three minutes, 3% sodium hypochlorite solution for two minutes, and then 75% ethanol for thirty seconds, they were rinsed five times with sterile water, and sterile filter paper was used to absorb water from the surface of the plant tissues. To find out if the plant tissue's surface was completely cleaned, the final rinse solution was put on a nutrient agar plate and incubated for two to three days at 28°C. For the subsequent stage of DNA extraction, the cleaned samples were kept in an ultra-low-temperature refrigerator set at -80°C. DNA Extraction of Endophytic Fungi Deoxyribonucleic acid (DNA) extraction was used to extract DNA from 3.0 g of pre-treated root, stem, and leaf samples. Protease K cleavage in conjunction with phenol-chloroform was used to extract DNA. 1.2 percent agarose gel electrophoresis was used to find the isolated DNA. PCR Amplification and Sequencing For high-throughput sequencing analysis, the ITS1 region sequence was chosen. PCR amplification was done in two steps to create the library. The ITS1 region universal primers ITS1F 5' -CTTGGTCATTTAGAGGAAGTAA-3' and ITS1R 5' -GCTGCGTTCTTCATCGATGC-3', along with the fusion primers containing barcode and partial sequencing primers, were used for PCR amplification using the purified DNA as a template. The results were detected by 1.2% agarose gel electrophoresis. 2% agarose gel electrophoresis was used to recover the samples with superior detection findings. Pre-denaturation at 94 °C for two minutes, denaturation at 94 °C for one minute, annealing at 50 °C for thirty seconds, and extension at 72 °C for thirty seconds, followed by 32 cycles, 72 °C extension for five minutes, 10 °C preservation, 5 × Buffer 10 μL, 10mM dNTP 1 μL, F/R inner primer (10μM), Phusion ultra-fidelity DNA polymerase 1U 1μL, ddH2O to 50μL, template 5ng-50ng. The AxyPrep DNA gel recovery kit (AXYGEN USA) was used to recover all PCR products, and the FTC-3000TM Real-Time PCR apparatus was used to quantify them. The reaction conditions for the second PCR amplification were pre-denaturation at 94 °C for 2 minutes, denaturation at 94 °C for 1 minute, annealing at 56 °C for 30 seconds, and extension at 72 °C for 30 seconds, for a total of 8 cycles. Extension of 72 °C 5 minutes, insulation at 10 °C, 5xBuffer 8 μL, dNTP (10 mM) 1 μL, F/R outer primer (10 μM), Phusion ultra-fidelity DNA polymerase 0.8 U 1 μL, 5 μL template, 40 μL ddH2O. To finish the library assembly, the adapters, sequencing primers, and barcodes needed for Illumina platform sequencing were added to both ends of the target segment. Microbase Biotechnology (Shangzhou) Co., Ltd. used the Novaseq 6000 SP 500 Cycle Reagent Kit (Illumina USA) to sequence the created library. Data Processing Barcodes were used to assign the raw data to the sample readings, and each sample's effective sequence was determined. A 50 bp window was selected, and the low quality was initially eliminated using Trimmomatic (version: 0.38), a program used for sequence quality control. The back-end base was disconnected from the window's initial location when the average quality value was less than 20, and the read with a length less than 50 bp following quality control was filtered out. The cutadapt software (version 1.16) was used to process the primers and sequencing adaptors. Based on the overlap relationship between PE readings, splicing operations were carried out using FLASH (version 1.2.11) software, and paired reads were combined into a sequence. To filter out sequences that don't fit the criteria and guarantee the precision and dependability of the results from the ensuing analysis, the minimum overlap length is set at 10 bp, and the highest mismatch ratio permitted by the overlap region of the splicing sequence is 0.2. Quality control filtering was applied to the combined reads to improve the quality and precision of the bioinformatics analysis results. The ambiguous base (ambiguous = 0), single base high repeat region (homologous = 8), too long (maxlength = 485) and too short (minlength = 200) sequences, as well as some chimeras produced during the PCR process, were eliminated to obtain the optimized sequence because PCR amplification is prone to non-specific amplification. After filtering out the OTU-annotated species categorization, non-research object sequences, and the singletons in the spliced long reads (the sequence corresponding to a single read), the optimized sequence was used as the data for the subsequent clustering OTU analysis. UPARSE software (USEARCH Version V8.1.1756, https://drive5.com/usearch/manual8.1/uparse_pipeline.html ) was used to do OTU (operational taxonomic unit) clustering. To acquire OTU representative sequences, clustering operations with 97% similarity were carried out using UPARSE. UCHIME eliminated the chimeras that resulted from PCR amplification in the OTU representative sequence. The abundance statistics table of each sample in each OTU was created after all sequences were aligned to the OTU representative sequence using the USEARCH_global technique. The classification.seqs function in the Mothur (version: 1.39.5) software was used to match the OTU representative sequence with the UNITE database of ITS fungi to implement species annotation. A confidence threshold of 0.6 was established. Only the remaining OTUs were kept for further analysis after the species whose annotation results were outside the purview of the study and the OTUs lacking annotation results were eliminated. R (Version 3.6.3) was used to conduct statistical and visual analysis, including heatmaps, VENN plots, dilution curves, and community structure histograms. For α diversity analysis (using species richness statistics like Chao and Ace and species diversity statistics like Shannon and Simpson) and β diversity analysis (using the Bray-Curtis method based on OTU), R (version 3.6.3) language vegan was utilized. Python (2.7.13) and LEfSE (1.0) were used to investigate the variations in prevalent endophytic fungus amongst LEfSe groups. Data processing, species annotation, and classification were followed by functional prediction and annotation, functional analysis, PCoA diagram construction, and differential function analysis using LEfSE. OTU classification was added based on the phylum, family, genus, and species classification technique. The FUNGuild database was used for fungal categorization and functional analysis. It is a tool for classifying and analyzing fungal communities using ecological relationships based on data from reliable websites or currently published research [5] . Sequence Accession Numbers The NCBI (National Center for Biotechnology Information) database received the raw high-throughput sequencing data using the BioProject number PRJNA1197658 and the Biosample numbers SAMN45811727, SAMN45811728, and SAMN45811729. Results Endophytic Fungi Sequencing Results Nine test samples yielded 212,783 valid sequences in total after quality control and filtering. In total, 51,430,286 optimized bases were found, with 210,896 optimized sequences. The three sets of optimized sequences ranged in length from 200 to 300 bp. Whether the quantity of sample sequencing data is appropriate can be explained using it.This is shown in Figure 1, the dilution curves often become flat as the number of sequences rises, suggesting that the sequencing data is credible [6] . A total of 539 OTUs were found in S. tetrandra 's roots, stems, and leaves, as seen in Figure 2. There were 105 OTUs that were identical between roots and stems, 162 OTUs between roots and leaves, and 120 OTUs between stems and leaves. It is evident that the species composition of endophytic fungus differs most between stems and roots and least between stems and leaves [7] . Diversity of Endophytic Fungi in Different Tissues The richness and diversity of endophytic fungi in roots were higher than those in stems and leaves, as seen by Table 1's α diversity index, which also indicates that the Chao, Ace, and Shannon indices of these fungi were higher in roots than in stems and leaves [8] . Table 1 The four indexes with a coverage rate of 99.99% reflecting community richness and uniformity of the different samples Sample Chao Ace Shannon Simpson fraction of coverage FG 226.43±2.01 a 224.63±0.70 a 1.94±0.24 a 0.37±0.01 a 99.99% FJ 98.69±1.38 c 99.38±1.08 c 1.48±0.03 c 0.32±0.02 a 99.99% FY 172.94±0.64 b 175.24±1.33 b 1.79±0.11 b 0.27±0.01 b 99.98% The three groups of S. tetrandra root, stem, and leaf samples were clearly divided, as demonstrated by PCoA analysis(Fig.3), and the genetic distance was great. This suggests that the origins of fungal flora in the three groups were different, and there were significant differences between them. Community Structure at Phylum Level As illustrated in Figure 4(A), Ascomycota accounted for 95.58%, 95.89%, and 98.85% of the endophytic fungi found in roots, leaves, and stems, respectively. Basidiomycota accounted for 2.79% of roots and 3.59% of leaves, respectively. Mortierellomycota , Glomeromycota , Mucoromycota , Chytridiomycota , Rhodomycota , Calcarisporiellomycota , and Oleaginaceae were among the biological gene sequences found in root samples. In root samples, unclassified endophytic fungi made up 0.04%. In roots, leaves, and stems, the unclassified phylum represented for 0.33% to 1.02%. The heat map indicates that the phylum Ascomycota is the most prevalent in the fungal community, with the highest abundance in stems [9] . As illustrated in Figure 4(A), Ascomycota accounted for 95.58%, 95.89%, and 98.85% of the endophytic fungi found in roots, leaves, and stems, respectively. Basidiomycota accounted for 2.79% of roots and 3.59% of leaves, respectively. Mortierellomycota , Glomeromycota , Mucoromycota , Chytridiomycota , Rhodomycota , Calcarisporiellomycota , and Oleaginaceae were among the biological gene sequences found in root samples. In root samples, unclassified endophytic fungi made up 0.04%. In roots, leaves, and stems, the unclassified phylum represented 0.33% to 1.02%. The heat map indicates that the phylum Ascomycota is the most prevalent in the fungal community, with the highest abundance in stems [9] . Community Structure at Genus Level According to Figure 4(B), the top 5 genera in the root were Cladosporium ( 58.98 % ), Alternaria ( 13.81 % ), Others ( 7.86 % ), Selenophoma ( 6.35 % ), and Erysiphe ( 3.55 % ). The top 5 genera in leaves were Cladosporium ( 42.24 % ), Erysiphe ( 26.73 % ), Selenophoma ( 12.75 % ), Alternaria ( 8.06 % ), Others ( 2.60 % ), Nigrospora ( 2.38 % ). The top 5 genera in stems were Cladosporium ( 47.81 % ), Botryosphaeria ( 22.10 % ), Selenophoma ( 21.52 % ), Alternaria ( 2.69 % ) and Others ( 1.71 % ). According to the heat map, Cladosporium was the dominant genus of endophytic fungi in the three tissues, accounting for 58.98 %, 42.24 % and 47.81 % of roots, leaves and stems, respectively. The second was Selenastrum , but the abundance of different tissues was different, and the content was the highest in stems ( 21.52 % ). Followed by leaf ( 12.75 % ) ; it was the lowest in root endophytic fungi ( 6.35 % ). Others ( 7.86 % ) in roots and Others ( 1.71 % ) in stems did not represent any genus. In addition to Cladosporium and Sphaerothecium , the proportion of Botryosphaeria in stems was also high, accounting for 22.10 %, while the proportion in roots was very low, accounting for 0.22 %. As the dominant genus of roots and leaves, powdery mildew accounted for 3.55 % and 26.73 % of endophytic fungi in roots and leaves, respectively. Functional Prediction Analysis of Endophytic Fungi in Different Tissues The FUNGuild database was utilized to forecast the role of endophytic fungi in various S. tetrandra tissues. As illustrated in Fig. 5(A), the endophytic fungi of S. tetrandra can be categorized into eight ecological function groups: symbiotic type, saprophytic type, saprophytic-symbiotic type, pathological parasitic type, pathological parasitic-septic type, pathological parasitic-skeptic-symbiotic type, pathological parasitic-symbiotic type, and undefined. In roots, leaves, and stems, undefinable colonies made up 62.96%, 46.01%, and 50.0% of the total. Furthermore, the percentage of roots, leaves, and stems that were saprophytic was 10.50%, 15.73%, and 24.02%, respectively. In roots, leaves, and stems, pathological parasitism-septicism-symbiosis accounted for 19.63%, 8.90%, and 25.25%, respectively. The highest percentage of pathological parasites was found in leaves (27.07%), followed by roots (4.84%) and stems (0.38%). It demonstrates that a comparatively large percentage of tissues with strong development are of the pathogenic parasitic type. The fundamental functional groups—pathological parasitism-saprophytic, pathological parasitism-symbiosis, saprophytic-symbiosis, and symbiotic—are the most prevalent in addition to the four nutritional types mentioned above. With the exception of the undefined, the top three ecological function groups in the root were Animal Pathogen-Endophyte-Plant Pathogen-Wood Saprotroph (13.81%), Undefined Saprotroph (8.12%), and Endophyte-Fungal Parasite-Lichen Parasite-Plant Pathogen-Wood Saprotroph (2.77%), as shown by the Guild's detailed classification in Figure 5 (B). Plant Pathogen (26.97%), Undefined Saprotroph (15.63%), and Fungal Parasite-Litter Saprotroph (1.58%) were the top three ecological functional categories in leaves. Undefined Saprotroph (23.00%), Endophyte-Plant Pathogen-Wood Saprotroph (22.10%), and Animal Pathogen-Endophyte-Plant Pathogen-Wood Saprotroph (2.69%) were the top three ecological functional groups in stems. Discussion Analysis of Diversity and Richness of Endophytic Fungi in Different Tissues According to related research, the host plant community will exhibit diversity due to varrious plant species, organizational structures, and functions [10] . The diversity of endophytic fungi in Huperzia serrata was also studied, revealing differences in the community structure and diversity of endophytic fungi in different tissues [11] . While the diversity and spatial distribution of endophytic fungi in Cinnamomum longepaniculatum were studied, it was discovered that the richness and diversity of fungal communities in seeds were the highest, followed by branches, leaves, and roots [12] . Three distinct S. tetrandra tissues were colonized by a large number of endophytic fungi in this investigation; however, the distribution of these fungi varied per organ and tissue. The endophytic fungi in three distinct S. tetrandra tissues were examined in this study, and it was discovered that there were clear variations in the endophytic fungal dispersion by organ tissue. The Chao index (226.43 ± 2.01a), Ace index (224.63 ± 0.70a), and Shannon index (1.94) of endophytic fungi in roots were higher than those in stems and leaves, according to the α diversity index (see Table 1). This suggests that the richness and diversity of endophytic fungi in roots were higher than those in stems and leaves. The rhizosphere environment is complicated from the standpoint of the microbial colonization environment. Numerous organic materials, including sugars, amino acids, organic acids, and others, are secreted on the root's surface and surrounding area. These secretions give the rhizosphere and rhizosphere fungus enough carbon, nitrogen, and other nutrients to draw in more rhizosphere microorganisms for colonization [13–14] , which in turn causes more microorganisms to colonize the roots. They colonize both whole plants and roots after germination, and the abundance and diversity of endophytic fungi can vary depending on the properties of the soil [15] . However, some research has shown that temperature, geographic location, and other variables all affect the variety of endophytic fungus in roots [16] . Conversely, the types and amounts of nutrients that are available are restricted, and the surface of stems and leaves is comparatively smooth. Microorganisms' species richness is limited by the environmental conditions, which have higher needs for their survival adaptability. Using leaves as an example, germs enter the leaves horizontally after first colonizing their surface through stomata, water holes, lesions, etc. However, the number of microorganisms in leaves declines as a result of UV radiation, as well as a shortage of water and nutrients [17] . Numerous parasitic and coexisting connections exist between microorganisms in roots and soils when considering the interaction between microorganisms and plants. Certain fungi that promote rhizosphere growth, for instance, can have a strong relationship with roots and aid in their ability to absorb nutrients, create plant hormones, and fend off disease. More plants can participate and create a more varied microbial community thanks to these intricate network relationships. However, it is challenging to create a diversified microbial community since the microbial sources and interactions of stems and leaves are somewhat straightforward. Numerous microorganisms that resemble rhizosphere growth-promoting fungi may be present in S. tetrandra roots. These microorganisms interact with the roots of S. tetrandra and support the diversity of endophytic fungal communities. On the other hand, it is challenging to create a diversified microbial population, and the microbial sources and interactions of stems and leaves are somewhat straightforward. The roots have different tissue levels from the standpoint of niche biochemistry. The physical and chemical characteristics of each level—such as pH, oxygen concentration, osmotic pressure, and so forth—have gradient variations from the epidermis to the cortex to the vascular bundle, offering a range of habitats for various microbial populations. The variety of microbial communities that can be supported is limited since the tissue levels in stems and leaves are rather homogeneous. The large diversity of endophytic fungi in T. tetrandra roots is consistent with the fact that this diversified niche can support a greater variety of endophytic fungal species. The variety of microbial communities that can be supported is limited since the tissue levels in stems and leaves are rather homogeneous. Analysis of Endophytic Fungal Community Composition in Different Tissues The community structure of endophytic fungi in S. tetrandra 's roots, stems, and leaves was significantly different, according to cluster heat map analysis. Ascomycota and Basidiomycota are the two most prevalent phyla among Chinese mangrove ferns, making up about 75% of all strains. [18] . Because of their diverse ecological settings and host specificity, Ascomycota is regarded as one of the most prevalent fungi in the world [19] . The majority of the phylum Ascomycota , which dominated this investigation, were saprophytic fungus. It contributes significantly to the cycling of nutrients and possesses high material degradation and synthesis capabilities. Its symbiotic relationship with plants is the easiest [20] . Consequently, the metabolism and nutrition cycling of S. tetrandra 's many tissues greatly depend on this phylum. Basidiomycetes are also comparatively prevalent in the roots and leaves in this investigation. Because of their healing capabilities, basidiomycetes can restore contaminated and deteriorated soils [21] . Ascomycota and Basidiomycota are the two groups of endophytic fungi that infect higher plants the most frequently [22–23] . The predominant genus is Cladosporium , with the majority of its strains being saprophytic [24] , but Sphaerothecium also makes up a sizable fraction. Powdery mildew is more prevalent in the roots and leaves, botryosphaeria is more prevalent in the stem, and the root is quite low. During long-term co-evolution with plants, the majority of plant endophytic fungus produce metabolites that are identical to or comparable to those of their host plants. Alkaloids, flavonoids, steroids, and other metabolites are abundant in S. tetrandra [1] .Research has revealed that S. tetrandra 's predominant metabolites possess biological activities such as antioxidant and anti-tumor properties [25] . Such fungi may exist because of S. tetrandra 's abundant metabolites. Functional Prediction Analysis of Endophytic Fungi in Different Tissues The functional classes of S. tetrandra 's endophytic fungus were categorized using the FUNGuild software. Pathological parasitic type, pathological parasitic-saprophytic type, pathological parasitic-saprophytic-symbiotic type, pathological parasitic-symbiotic type, saprophytic type, saprophytic-symbiotic type, symbiotic type, and undefined were the eight ecological functional groups that were identified. The undefined functional groups made up 62.96%, 46.01%, and 50.0% of the eight functional groups in the base, leaf, and stem, respectively. On the one hand, it demonstrates the lack of thoroughness in the FUNGuild database study. It also indicates that there are many endophytic fungal resources in S. tetrandra that need to be unearthed, which is very valuable for research. In S. tetrandra , the relative abundance of saprophytic functional groups was greater than 10%, with the exception of undefined functional groups. Because saprophytic fungi break down organic matter for food, they contribute significantly to ecosystems' material cycles. The soil contains a wide variety of saprophytic fungus. The soil and the rhizosphere of S. tetrandra are intimately associated. With the help of the plants' ability to transfer nutrients, saprophytic fungi can eventually infiltrate the roots and then selectively colonize the stems and leaves. Saprophytic fungus, for instance, can break down organic materials like litter into inorganic stuff and then reintroduce it into the soil to supply nutrients for plant development in forest ecosystems. It's possible that the saprophytic endophytic fungus in S. tetrandra participate in comparable mechanisms to encourage plants to absorb and repurpose nutrients. In roots and stems, the pathoparasitic-saprophytic-symbiotic type was more than 15% abundant. The root, which is S. tetrandra 's vegetative organ, is in charge of taking in water, soluble small molecules, and inorganic ions. Both of them are nutrient-rich, and the stem contains a transport tissue for carrying nutrients. This may be why functional fungi make up a comparatively large percentage of roots and stems [29] . This type of fungus can change its mode of nourishment depending on the environment and has a number of survival strategies. In addition to obtaining nourishment through symbiosis with plants, it can also, in some circumstances, engage in saprophytic life and even become parasitic. By developing a symbiotic association with plant roots, certain mycorrhizal fungi aid in nutrient absorption while promoting saprophytic growth on plant waste. Because of this trait, they are particularly important for S. tetrandrae 's growth, development, and ecological adaptation. They may also play a role in controlling the balance of nutrients and plant physiological metabolism. Only 4.84% of roots and 0.38% of stems had the pathogenic parasite kind, which made up the largest percentage of leaves (27.07%). The leaves have a comparatively high percentage of pathogenic parasite fungus because they grow quickly and are exposed to the natural environment, which makes them more susceptible to invasion by outside diseases. It is well recognized that a variety of plant diseases, including apple anthracnose, common wheat rust, and others, are brought on by particular parasite fungi. Consequently, it is hypothesized that the pathogenic fungi that cause plant illness and pollution during tissue culture are most likely the pathological fungi seen in S. tetrandra leaves. This implies that in order to guarantee the plants' healthy growth during the planting and tissue culture processes of S. tetrandra , attention must be paid to the prevention and management of leaf diseases. Conclusion The findings demonstrated that endophytic fungi were highly diverse across all tissues, with roots having the greatest diversity and richness of endophytic fungi. The dominating genus was Cladosporium, and the major flora was Ascomycota. According to the functional prediction, there were eight functional ecological groups among the endophytic fungus, with a significant percentage of undefined functional groupings. The abundance of materials that needed to be dug up might be attributed to variations in the environment and nutrition of the tissues. The structure and diversity of endophytic fungi in various S. tetrandra tissues were demonstrated in this work, which also served as a foundation for future research and the identification of strain resources. Abbreviations OTU Optical taxonomic unit PCR Ribosomal database project PCoA Principal coordinate analysis LDA Latent Dirichlet Allocation Declarations Author Contributions LY and SL: designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. QC and HG: Make changes to the document. WL: Responsible for editing tables and icons. QC: and DH: supervised the research and critically revised the manuscript. All authors read and approved the manuscript. Acknowledgements We would like to thank Microbase Biotechnology (Shanghai) Co., Ltd. for providing the platform and technical support for high-throughput sequencing and bioinformatics analysis. Funding The research was supported by the Henan province science and technology research ( 242102311200 ) ; natural Science Foundation of Henan Province ( 232300420065 ) ; Supported by the Science and Technology Major Projects in Henan Science and Technology Agency ( 241111311400 ) ; youth Fund Project of Xinyang College of Agriculture and Forestry ( QN2023026 ). Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information fles. Conflict of interest The authors declare that they have no competing interests. Ethical Approval Not applicable. Consent to Participate Not applicable. Consent for Publication Not applicable. 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Microorganisms 7(9):332.https://doi.org/10.3390/microorganisms7090332 Pang B, Yin D, Zhai Y, He A, Qiu L, Liu Q, Ma N, Shen H, Jia Q, Liang Z, Wang D (2022) Diversity of endophytic fungal community in Huperzia serrata from different ecological areas and their correlation with Hup A content. BMC Microbiol 22(1):191.https://doi.org/10.1186/s12866-022-02605-y Zhou W, Wei Q, Feng R, Liu Y, Liang H, Li J, Yan K (2021) Diversity and spatial distribution of endophytic fungi in Cinnamomum longepaniculatum of Yibin, China. Arch Microbiol 203(6):3361-3372.https://doi.org/10.1007/s00203-021-02325-3 Zhong F, Fan X, Ji W, Hai Z, Hu N, Li X, Liu G, Yu C, Chen Y, Lian B, Wei H, Zhang J (2022) Soil Fungal Community Composition and Diversity of Culturable Endophytic Fungi from Plant Roots in the Reclaimed Area of the Eastern Coast of China. J Fungi (Basel) 8(2):124.https://doi.org/10.3390/jof8020124 Chen, M.; Chen, J.; Liu, J., Wu, M.; Yan, Q.; Li, P..Huang, L.;, Xiao, X (2021) Diversity analysis of rhizosphere soil fungi and endophytic fungi in Ampelocalamus luodianensis.Acta Ecol 41,4120-4130.https://doi.org/10.5846/stxb201912032619 Yang H, Ye W, Ma J, Zeng D, Rong Z, Xu M, Wang Y, Zheng X (2018) Endophytic fungal communities associated with field-grown soybean roots and seeds in the Huang-Huai region of China. PeerJ 6:e4713.https://doi.org/10.7717/peerj.4713 Wang B, Chen C, Xiao Y, Chen K, Wang J, Zhou G (2023) Temperature thresholds drive the biogeographic pattern of root endophytic fungal diversity in the Qinghai-Tibet Plateau. Sci Total Environ 889:164270.https://doi.org/10.1016/j.scitotenv.2023.164270 Hardoim, P.R.; van Overbeek, L.S.; Berg, G.; Pirttilä, A.M.; Compant, S.; Campisano, A.; Doring, M.; Sessitsch, A (2015) The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes. Microbiol. Mol. Biol. Rev 79, 293-320.https://doi.org/10.1128/MMBR.00050-14 Zhu H, Zeng W, Chen M, He D, Cheng X, Yu J, Liu Y, Wu Y, Yang D (2024) Endophytic Fungal Diversity of Mangrove Ferns Acrostichum speciosum and A. aureum in China. Plants (Basel) 13(5):685.https://doi.org/10.3390/plants13050685 Sharma M, Bharti S, Goswami A, Mallubhotla S (2023) Diversity, Antimicrobial, Antioxidant, and Anticancer Activity of Culturable Fungal Endophyte Communities in Cordia dichotoma. Molecules 28(19):6926.https://doi.org/10.3390/molecules28196926 Sun ZB, Li SD, Ren Q, Xu JL, Lu X, Sun MH (2020) Biology and applications of Clonostachys rosea. J Appl Microbiol 129(3):486-495.https://doi.org/10.1111/jam.14625 Traxler L, Krause K, Kothe E (2024) Basidiomycetes to the rescue: Mycoremediation of metal-organics co-contaminated soils. Adv Appl Microbiol 129:83-113.https://doi.org/10.1016/bs.aambs.2024.06.001 Jin, H.; Yang, X.; Lu, D.; Li, C.; Yan, Z.; Li, X.; Zeng, L.; Qin, B (2015) Phylogenic diversity and tissue specificity of fungal endophytes associated with the pharmaceutical plant, Stellera chamaejasme L. revealed by a cultivation-independent approach. Antonie Leeuwenhoek 108:835-850.https://doi.org/10.1007/s10482-015-0538-8 Pawłowska, J.; Wilk, M.; Śliwińska-Wyrzychowska AMętrak, M.; Wrzosek, M (2014) The diversity of endophytic fungi in the above-ground tissue of two Lycopodium species in Poland. Symbiosis 63:87-97.https://doi.org/10.1007/s13199-014-0291-1 Bensch K, Groenewald JZ, Dijksterhuis J, Starink-Willemse M, Andersen B, Summerell BA, Shin HD, Dugan FM, Schroers HJ, Braun U, Crous PW (2010) Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales). Stud Mycol 67:1-94.https://doi.org/10.3114/sim.2010.67.01 Li Y, Wang Y, Wang H, Shi T, Wang B (2014) The Genus Cladosporium: A Prospective Producer of Natural Products. Int J Mol Sci 25(3):1652.https://doi.org/10.3390/ijms25031652 Kubicek CP, Starr TL, Glass NL (2014) Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annu Rev Phytopathol 52:427-51.https://doi.org/10.1146/annurev-phyto-102313-045831 Van der Nest MA, Steenkamp ET, McTaggart AR, Trollip C, Godlonton T, Sauerman E, Roodt D, Naidoo K, Coetzee MP, Wilken PM, Wingfield MJ, Wingfield BD (2015) Saprophytic and pathogenic fungi in the Ceratocystidaceae differ in their ability to metabolize plant-derived sucrose. BMC Evol Biol 15:273.https://doi.org/10.1186/s12862-015-0550-7 Ngugi HK, Scherm H (2006) Mimicry in plant-parasitic fungi. FEMS Microbiol Lett 257(2):171-6.https://doi.org/10.1111/j.1574-6968.2006.00168.x Hou QZ, Chen DW, Wang YP, Ehmet N, Ma J, Sun K (2022) Analysis of endophyte diversity of two Gentiana plants species and the association with secondary metabolite. BMC Microbiol. 22(1):90.https://doi.org/10.1186/s12866-022-02510-4 Additional Declarations No competing interests reported. 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Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIie3Qv0sDMRTA8SeBdDnr+g7xx58QCdT63+QodArq2KHYSMHV1UH8GzJV3N5xcF1Out7Ys4tDh4OuHTSC0CnpKJjvkCG8DyQPIBb7qy0FB2Cgiu0IT4/2IsqRjmkoqfoyNXsRdyT5ig4fRpkNETF/Lz7VbTd7xYGi9AUPLLDmo/aR6nrY/35Y9vY0VHQxQ3YJXErtI6R7whFba0XZDPmVSfixlyzWv+SmpfwZE0EhUmu5/CEVqfzeIAZJWq97bsnSzo0qoESRTgN/6S60bNtteWILGGxgfDd57EyblY+cE3AEKHeumGfcdWaAtQDjwFgsFov9674AcwtV9qrBIZwAAAAASUVORK5CYII=","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":true,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Yang","suffix":""},{"id":459772969,"identity":"d54d4b00-a6d1-478c-a097-ebf4253c4c16","order_by":1,"name":"Shaogang Li","email":"","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Shaogang","middleName":"","lastName":"Li","suffix":""},{"id":459772971,"identity":"66446af4-d30b-4790-a8de-eb1ab2b8de3b","order_by":2,"name":"Qian Cao","email":"","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Cao","suffix":""},{"id":459772973,"identity":"e8b071f4-3e73-4212-a32f-cf75b4cd50a2","order_by":3,"name":"Han Gao","email":"","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Gao","suffix":""},{"id":459772975,"identity":"31a10729-bdcb-4a71-9957-4a8426a56452","order_by":4,"name":"Wei Luo","email":"","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Luo","suffix":""},{"id":459772977,"identity":"f079726f-7746-452e-b0cf-1c09f3a184a9","order_by":5,"name":"Qiong Chen","email":"","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Chen","suffix":""},{"id":459772979,"identity":"76a2398b-84ac-4c7e-b394-e70898c7be0f","order_by":6,"name":"Dingxuan He","email":"","orcid":"","institution":"Xinyang College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Dingxuan","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-05-18 13:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6691995/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6691995/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83276860,"identity":"4e9ed0bc-499d-4cac-8fc5-2ca1188585ff","added_by":"auto","created_at":"2025-05-22 09:20:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93523,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent tissue dilution curves\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/9177954d590043fa32b9abe4.png"},{"id":83276863,"identity":"965622e9-2d8f-4783-8538-f4a8ea1fdcaf","added_by":"auto","created_at":"2025-05-22 09:20:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35789,"visible":true,"origin":"","legend":"\u003cp\u003eThe Venn diagram shows the unique and shared OTUs of the three tissue samples\u003c/p\u003e\n\u003cp\u003eNote：Different lowercase letters in same column indicate significant differences between different tissues at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 level.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/4c751361419ab749209b3a85.png"},{"id":83277584,"identity":"818ab8bb-da82-475b-adcd-c71bc951080c","added_by":"auto","created_at":"2025-05-22 09:28:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35120,"visible":true,"origin":"","legend":"\u003cp\u003ePCoA analysis of endophytic fungal communities in different tissues\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/336ade7e0523c7c8832e9a5b.png"},{"id":83277586,"identity":"e342724c-0662-4224-8bb9-39ef3c8aa55a","added_by":"auto","created_at":"2025-05-22 09:28:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":635192,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance and heat map analysis of endophytic phyla and genera in different tissues (A: phylum level; B: Genus level)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/8833949cd20864c64daee51c.png"},{"id":83276888,"identity":"a33c8d19-b8ad-4374-b64f-9adb4cb28146","added_by":"auto","created_at":"2025-05-22 09:20:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":522808,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional classification and proportion of endophytic fungi in different tissues ( A : nutritional classification B :Guild detailed classification )\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/8ba214f55127b799d06e2bca.png"},{"id":86425201,"identity":"d96dbb9b-b299-4e5f-9bc4-6bfa3dcf6ae2","added_by":"auto","created_at":"2025-07-10 13:17:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2069154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/1468dabb-7863-4d49-aa31-3a7a70b65ec4.pdf"},{"id":83277583,"identity":"2e57b05e-4a15-411d-9c3d-8c8e1ace547a","added_by":"auto","created_at":"2025-05-22 09:28:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":40867,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-6691995/v1/476eae41dc673b3238334961.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of the Endophytic Fungi Community Structure and Function of Stephania tetrandra Based on High-throughput Sequencing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe root of \u003cem\u003eStephania tetrandra\u003c/em\u003e is its therapeutic component; this information was initially documented in \"\u003cem\u003eShennong's Herbal Classic\u003c/em\u003e.\" It is well-known for its detumescence and diuresis, which help to expel wind and ease pain \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.The investigation of \u003cem\u003eS. tetrandra\u003c/em\u003e's chemical makeup and potential usefulness in medicine are currently the primary areas of study. A class of fungus known as endophytic fungi inhabits plants for a specific amount of time or at all stages and does not clearly harm their host plants \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.Research on its pharmacological effects and therapeutic components has long been popular. Endophytic fungi and host plants have a special and intimate symbiotic relationship in the intricate system of plant-environment interaction, which has a significant impact on the physiological and biochemical traits, growth and development process, and environmental adaptability of plants \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe detection range and resolution constraints of conventional microbial research methods can be overcome by high-throughput sequencing technology, which can thoroughly examine complex microbial communities with high precision and high sensitivity \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to shedding light on the internal mechanism of plant-microbe interaction, a thorough study of the endophytic fungal community structure in \u003cem\u003eS. tetrandra\u003c/em\u003e is crucial for comprehending the organism's growth, development, and adaptability mechanisms. The quality of Chinese medicinal materials may be impacted by these endophytic fungi's role in the synthesis and control of \u003cem\u003eS. tetrandra\u003c/em\u003e's therapeutic components. It might also be crucial for the development of plant tolerance to biotic and abiotic stressors, which offers fresh concepts for disease prevention and ecological planting. The growth of biotechnology and the pharmaceutical sector may benefit from the discovery of novel bioactive compounds and enzymes that endophytic fungi themselves may provide.To provide a new theoretical foundation for the thorough investigation, development, and use of \u003cem\u003eS. tetrandra\u003c/em\u003e, this study employed high-throughput sequencing technology to examine the community structure of endophytic fungi in various tissues of the species and performed functional prediction based on this information.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eSource of Sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn Xinyang City, Henan Province, China, fresh S. texandra was gathered (GPS coordinates: 114 \u0026deg; 01\u0026apos;-114 \u0026deg; 06\u0026apos;E, 31 \u0026deg;46\u0026apos;-31\u0026deg;52\u0026apos;N). The experiment included samples from three different plants, with three copies for each type of sample: root samples (FG): FG1, FG2, FG3; leaf samples (FY): FY1, FY2, FY3; and stem samples (FJ): FJ1, FJ2, FJ3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Surface Sterilization and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRinse the new plant\u0026apos;s roots, stems, and leaves. Then, take 5.0 g of each tissue material, wash it three times with sterile water in an ultra-clean bench, and use sterile filter paper to absorb the moisture from the surface. After soaking in 75% ethanol for three minutes, 3% sodium hypochlorite solution for two minutes, and then 75% ethanol for thirty seconds, they were rinsed five times with sterile water, and sterile filter paper was used to absorb water from the surface of the plant tissues. To find out if the plant tissue\u0026apos;s surface was completely cleaned, the final rinse solution was put on a nutrient agar plate and incubated for two to three days at 28\u0026deg;C. For the subsequent stage of DNA extraction, the cleaned samples were kept in an ultra-low-temperature refrigerator set at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Extraction of Endophytic Fungi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeoxyribonucleic acid (DNA) extraction was used to extract DNA from 3.0 g of pre-treated root, stem, and leaf samples. Protease K cleavage in conjunction with phenol-chloroform was used to extract DNA. 1.2 percent agarose gel electrophoresis was used to find the isolated DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR Amplification and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor high-throughput sequencing analysis, the ITS1 region sequence was chosen. PCR amplification was done in two steps to create the library. The ITS1 region universal primers ITS1F 5\u0026apos; -CTTGGTCATTTAGAGGAAGTAA-3\u0026apos; and ITS1R 5\u0026apos; -GCTGCGTTCTTCATCGATGC-3\u0026apos;, along with the fusion primers containing barcode and partial sequencing primers, were used for PCR amplification using the purified DNA as a template. The results were detected by 1.2% agarose gel electrophoresis. 2% agarose gel electrophoresis was used to recover the samples with superior detection findings. Pre-denaturation at 94 \u0026deg;C for two minutes, denaturation at 94 \u0026deg;C for one minute, annealing at 50 \u0026deg;C for thirty seconds, and extension at 72 \u0026deg;C for thirty seconds, followed by 32 cycles, 72 \u0026deg;C extension for five minutes, 10 \u0026deg;C preservation, 5 \u0026times; Buffer 10 \u0026mu;L, 10mM dNTP 1 \u0026mu;L, F/R inner primer (10\u0026mu;M), Phusion ultra-fidelity DNA polymerase 1U 1\u0026mu;L, ddH2O to 50\u0026mu;L, template 5ng-50ng. The AxyPrep DNA gel recovery kit (AXYGEN USA) was used to recover all PCR products, and the FTC-3000TM Real-Time PCR apparatus was used to quantify them. The reaction conditions for the second PCR amplification were pre-denaturation at 94 \u0026deg;C for 2 minutes, denaturation at 94 \u0026deg;C for 1 minute, annealing at 56 \u0026deg;C for 30 seconds, and extension at 72 \u0026deg;C for 30 seconds, for a total of 8 cycles. Extension of 72 \u0026deg;C 5 minutes, insulation at 10 \u0026deg;C, 5xBuffer 8 \u0026mu;L, dNTP (10 mM) 1 \u0026mu;L, F/R outer primer (10 \u0026mu;M), Phusion ultra-fidelity DNA polymerase 0.8 U 1 \u0026mu;L, 5 \u0026mu;L template, 40 \u0026mu;L ddH2O. To finish the library assembly, the adapters, sequencing primers, and barcodes needed for Illumina platform sequencing were added to both ends of the target segment. Microbase Biotechnology (Shangzhou) Co., Ltd. used the Novaseq 6000 SP 500 Cycle Reagent Kit (Illumina USA) to sequence the created library.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBarcodes were used to assign the raw data to the sample readings, and each sample\u0026apos;s effective sequence was determined. A 50 bp window was selected, and the low quality was initially eliminated using Trimmomatic (version: 0.38), a program used for sequence quality control. The back-end base was disconnected from the window\u0026apos;s initial location when the average quality value was less than 20, and the read with a length less than 50 bp following quality control was filtered out. The cutadapt software (version 1.16) was used to process the primers and sequencing adaptors. Based on the overlap relationship between PE readings, splicing operations were carried out using FLASH (version 1.2.11) software, and paired reads were combined into a sequence. To filter out sequences that don\u0026apos;t fit the criteria and guarantee the precision and dependability of the results from the ensuing analysis, the minimum overlap length is set at 10 bp, and the highest mismatch ratio permitted by the overlap region of the splicing sequence is 0.2. Quality control filtering was applied to the combined reads to improve the quality and precision of the bioinformatics analysis results. The ambiguous base (ambiguous = 0), single base high repeat region (homologous = 8), too long (maxlength = 485) and too short (minlength = 200) sequences, as well as some chimeras produced during the PCR process, were eliminated to obtain the optimized sequence because PCR amplification is prone to non-specific amplification. After filtering out the OTU-annotated species categorization, non-research object sequences, and the singletons in the spliced long reads (the sequence corresponding to a single read), the optimized sequence was used as the data for the subsequent clustering OTU analysis. UPARSE software (USEARCH Version V8.1.1756, \u003ca href=\"https://drive5.com/usearch/manual8.1/uparse_pipeline.html\" target=\"_blank\"\u003ehttps://drive5.com/usearch/manual8.1/uparse_pipeline.html\u003c/a\u003e) was used to do OTU (operational taxonomic unit) clustering. To acquire OTU representative sequences, clustering operations with 97% similarity were carried out using UPARSE. UCHIME eliminated the chimeras that resulted from PCR amplification in the OTU representative sequence. The abundance statistics table of each sample in each OTU was created after all sequences were aligned to the OTU representative sequence using the USEARCH_global technique. The classification.seqs function in the Mothur (version: 1.39.5) software was used to match the OTU representative sequence with the UNITE database of ITS fungi to implement species annotation. A confidence threshold of 0.6 was established. Only the remaining OTUs were kept for further analysis after the species whose annotation results were outside the purview of the study and the OTUs lacking annotation results were eliminated. R (Version 3.6.3) was used to conduct statistical and visual analysis, including heatmaps, VENN plots, dilution curves, and community structure histograms. For \u0026alpha; diversity analysis (using species richness statistics like Chao and Ace and species diversity statistics like Shannon and Simpson) and \u0026beta; diversity analysis (using the Bray-Curtis method based on OTU), R (version 3.6.3) language vegan was utilized. Python (2.7.13) and LEfSE (1.0) were used to investigate the variations in prevalent endophytic fungus amongst LEfSe groups. Data processing, species annotation, and classification were followed by functional prediction and annotation, functional analysis, PCoA diagram construction, and differential function analysis using LEfSE.\u003c/p\u003e\n\u003cp\u003eOTU classification was added based on the phylum, family, genus, and species classification technique. The FUNGuild database was used for fungal categorization and functional analysis. It is a tool for classifying and analyzing fungal communities using ecological relationships based on data from reliable websites or currently published research \u003csup\u003e[5]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence Accession Numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NCBI (National Center for Biotechnology Information) database received the raw high-throughput sequencing data using the BioProject number PRJNA1197658 and the Biosample numbers SAMN45811727, SAMN45811728, and SAMN45811729.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEndophytic Fungi Sequencing Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNine test samples yielded 212,783 valid sequences in total after quality control and filtering. In total, 51,430,286 optimized bases were found, with 210,896 optimized sequences. The three sets of optimized sequences ranged in length from 200 to 300 bp. Whether the quantity of sample sequencing data is appropriate can be explained using it.This is shown in Figure 1, the dilution curves often become flat as the number of sequences rises, suggesting that the sequencing data is credible \u003csup\u003e[6]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA total of 539 OTUs were found in \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s roots, stems, and leaves, as seen in Figure 2. There were 105 OTUs that were identical between roots and stems, 162 OTUs between roots and leaves, and 120 OTUs between stems and leaves. It is evident that the species composition of endophytic fungus differs most between stems and roots and least between stems and leaves \u003csup\u003e[7]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiversity of Endophytic Fungi in Different Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe richness and diversity of endophytic fungi in roots were higher than those in stems and leaves, as seen by Table 1\u0026apos;s \u0026alpha; diversity index, which also indicates that the Chao, Ace, and Shannon indices of these fungi were higher in roots than in stems and leaves \u003csup\u003e[8]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTable 1 The four indexes with a coverage rate of 99.99% reflecting community richness and uniformity of the different samples\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"549\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChao\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eAce\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eShannon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eSimpson\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003efraction of coverage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003eFG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e226.43\u0026plusmn;2.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e224.63\u0026plusmn;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.94\u0026plusmn;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.37\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.99%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003eFJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e98.69\u0026plusmn;1.38\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.38\u0026plusmn;1.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.48\u0026plusmn;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.32\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.99%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003eFY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e172.94\u0026plusmn;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e175.24\u0026plusmn;1.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.79\u0026plusmn;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.27\u0026plusmn;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.98%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe three groups of \u003cem\u003eS. tetrandra\u003c/em\u003e root, stem, and leaf samples were clearly divided, as demonstrated by PCoA analysis(Fig.3), and the genetic distance was great. This suggests that the origins of fungal flora in the three groups were different, and there were significant differences between them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommunity Structure at Phylum Level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 4(A), \u003cem\u003eAscomycota\u003c/em\u003e accounted for 95.58%, 95.89%, and 98.85% of the endophytic fungi found in roots, leaves, and stems, respectively. \u003cem\u003eBasidiomycota\u003c/em\u003e accounted for 2.79% of roots and 3.59% of leaves, respectively. \u003cem\u003eMortierellomycota\u003c/em\u003e, \u003cem\u003eGlomeromycota\u003c/em\u003e, \u003cem\u003eMucoromycota\u003c/em\u003e, \u003cem\u003eChytridiomycota\u003c/em\u003e, \u003cem\u003eRhodomycota\u003c/em\u003e, \u003cem\u003eCalcarisporiellomycota\u003c/em\u003e, and \u003cem\u003eOleaginaceae\u003c/em\u003e were among the biological gene sequences found in root samples. In root samples, unclassified endophytic fungi made up 0.04%. In roots, leaves, and stems, the unclassified phylum represented for 0.33% to 1.02%. The heat map indicates that the phylum \u003cem\u003eAscomycota\u003c/em\u003e is the most prevalent in the fungal community, with the highest abundance in stems \u003csup\u003e[9]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 4(A), \u003cem\u003eAscomycota\u0026nbsp;\u003c/em\u003eaccounted for 95.58%, 95.89%, and 98.85% of the endophytic fungi found in roots, leaves, and stems, respectively. \u003cem\u003eBasidiomycota\u003c/em\u003e accounted for 2.79% of roots and 3.59% of leaves, respectively. \u003cem\u003eMortierellomycota\u003c/em\u003e, \u003cem\u003eGlomeromycota\u003c/em\u003e, \u003cem\u003eMucoromycota\u003c/em\u003e, \u003cem\u003eChytridiomycota\u003c/em\u003e, \u003cem\u003eRhodomycota\u003c/em\u003e, \u003cem\u003eCalcarisporiellomycota\u003c/em\u003e, and \u003cem\u003eOleaginaceae\u003c/em\u003e were among the biological gene sequences found in root samples. In root samples, unclassified endophytic fungi made up 0.04%. In roots, leaves, and stems, the unclassified phylum represented 0.33% to 1.02%. The heat map indicates that the phylum \u003cem\u003eAscomycota\u003c/em\u003e is the most prevalent in the fungal community, with the highest abundance in stems \u003csup\u003e[9]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommunity Structure at Genus Level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Figure 4(B), the top 5 genera in the root were \u003cem\u003eCladosporium\u003c/em\u003e ( 58.98 % ), \u003cem\u003eAlternaria\u003c/em\u003e ( 13.81 % ), Others ( 7.86 % ), \u003cem\u003eSelenophoma\u003c/em\u003e ( 6.35 % ), and \u003cem\u003eErysiphe\u003c/em\u003e ( 3.55 % ). The top 5 genera in leaves were \u003cem\u003eCladosporium\u003c/em\u003e ( 42.24 % ), \u003cem\u003eErysiphe\u003c/em\u003e ( 26.73 % ), \u003cem\u003eSelenophoma\u003c/em\u003e ( 12.75 % ), \u003cem\u003eAlternaria\u003c/em\u003e ( 8.06 % ), Others ( 2.60 % ), \u003cem\u003eNigrospora\u003c/em\u003e ( 2.38 % ). The top 5 genera in stems were \u003cem\u003eCladosporium\u003c/em\u003e ( 47.81 % ), \u003cem\u003eBotryosphaeria\u003c/em\u003e ( 22.10 % ), \u003cem\u003eSelenophoma\u003c/em\u003e ( 21.52 % ), \u003cem\u003eAlternaria\u003c/em\u003e ( 2.69 % ) and Others ( 1.71 % ). According to the heat map, \u003cem\u003eCladosporium\u003c/em\u003e was the dominant genus of endophytic fungi in the three tissues, accounting for 58.98 %, 42.24 % and 47.81 % of roots, leaves and stems, respectively. The second was \u003cem\u003eSelenastrum\u003c/em\u003e, but the abundance of different tissues was different, and the content was the highest in stems ( 21.52 % ). Followed by leaf ( 12.75 % ) ; it was the lowest in root endophytic fungi ( 6.35 % ). Others ( 7.86 % ) in roots and Others ( 1.71 % ) in stems did not represent any genus. In addition to \u003cem\u003eCladosporium\u003c/em\u003e and \u003cem\u003eSphaerothecium\u003c/em\u003e, the proportion of \u003cem\u003eBotryosphaeria\u003c/em\u003e in stems was also high, accounting for 22.10 %, while the proportion in roots was very low, accounting for 0.22 %. As the dominant genus of roots and leaves, powdery mildew accounted for 3.55 % and 26.73 % of endophytic fungi in roots and leaves, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Prediction Analysis of Endophytic Fungi in Different Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FUNGuild database was utilized to forecast the role of endophytic fungi in various \u003cem\u003eS. tetrandra\u003c/em\u003e tissues. As illustrated in Fig. 5(A), the endophytic fungi of \u003cem\u003eS. tetrandra\u003c/em\u003e can be categorized into eight ecological function groups: symbiotic type, saprophytic type, saprophytic-symbiotic type, pathological parasitic type, pathological parasitic-septic type, pathological parasitic-skeptic-symbiotic type, pathological parasitic-symbiotic type, and undefined. In roots, leaves, and stems, undefinable colonies made up 62.96%, 46.01%, and 50.0% of the total. Furthermore, the percentage of roots, leaves, and stems that were saprophytic was 10.50%, 15.73%, and 24.02%, respectively. In roots, leaves, and stems, pathological parasitism-septicism-symbiosis accounted for 19.63%, 8.90%, and 25.25%, respectively. The highest percentage of pathological parasites was found in leaves (27.07%), followed by roots (4.84%) and stems (0.38%). It demonstrates that a comparatively large percentage of tissues with strong development are of the pathogenic parasitic type. The fundamental functional groups\u0026mdash;pathological parasitism-saprophytic, pathological parasitism-symbiosis, saprophytic-symbiosis, and symbiotic\u0026mdash;are the most prevalent in addition to the four nutritional types mentioned above.\u003c/p\u003e\n\u003cp\u003eWith the exception of the undefined, the top three ecological function groups in the root were Animal Pathogen-Endophyte-Plant Pathogen-Wood Saprotroph (13.81%), Undefined Saprotroph (8.12%), and Endophyte-Fungal Parasite-Lichen Parasite-Plant Pathogen-Wood Saprotroph (2.77%), as shown by the Guild\u0026apos;s detailed classification in Figure 5 (B). Plant Pathogen (26.97%), Undefined Saprotroph (15.63%), and Fungal Parasite-Litter Saprotroph (1.58%) were the top three ecological functional categories in leaves. Undefined Saprotroph (23.00%), Endophyte-Plant Pathogen-Wood Saprotroph (22.10%), and Animal Pathogen-Endophyte-Plant Pathogen-Wood Saprotroph (2.69%) were the top three ecological functional groups in stems.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eAnalysis of Diversity and Richness of Endophytic Fungi in Different Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to related research, the host plant community will exhibit diversity due to varrious plant species, organizational structures, and functions \u003csup\u003e[10]\u003c/sup\u003e. The diversity of endophytic fungi in \u003cem\u003eHuperzia serrata\u003c/em\u003e was also studied, revealing differences in the community structure and diversity of endophytic fungi in different tissues \u003csup\u003e[11]\u003c/sup\u003e. While the diversity and spatial distribution of endophytic fungi in \u003cem\u003eCinnamomum longepaniculatum\u0026nbsp;\u003c/em\u003ewere studied, it was discovered that the richness and diversity of fungal communities in seeds were the highest, followed by branches, leaves, and roots \u003csup\u003e[12]\u003c/sup\u003e. Three distinct \u003cem\u003eS. tetrandra\u003c/em\u003e tissues were colonized by a large number of endophytic fungi in this investigation; however, the distribution of these fungi varied per organ and tissue. The endophytic fungi in three distinct \u003cem\u003eS. tetrandra\u003c/em\u003e tissues were examined in this study, and it was discovered that there were clear variations in the endophytic fungal dispersion by organ tissue. The Chao index (226.43 \u0026plusmn; 2.01a), Ace index (224.63 \u0026plusmn; 0.70a), and Shannon index (1.94) of endophytic fungi in roots were higher than those in stems and leaves, according to the \u0026alpha; diversity index (see Table 1). This suggests that the richness and diversity of endophytic fungi in roots were higher than those in stems and leaves.\u003c/p\u003e\n\u003cp\u003eThe rhizosphere environment is complicated from the standpoint of the microbial colonization environment. Numerous organic materials, including sugars, amino acids, organic acids, and others, are secreted on the root\u0026apos;s surface and surrounding area. These secretions give the rhizosphere and rhizosphere fungus enough carbon, nitrogen, and other nutrients to draw in more rhizosphere microorganisms for colonization \u003csup\u003e[13\u0026ndash;14]\u003c/sup\u003e, which in turn causes more microorganisms to colonize the roots. They colonize both whole plants and roots after germination, and the abundance and diversity of endophytic fungi can vary depending on the properties of the soil \u003csup\u003e[15]\u003c/sup\u003e. However, some research has shown that temperature, geographic location, and other variables all affect the variety of endophytic fungus in roots \u003csup\u003e[16]\u003c/sup\u003e. Conversely, the types and amounts of nutrients that are available are restricted, and the surface of stems and leaves is comparatively smooth. Microorganisms\u0026apos; species richness is limited by the environmental conditions, which have higher needs for their survival adaptability. Using leaves as an example, germs enter the leaves horizontally after first colonizing their surface through stomata, water holes, lesions, etc. However, the number of microorganisms in leaves declines as a result of UV radiation, as well as a shortage of water and nutrients \u003csup\u003e[17]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNumerous parasitic and coexisting connections exist between microorganisms in roots and soils when considering the interaction between microorganisms and plants. Certain fungi that promote rhizosphere growth, for instance, can have a strong relationship with roots and aid in their ability to absorb nutrients, create plant hormones, and fend off disease. More plants can participate and create a more varied microbial community thanks to these intricate network relationships. However, it is challenging to create a diversified microbial community since the microbial sources and interactions of stems and leaves are somewhat straightforward. Numerous microorganisms that resemble rhizosphere growth-promoting fungi may be present in \u003cem\u003eS. tetrandra\u003c/em\u003e roots. These microorganisms interact with the roots of \u003cem\u003eS. tetrandra\u003c/em\u003e and support the diversity of endophytic fungal communities. On the other hand, it is challenging to create a diversified microbial population, and the microbial sources and interactions of stems and leaves are somewhat straightforward.\u003c/p\u003e\n\u003cp\u003eThe roots have different tissue levels from the standpoint of niche biochemistry. The physical and chemical characteristics of each level\u0026mdash;such as pH, oxygen concentration, osmotic pressure, and so forth\u0026mdash;have gradient variations from the epidermis to the cortex to the vascular bundle, offering a range of habitats for various microbial populations. The variety of microbial communities that can be supported is limited since the tissue levels in stems and leaves are rather homogeneous. The large diversity of endophytic fungi in T. tetrandra roots is consistent with the fact that this diversified niche can support a greater variety of endophytic fungal species. The variety of microbial communities that can be supported is limited since the tissue levels in stems and leaves are rather homogeneous.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Endophytic Fungal Community Composition in Different Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe community structure of endophytic fungi in \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s roots, stems, and leaves was significantly different, according to cluster heat map analysis. Ascomycota and Basidiomycota are the two most prevalent phyla among Chinese mangrove ferns, making up about 75% of all strains. \u003csup\u003e[18]\u003c/sup\u003e. Because of their diverse ecological settings and host specificity, \u003cem\u003eAscomycota\u003c/em\u003e is regarded as one of the most prevalent fungi in the world \u003csup\u003e[19]\u003c/sup\u003e. The majority of the phylum \u003cem\u003eAscomycota\u003c/em\u003e, which dominated this investigation, were saprophytic fungus. It contributes significantly to the cycling of nutrients and possesses high material degradation and synthesis capabilities. Its symbiotic relationship with plants is the easiest \u003csup\u003e[20]\u003c/sup\u003e. Consequently, the metabolism and nutrition cycling of \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s many tissues greatly depend on this phylum. \u003cem\u003eBasidiomycetes\u003c/em\u003e are also comparatively prevalent in the roots and leaves in this investigation. Because of their healing capabilities, basidiomycetes can restore contaminated and deteriorated soils \u003csup\u003e[21]\u003c/sup\u003e. \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e are the two groups of endophytic fungi that infect higher plants the most frequently \u003csup\u003e[22\u0026ndash;23]\u003c/sup\u003e. The predominant genus is \u003cem\u003eCladosporium\u003c/em\u003e, with the majority of its strains being saprophytic \u003csup\u003e[24]\u003c/sup\u003e, but \u003cem\u003eSphaerothecium\u003c/em\u003e also makes up a sizable fraction. Powdery mildew is more prevalent in the roots and leaves, botryosphaeria is more prevalent in the stem, and the root is quite low. During long-term co-evolution with plants, the majority of plant endophytic fungus produce metabolites that are identical to or comparable to those of their host plants. Alkaloids, flavonoids, steroids, and other metabolites are abundant in \u003cem\u003eS. tetrandra\u003c/em\u003e \u003csup\u003e[1]\u003c/sup\u003e.Research has revealed that \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s predominant metabolites possess biological activities such as antioxidant and anti-tumor properties \u003csup\u003e[25]\u003c/sup\u003e. Such fungi may exist because of \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s abundant metabolites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Prediction Analysis of Endophytic Fungi in Different Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functional classes of \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s endophytic fungus were categorized using the FUNGuild software. Pathological parasitic type, pathological parasitic-saprophytic type, pathological parasitic-saprophytic-symbiotic type, pathological parasitic-symbiotic type, saprophytic type, saprophytic-symbiotic type, symbiotic type, and undefined were the eight ecological functional groups that were identified. The undefined functional groups made up 62.96%, 46.01%, and 50.0% of the eight functional groups in the base, leaf, and stem, respectively. On the one hand, it demonstrates the lack of thoroughness in the FUNGuild database study. It also indicates that there are many endophytic fungal resources in \u003cem\u003eS. tetrandra\u003c/em\u003e that need to be unearthed, which is very valuable for research.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eS. tetrandra\u003c/em\u003e, the relative abundance of saprophytic functional groups was greater than 10%, with the exception of undefined functional groups. Because saprophytic fungi break down organic matter for food, they contribute significantly to ecosystems\u0026apos; material cycles. The soil contains a wide variety of saprophytic fungus. The soil and the rhizosphere of \u003cem\u003eS. tetrandra\u003c/em\u003e are intimately associated. With the help of the plants\u0026apos; ability to transfer nutrients, saprophytic fungi can eventually infiltrate the roots and then selectively colonize the stems and leaves. Saprophytic fungus, for instance, can break down organic materials like litter into inorganic stuff and then reintroduce it into the soil to supply nutrients for plant development in forest ecosystems. It\u0026apos;s possible that the saprophytic endophytic fungus in \u003cem\u003eS. tetrandra\u003c/em\u003e participate in comparable mechanisms to encourage plants to absorb and repurpose nutrients.\u003c/p\u003e\n\u003cp\u003eIn roots and stems, the pathoparasitic-saprophytic-symbiotic type was more than 15% abundant. The root, which is \u003cem\u003eS. tetrandra\u003c/em\u003e\u0026apos;s vegetative organ, is in charge of taking in water, soluble small molecules, and inorganic ions. Both of them are nutrient-rich, and the stem contains a transport tissue for carrying nutrients. This may be why functional fungi make up a comparatively large percentage of roots and stems\u003csup\u003e[29]\u003c/sup\u003e. This type of fungus can change its mode of nourishment depending on the environment and has a number of survival strategies. In addition to obtaining nourishment through symbiosis with plants, it can also, in some circumstances, engage in saprophytic life and even become parasitic. By developing a symbiotic association with plant roots, certain mycorrhizal fungi aid in nutrient absorption while promoting saprophytic growth on plant waste. Because of this trait, they are particularly important for \u003cem\u003eS. tetrandrae\u003c/em\u003e\u0026apos;s growth, development, and ecological adaptation. They may also play a role in controlling the balance of nutrients and plant physiological metabolism.\u003c/p\u003e\n\u003cp\u003eOnly 4.84% of roots and 0.38% of stems had the pathogenic parasite kind, which made up the largest percentage of leaves (27.07%). The leaves have a comparatively high percentage of pathogenic parasite fungus because they grow quickly and are exposed to the natural environment, which makes them more susceptible to invasion by outside diseases. It is well recognized that a variety of plant diseases, including apple anthracnose, common wheat rust, and others, are brought on by particular parasite fungi. Consequently, it is hypothesized that the pathogenic fungi that cause plant illness and pollution during tissue culture are most likely the pathological fungi seen in \u003cem\u003eS. tetrandra\u003c/em\u003e leaves. This implies that in order to guarantee the plants\u0026apos; healthy growth during the planting and tissue culture processes of \u003cem\u003eS. tetrandra\u003c/em\u003e, attention must be paid to the prevention and management of leaf diseases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings demonstrated that endophytic fungi were highly diverse across all tissues, with roots having the greatest diversity and richness of endophytic fungi. The dominating genus was Cladosporium, and the major flora was Ascomycota. According to the functional prediction, there were eight functional ecological groups among the endophytic fungus, with a significant percentage of undefined functional groupings. The abundance of materials that needed to be dug up might be attributed to variations in the environment and nutrition of the tissues. The structure and diversity of endophytic fungi in various S. tetrandra tissues were demonstrated in this work, which also served as a foundation for future research and the identification of strain resources.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOTU \u0026nbsp; \u0026nbsp;Optical taxonomic unit\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp; \u0026nbsp;Ribosomal database project\u003c/p\u003e\n\u003cp\u003ePCoA \u0026nbsp; Principal coordinate analysis\u003c/p\u003e\n\u003cp\u003eLDA \u0026nbsp; \u0026nbsp; Latent Dirichlet Allocation\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp; LY and SL: designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. QC and HG: Make changes to the document. WL: Responsible for editing tables and icons. QC: and DH: supervised the research and critically revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWe would like to thank Microbase Biotechnology (Shanghai) Co., Ltd. for providing the platform and technical support for high-throughput sequencing and bioinformatics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp; \u0026nbsp;The research was supported by the Henan province science and technology research ( 242102311200 ) ; natural Science Foundation of Henan Province ( 232300420065 ) ; Supported by the Science and Technology Major Projects in Henan Science and Technology Agency ( 241111311400 ) ; youth Fund Project of Xinyang College of Agriculture and Forestry ( QN2023026 ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information fles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp; The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003eNot applicable. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJiang Y, Liu M, Liu H, Liu S (2020) A critical review: traditional uses, phytochemistry, pharmacology and toxicology of Stephania tetrandra S. 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BMC Microbiol. 22(1):90.https://doi.org/10.1186/s12866-022-02510-4\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"endophytic fungal diversity, function prediction, high-throughput sequencing, organizational differences, Stephania tetrandra","lastPublishedDoi":"10.21203/rs.3.rs-6691995/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6691995/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnalyzing the endophytic fungi in various \u003cem\u003eStephania tetrandra\u003c/em\u003e tissues is crucial for investigating strain resources, enhancing \u003cem\u003eS. tetrandra\u003c/em\u003e cultivation, and carrying out further studies. In order to investigate the variety, community structure, and function of endophytic fungus in various tissues, the three distinct tissues of \u003cem\u003eS. tetrandra\u003c/em\u003e's roots, stem, and leaves were sequenced and analyzed using Illumina MiSeq high-throughput sequencing technology. Findings: 539 OTUs and 212,783 valid sequences throughout 11 phyla, 29 classes, 64 orders, 135 families, 222 genera, and 286 species were found. The major phylum and genus were \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eCladosporium\u003c/em\u003e. Eight functional ecological groups were found in the endophytic fungi of \u003cem\u003eS. tetrandra\u003c/em\u003e, according to the FUNGuild software analysis. The undefined functional groups made up a comparatively large percentage of the various tissues of \u003cem\u003eS. tetrandra\u003c/em\u003e, which required more research. This work established a foundation for the development and use of \u003cem\u003eS. tetrandra\u003c/em\u003e resources by revealing the diversity and structure of endophytic fungi in the roots, stems, and leaves of various \u003cem\u003eS. tetrandra\u003c/em\u003e tissues.\u003c/p\u003e","manuscriptTitle":"Analysis of the Endophytic Fungi Community Structure and Function of Stephania tetrandra Based on High-throughput Sequencing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 09:20:06","doi":"10.21203/rs.3.rs-6691995/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e29d642e-8bef-4fca-95b3-260d622af63b","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-10T13:08:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 09:20:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6691995","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6691995","identity":"rs-6691995","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}