{"paper_id":"01da637c-15c8-4edd-9afb-72676d8da8a6","body_text":"Vitex rotundifolia L. f. Engineered Microbial Interaction Reverse Desertification: Nitrogen-Fixing Consortia Drive Ecosystem Restoration in Sandy Wetlands of Poyang Lake | 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 Vitex rotundifolia L. f. Engineered Microbial Interaction Reverse Desertification: Nitrogen-Fixing Consortia Drive Ecosystem Restoration in Sandy Wetlands of Poyang Lake Haijing Xiao, Min Guo, Yiqing Luo, Yiyun Xu, Zhaoqi Xie, Chunsong Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7181804/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 Background: The sandy areas of Poyang Lake are a typical region of wind erosion desertification in humid areas, and their ecological restoration urgently requires efficient, low-cost plant and microbial co-management measures. Vitex rotundifolia L. f., a local dominant pioneer shrub, has both medicinal and ecological value, but its mechanism of influence on sandy soil microorganisms remains unclear. To systematically assess how Vitex rotundifolia L. f. drives sand dune ecological restoration by altering soil physical and chemical properties and microbial community structure. Results: The total nitrogen (TN) and alkali-hydrolyzable nitrogen (AN) in the rhizosphere soil of Vitex rotundifolia L. f. grown for more than 3 years were 17-fold and 1.2-fold higher than those of bare sand, respectively. The activities of urease (UA) and nitrogenase (NA) significantly increased, while the moisture content slightly decreased. The bacterial α-diversity first increased and then slightly decreased, while fungal diversity decreased with increasing growth years. Cyanobacteria in the rhizosphere soil surged by 122-fold, while Actinobacteria and Proteobacteria increased by 1.3-fold and 1.4-fold, respectively. Fungi were dominated by Ascomycota and Basidiomycota. Functional prediction showed that nitrogen fixation, amino acid metabolism, and membrane transport pathways were significantly enriched. The proportion of wood-decaying fungi and ectomycorrhizal fungi increased, while pathogenic bacteria significantly decreased. Conclusion: Vitex rotundifolia L. f. can significantly accelerate soil formation and ecological restoration of wet sand dunes in Poyang Lake by promoting nitrogen accumulation, constructing beneficial microbial communities, and forming biological crusts, providing a new strategy for low-cost and large-scale restoration of sandy land in humid areas. Vitex rotundifolia L. f. microbiome analysis function prediction Poyang Lake sand hills Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Background Desertification in humid areas means land degradation processes occurring in humid areas resulting from various factors, including climatic variations and human activities [ 1 , 2 , 3 , 4 ]. The historical development of desertification in humid areas has been closely correlated with anthropogenic activities during the past one to two centuries [ 5 , 6 ]. It has occurred across multiple regions, including South Asia, Southeast Asia, the Mediterranean coast, several sub-Saharan African countries, and extensive areas of southwestern China, exerting substantial negative impacts on both environmental systems and socioeconomic conditions at local and regional scales [ 7 , 8 , 9 , 10 ]. The desertified land area in humid southern China is estimated at 198,000 km 2 [ 10 ]. It exhibits a patchy distribution across the hilly and mountainous terrain of southern China, primarily occurring in areas with weathering crusts developed from red clay, quaternary red clay, granite, and limestone formations, as well as within alluvial floodplains, fluvial sandy terraces along lower river reaches, and lacustrine shoreline environments [ 1 , 11 ]. The main types of desertification in China's humid areas include: water erosion-induced desertification, characterized by eroded badlands and rocky slopes, and wind erosion-induced desertification, marked by wind-eroded lands and mobile sand dunes [ 12 ]. The Poyang Lake periphery experiences severe land desertification, locally referred to as \"sand hills\", which belongs to the second desertification type (wind erosion-induced desertification) [ 13 , 14 ]. The desertification area around Poyang Lake spans up to 38,900 km 2 , accounting for 19.65% of the desertification in China's humid areas [ 13 , 14 , 15 ]. It is primarily distributed in the lakeside areas of Poyang Lake and the delta regions of the \"Five Rivers\" (the Gan, Fu, Xin, Rao, and Xiu River) [ 16 , 17 ]. Severe wind and sand erosion has buried farmland, destroyed arable land, exacerbated poverty among local residents, intensified human-land conflicts, caused ecological degradation, and seriously threatening regional ecological and food security [ 18 ]. Vitex rotundifolia L. f. is a deciduous shrub or small tree of the Lamiaceae family in the Vitex L., and evolved from Vitex trifolia L. [ 19 ]. It has been used as medicine for over 2,000 years, spanning the Middle Ages and the Renaissance periods [ 19 , 20 ]. Vitex rotundifolia L. f. has been listed in the prestigious Pharmacopoeia Londinensis which dates back to the year 1618, and is indicated for the management of traumatic injuries and inflammatory disorders [ 21 , 22 , 23 , 24 ]. In China, the mature dried fruits of Vitex rotundifolia L. f. or Vitex trifolia L. are known as \"Manjingzi\" [ 19 ]. This herbal medicine was first documented in the Divine Farmer’s Classic of Materia Medica and is currently included in the Pharmacopoeia of the People's Republic of China [ 25 , 26 , 27 ]. It is widely used in Traditional Chinese Medicine (TCM) for treating various conditions, including premenstrual syndrome, headache disorders, migraines, colds, and eye pain [ 27 , 28 , 29 , 30 ]. Vitex rotundifolia L. f. thrives in beach and dune habitats, widely distributed in desert areas and coastal regions. with mainly found in in provinces such as Jiangxi, Fujian, Shandong, Anhui and Zhejiang [ 31 ]. The stems develop nodal root systems that enable the formation of dense clonal clusters, extending over 10 meters from the mother plants. Both roots and stems exhibit rapid regeneration rapidly [ 31 ]. Vitex rotundifolia L. f. demonstrates remarkable adaptability to sandy and saline-alkali environments, and possesses ecological functions such as windbreak stabilization, sand fixation, water storage, and moisture retention [ 19 , 32 ]. Recognized as a pioneer species for sandy ecosystem rehabilitation and coastal landscaping, it represents a multifunctional resource with integrated social, ecological, and medicinal values [ 33 ]. The vegetation of the sand hills in Poyang Lake has the characteristics of a subtropical desert [ 13 ]. The shrub layer vegetation in Poyang Lake's sand hills ecosystem is primarily constructed by dominant and pioneer species including Vitex rotundifolia L. f., Artemisia capillaris Thunb., and Lespedeza bicolor Turcz.. Among these, Vitex rotundifolia L. f. holds a significant position on different types of sand dunes (flowing sand dunes, semi-flowing sand dunes, and fixed sand dunes) [ 16 ]. Our field observations reveal that Vitex rotundifolia L. f. exhibits a patchy distribution pattern across the sand hills. The rhizosphere soil within these patches displays significantly darker coloration compared to bare sand, demonstrating this species' ability to improve edaphic conditions in dune systems. The research on Vitex rotundifolia L. f. has primarily focused on traditional medicine, phytochemistry, and pharmacology [ 27 ]., with relatively few studies investigating its mechanisms for soil amelioration and ecological restoration. Plants can modify the nitrogen cycle in the soil, transforming inherent and unchanging componentsinto available nutrients, and altering the structure and function of the soil microbial communities. Meanwhile, soil microorganisms regulating the nitrogen cycle through decomposition of soil organic matter and plant residues [ 34 , 35 , 36 ]. Therefore, understanding the compositional composition and function characteristics of soil microbial communities is crucial for studying Vitex rotundifolia L. f. growth, contributing to a deeper exploration of the microbial ecological mechanisms underlying variations in Poyang Lake's sand hills [ 37 , 38 , 39 ]. This study employed 16S rRNA and ITS sequencing techniques to investigate the diversity and composition of bacterial and fungal communities in the soil of the sand hills in Poyang Lake. A comparative microbiome analysis was performed across three chronosequence stages: (1) non- Vitex rotundifolia L. f. areas, (2) 1-year Vitex rotundifolia L. f. growth areas, and (3) multi-year Vitex rotundifolia L. f. growth areas, to assess successional microbial shifts. The relationship between soil physicochemical properties (environmental factors and enzyme activities) related to the nitrogen cycle and microorganisms was studied. The functions of soil microorganisms were predicted to identify metabolic pathways related to different growth areas of Vitex rotundifolia L. f.. This study aimed to provide insights into the ecological mechanisms of growth of Vitex rotundifolia L. f., providing scientific data for standardized cultivation in Poyang Lake's desertified areas, and offering governance approaches for the ecological restoration of deserts in humid regions, thus achieving the goal of ecological conservation (lucid waters and lush mountains) and sustainable socio-economic benefits (golden and silver mountains). 2. Methods 2.1 Study sites description In this study, sandy soil from the experimental area of Liaohua Town, Xingzi County, Jiujiang City, Jiangxi Province (115°48′ to 116°10′E, 29°8′ to 29°36′N) was used as the material. The test area is located on the left bank of the floodway connecting Poyang Lake to the Yangtze River. It belongs to the mid-subtropical humid zone, characterized by high temperatures, abundant rainfall, distinct dry and wet seasons, and an uneven distribution of seasonal precipitation throughout the year [ 40 ]. The average annual temperature is 16.5 ~ 17.8℃, while the average and highest surface temperatures are 21.3℃ and 69.5℃, respectively [ 41 ]. The annual evaporation is 1880 mm, and the annual precipitation ranges from 1300 ~ 1600 mm. Precipitation mainly occurs in spring and summer, with limited rainfall in autumn and winter. Cold north winds are common in winter, with wind speeds reaching up to 17 m/s, resulting in lake shrinkage and the exposure of lakebed sand [ 40 , 41 ]. 2.2 Soil sampling Two sampling areas, L and X, were established in the experimental area, and sampling was conducted in October 2023 (Fig. 1 ). Area L consisted of a Vitex rotundifolia L. f. community with more than three years of growth. Area X was prepared in October 2022 by leveling 20 acres of sandy land and planting Vitex rotundifolia L. f.. Thus, Area X consists of a Vitex rotundifolia L. f. community that has been growing for less than two years. Blank control (LCK, XCK) samples were collected from sandy areas (L area, X area) without the growth of Vitex rotundifolia L. f.. The root-surrounding soil (LGZ, XGZ) samples were collected from soil within 5–20 cm around the roots of Vitex rotundifolia L. f.. The rhizosphere soil samples (LGJ, XGJ) were collected by gently brushing the tightly adhered soil from the roots of Vitex rotundifolia L. f. using a sterile soft-bristled brush. Samplings were selected five-point sampling method, soil samples from five different locations were mixed and transferred into sterile plastic bags. The samples were divided into two parts, one part was air-dried for the determination of soil pH, moisture content (MC), total nitrogen (TN), Alkali-hydrolyzable nitrogen (AN), urease activity (UA) and nitrogenase activity (NA) content, and the other part was kept in sterile centrifugal tubes and frozen in -80 ℃ refrigerator for spare. 2.3 Soil physicochemical indicators and enzyme activity analysis Soil water content was determined using the drying method. pH was determined using pH 368 (resolution 0.01 pH). TN was determined using the Semi-micro kjeldahl method [ 42 , 43 ]. AN was determined using the alkali diffusion method [ 44 ]. UA was determined using the indophenol blue colorimetric method [ 45 ]. NA was determined using the microbial nitrogenase ELISA kit [ 46 ]. 2.4 DNA extraction, amplification, and sequencing Nucleic acids were extracted from samples using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA) kit. The DNA concentration was then detected using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Scientific, USA). We used an ABI GeneAmp® 9700 thermal cycler for DNA fragment amplification. The 16s rRNA V3-V4 region was selected for bacterial PCR amplification with primer sequences 341F (5'-CCTACGGGGNGGCWGCAG-3'), 805R (5'-GACTACHVGGGGTATCTAATCC-3 '). The ITS rDNA ITS region was selected for PCR amplification of the fungus, and the primer sequences were ITS1F (5'-GGAAGTAAAAGTCGTAACAAGG-3'), ITS2R (5'-GCTGCGTTCTTCATCGATGC-3 '). The PCR reflection parameters were: the DNA was denatured at 98°C for 5 min in a single cycle; then denatured at 98°C for 30 s, annealed at 52°C for 30 s, and extended at 72°C for 45 s, for a total of 28 cycles; and finally, the product was kept at 72°C for 5 min to make the product extension complete and stored at 12°C. Finally, the amplification products were screened by 2% agarose gel electrophoresis, and the target fragments were recovered by sorted magnetic bead recovery method. Nova Seq 6000 SP Reagent Kit (500 cycles) was utilized to perform 2×250bp double-end sequencing to obtain raw sequencing data. 2.5 Statistical analysis Raw microbiome sequencing data were spliced and optimized using Fastp v0.19.6 and FLASH v1.2.7 software. The optimized data were then processed using the DADA2 method for sequence noise reduction, resulting in the identification of amplicon sequence variants (ASVs) within the samples. The representative sequences of the ASVs were classified and annotated with QIIME2 v2022.2 software, utilizing the bacterial 16S rRNA gene Silva database and the fungal ITS UNITE database as reference sources, respectively. Figs were performed applying Excel 2019, Origin 2018 and GraphPad Prism 9. One-way ANOVA was carried out using SPSS version 25.0, and the significance of differences was assessed using the Least Significant Difference method. Differences in the values with P < 0.05 (*) were considered statistically significant. LEfSe analyses were performed using an online data analysis tool ( http://huttenhower.sph.harvard.edu/galaxy/ ), and the LDA discriminant value of 4.0 was selected. Redundancy analysis (RDA) was used to elucidate the overall relationships between microbial communities and related factors. Functional differences among the microbial communities were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Fungal functional guild assignment for each ASV was performed using FUNGuild version 1.0. 3. Results 3.1 Physicochemical properties of the soil samples The growth of Vitex rotundifolia L. f. has changed the Physicochemical properties of the sand hills in Poyang Lake (Table 1 , Table S1 ). Soil water content was significantly lower in plots grown with Vi tex rotundifolia L. f. than without growing in plots (P < 0.05). The root-surrounding soil and rhizosphere soil exhibited decreasing water content with increasing growth duration. Soils planted with Vitex rotundifolia L. f. showed significantly higher values of pH, TN, AN, and UA compared to non-planted control soils. Furthermore, these parameters in root-surrounding and rhizosphere soils showed increases with extended growth duration. Table 1 Soil physicochemical properties of Vitex rotundifolia L. f. with different stand ages and locations. Index LGZ LGJ XGZ XGJ LCK XCK Moisture Content (%) 4.21 ± 0.15bc 3.57 ± 0.23c 4.29 ± 0.27b 3.92 ± 0.30bc 5.00 ± 0.22a 5.33 ± 0.27a pH 6.45 ± 0.17ab 6.90 ± 0.38a 6.05 ± 0.21bc 6.72 ± 0.20a 5.75 ± 0.21c 5.54 ± 0.24c TN (g/kg) 1.32 ± 0.05b 4.65 ± 0.16a 0.77 ± 0.01c 0.84 ± 0.03c 0.27 ± 0.02d 0.29 ± 0.01d AN (mg/kg) 53.70 ± 1.96a 53.57 ± 0.73a 48.73 ± 0.93b 53.91 ± 1.00a 45.90 ± 1.51c 43.68 ± 1.12c UA (U/g) 255.75 ± 4.11c 462.70 ± 15.67a 240.58 ± 7.17c 299.22 ± 11.78b 214.79 ± 6.13d 191.87 ± 5.20e NA (U/g) 2.49 ± 0.03d 2.17 ± 0.02c 1.68 ± 0.04b 2.40 ± 0.01d 1.47 ± 0.03a 1.60 ± 0.15a Note: Different letters in the same row indicate significant differences among different treatments (P < 0.05). 3.2 Diversity of the microbial community Microbiome sequencing of soil samples from areas L and X yielded a total of 24,172 bacterial ASVs and 6,456 fungal ASVs, with average sequence lengths of 221 bp and 220 bp, respectively. Shared species analysis was performed for common bacterial and fungal species among the six sampling sites (LGZ, LGJ, XGZ, XGJ, LCK, XCK), and a petal diagram was generated to visualize the results (Fig. 2 ). The proportions of species in bacteria and fungi were 78.9% and 21.1%, respectively, and shared bacterial and fungal species among the six sampling sites accounted for 0.22% and 1.17%, respectively. In the bacterial community, the number of ASVs shared and unique in area X (XGZ, XGJ) is the highest among all groups (Fig. 2 A). In the fungal community, the highest number of ASVs shared with other groups was found in area X (XGZ, XGJ), while the highest number of ASVs unique in LCK (Fig. 2 B). The Chao and Shannon indices were used to assess the alpha diversity of the microbial community, with the former reflecting species richness and the latter reflecting species diversity (Table 2 , Table S6-S9). In the bacterial community, both indices of the X area exhibited highest values than those of the L area (XCK > LCK, XGJ > LGJ, XGZ > LGZ), particularly in the Shannon index, which displayed significant differences across all regions (p < 0.05). Regarding the fungal community, both indices of the L area exhibited highest values than those of the X area (LCK > XCK, LGJ > XGJ, LGZ > XGZ). However, there were no significant differences in the Shannon and Chao indices of fungal communities among the different groups. These findings indicate that growth Vitex rotundifolia L. f. increased increased the species richness and diversity of bacterial and fungal communities (p < 0.05), but as the growth time increased, species richness and diversity decreased. Table 2 Alpha diversity of soil microorganisms Group Bacteria Fungi Chao1 index Shannon index Chao1 index Shannon index LGZ 2965.96 ± 78.59a 9.79 ± 0.25ab 565.06 ± 91.70bc 6.04 ± 0.15ab LGJ 1836 ± 83.69b 9.04 ± 0.19bc 488.75 ± 55.64c 5.44 ± 0.27ab XGZ 3345.33 ± 997.30a 10.28 ± 0.58a 699.23 ± 66.46ab 6.53 ± 0.44a XGJ 3476.66 ± 611.60a 10.16 ± 0.22a 624.59 ± 136.04abc 5.52 ± 0.55b LCK 1297.05 ± 30.56b 7.79 ± 0.21d 766.17 ± 108.42a 5.56 ± 0.28ab XCK 1697.05 ± 69.84c 8.80 ± 0.09c 437.04 ± 42.88c 5.42 ± 0.70b Note: The values in the table are presented as mean ± standard deviation. Different lowercase letters following the values in the same column indicate a significant difference ( p < 0.05). PCOA of the 16S rRNA sequencing data revealed that in the bacterial community 40.03% of the variation accounted for by PCoA1 and PCoA2 (Fig. 3 A), while PCOA of the ITS rDNA sequencing revealed that 28.99% of the variation accounted for by PCoA1 and PCoA2 in the fungal community composition (Fig. 3 B). According to the 16S rRNA data, LGJ was separated from XCK on the X-axis, and LGJ was separated from the other groups on the Y-axis. Based on ITS gene sequencing, LGJ was separated from XCK and LCK on the X-axis, LCK showed the widest span on the y-axis. These findings support the hypothesis that regional differences in soil microbial communities may be associated with the growth of Vitex rotundifolia L. f.. 3.3 Differences in the microbial relative abundance and community structure The relative abundances of soil bacterial communities were compared at the phylum level among the LGZ, LGJ, XGZ, XGJ, LCK and XCK samples (Fig. 4 , Table S2 ). The top 10 taxa accounted for 93.2% of all species, with Actinobacteria, Proteobacteria, Acidobacteriota, and Cyanobacteria being the most abundant phylum (Fig. 4 A). The relative abundances of Cyanobacteria were 22.7% and 15.5% in XGJ and LGJ, respectively, representing 104-fold and 122-fold increases compared to XCK and LCK (Fig. 4 F). The relative abundances of Actinobacteriota (Fig. 4 B) and Proteobacteria (Fig. 4 C) were consistently higher in root-surrounding soil and rhizosphere soil compared to the control group. Overall, the relative abundance of dominant microbial populations (Actinobacteria, Proteobacteria, and Cyanobacteria) increased by 125% in the root-surrounding soil (LGZ, XGZ) and 141% in the rhizoplane soil (LGJ, XGJ). In addition, the composition of the bacterial communities was measured at different taxonomic levels (Fig. 5 , Table S3). At the genus level, the relative abundances of Chloroplast , Acidothermus , and Mycobacterium were significantly higher in root-surrounding soil (LGZ, XGZ) and rhizosphere soils (LGJ, XGJ) compared to the control group (LCK, XCK) (Fig. 5 A). The relative abundance of Acidothermus (Fig. 5 D) and Mycobacterium (Fig. 5 F) increased with increasing growth duration. The relative abundances of soil fungal communities were compared at the phylum level among the LGZ, LGJ, XGZ, XGJ, LCK and XCK samples (Fig. 6 , Table S4). The top 10 taxa accounted for 81.2% of all species, with Ascomycota, Basidiomycota, Mucoromycota, and Chytridiomycota being the most abundant phylum (Fig. 6 A). The relative abundance of Ascomycota was the highest among all six groups, with the highest abundance at 82.8% in LGJ (Fig. 6 B). The relative abundance of Basidiomycota was highest at 43.5% in XGJ, representing 4.93-fold and 2.18-fold increases compared to XCK and LCK respectively, with significant differences observed across all groups (P < 0.05) (Fig. 6 C). The relative abundance of Mucoromycota was highest in root-surrounding soil, followed by rhizosphere soil (Fig. 6 D). The relative abundance of Mucoromycota increased with increasing cultivation duration, with LGZ showing significant differences compared to all other groups (Fig. 6 D). The composition of the fungal communities was measured at different taxonomic levels (Fig. 7 , Table S5). At the genus level, Purpureocillium and Gongronella were most abundant in root-surrounding soil (LGZ, XGZ), followed by rhizosphere soil (LGJ and XGJ) (Fig. 7 A). The relative abundance of Purpureocillium (Fig. 7 D) and Gongronella (Fig. 7 E) increased with increasing growth duration. Biomarkers were compared among the plots (Fig. 8 ). LEfSe is an algorithm used for identifying high-dimensional biomarkers from multiple taxa. In the cladogram of soil bacterial communities (Fig. 8 A, Table S10), LGJ contains the highest number of specific biomarkers, primarily composed of Actinobacteriota, Planctomycetota, and Acidobacteriota. Among these, WD2101_soil_group and Isosphaeraceae are involved in carbon fixation and cycling. The specific biomarkers in LGZ and XGJ include Rhizobiales, Chthoniobacterales, and Burkholderiaceae, which are related to nitrogen fixation. There are significantly more specific biomarkers in LCK than in XCK, mainly composed of Chloroflexi, Firmicutes, and Proteobacteria. In the cladogram of soil fungal communities (Fig. 8 B, Table S11), specific biomarkers mainly comprised Ascomycota, which are most abundant in root-surrounding soil (LGZ, XGZ). 3.4 Correlation between soil physicochemical properties and microorganisms The redundancy analysis (RDA) plot showed the overall relationships between genus populations, environmental factors, and enzyme activities (Fig. 9 ). The first two axes of the RDA explained 47.45% and 12.88% of the total bacteria variation (Fig. 9 A). The top two influential factors were moisture content (MC) and nitrogenase activity (NA) for microbial taxa at the bacterial genus level and moisture content (MC) and pH for microbial taxa at the bacterial species level (Fig. 9 B). Among the top ten genera in terms of relative abundance (Fig. 9 A), IMCC26256 and Subgroup_2 were positively correlated with MC, and Brybacter , Mycobacterium and Conexibacter were positively correlated with total nitrogen (TN). Thermonosporaceae_bacterium and Acidibacter_ferrireducens abundance was positively correlated with urease activity (UA). Actinobacterium_BGR abundance was positively correlated with nitrogenase activity (NA) (Fig. 9 B). For fungal genus (Fig. 9 C), RDA1 accounted for 18.58%, while RDA2 accounted for 10.93% of the total variations. Soil moisture content (MC) was the highest determinant factor followed by total nitrogen (TN) at the fungal genus level and species level (Fig. 9 D). Among the top ten genera in terms of relative abundance (Fig. 9 C), Calcarisporiella , Aspergillus and Talaromyces were positively correlated with MC, and Penicillium , Purpureocillium and Agaricus were positively correlated with total nitrogen (TN). Acremonium_pinkertoniae and Purpureocillium_lilacinum abundance was positively correlated with urease activity (UA) (Fig. 9 D). 3.4 Functional predictions of the microorganisms Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the abundance of functional genes is predicted. Functional predictions of the bacterial communities were performed using PICRUSt2, and FAPROTAX. Comparisons of our data with the KEGG pathway database suggested that metabolism was the main function of soil bacterial, accounting for 70.8% in all genes, followed by genetic information processing, environmental information processing, cellular processes, human diseases, and organismal systems (Fig. 10 A, Table S12). Furthermore, the experimental groups (LGJ, LGZ, XGJ, XGZ) exhibited significant differences compared to control group (LCK) in metabolism, environmental information processing, genetic information processing, and human diseases (P < 0.05) (Fig. 10 B, Table S13). Relatively abundant genes were found to be associated with metabolism of cofactors and vitamins and Metabolism of other amino acids in the L area (LGJ, LGZ); while genes associated with the lipid metabolism, carbohydrate metabolism, and metabolism of terpenoids and polyketides were more prevalent in the X area (XGJ, XGZ) (Fig. 10 C). Functional genes with statistical differences groups (p < 0.05), primarily concentrated between the experimental groups (LGJ, LGZ, XGJ) and the control group (LCK). Notably, 19 identical metabolic genes exhibited significant differences compared to LCK (p < 0.05), with highly significant differences (p < 0.01) in the following pathways: metabolism of other amino acids, transport and catabolism, translation, replication and repair, nucleotide metabolism, and membrane transport (Supplementary Fig S1 ). The metabolic and ecological functions of the bacteria were predicted using FAPROTAX software. The results (Supplementary Fig S2 ) showed that chemoheterotrophy and aerobic chemoheterotrophs were the most abundant in each sample. The abundance of chloroplasts were mainly concentrated in the rhizosphere soil (LGJ, XGJ), while cellulolysis was primarily concentrated in the L region (LGJ, LGZ). Animal parasites or symbionts, aromatic compound degradation, and human pathogens were all significantly enriched in LCK. Functional Guild (FUNGuild) is a tool used for the classification and analysis of fungal communities in a microecological guild. The fungal ASVs into distinct ecological locations after assigning them to various nutritional categories (Fig. 11 ). These ASVs belong to nine trophic modes (Fig. 11 A, Table S14): saprotroph (54.31%), pathotroph-saprotroph-symbiotroph (22.16%), pathotroph-symbiotroph (9.04%), pathotroph (8.48%), pathotroph-saprotroph (2.38%), pathotroph-saprotroph-symbiotroph (2.13%), symbiotroph (1.19%), saprotroph-symbiotroph (0.31%), and saprotroph-pathotroph-symbiotroph (0.01%). The relative abundance of pathotroph-symbiotroph was highest in the rhizosphere soil (LGJ, XGJ), showing statistically significant differences compared to all other groups (P < 0.05). A total of 73 KEGG metabolic pathways were annotated, with 52 having relative abundances greater than 0.01%, including pathways related to wood saprotroph, plant pathogen, fungal parasite, ectomycorrhizal and endophyte. The top 25 most abundant metabolic pathways, the results show: Wood Saprotroph relative abundances were higher in XGJ than in other groups, Ectomycorrhizal fungi were most abundant in XGZ, whereas LCK contained the highest abundances of both plant pathogens and dung saprotrophs (Fig. 11 B, Table S15). The top 20 functional groups in abundance were displayed, with other groups combined into 'Others'(Fig. 11 C). Among the 20 functional groups of fungi, 3 were saprotroph, including undefined Saprotroph, wood saprotroph and dung saprotroph, totaling 50.53%. Only two were pathotroph, including plant pathogen and animal pathogen, which accounted for 7.24%. Moreover, there were ten mixed trophic types of fungi, totaling 29.40%. 4. Discussion 4.1 The growth of Vitex rotundifolia L. f. accelerates the soil formation process and promotes nitrogen accumulation. Consistent with our first hypothesis, the growth of Vitex rotundifolia L. f. significantly increased soil pH, total nitrogen (TN), and alkaline-hydrolyzable nitrogen (AN) compared to bare sandy (Table 1 ), which is consistent with the previously reported mechanisms where pioneer plants improve soil nutrients through root exudates and litter inputs [ 47 ]. In root zones (LGJ) with more than 3 years of growth, TN increased by 17-fold, confirming the role of nodal rooting systems in capturing fine particles and enhancing organic matter deposition [ 32 ]. Elevated urease and nitrogenase activities further indicate a shift from nitrogen-limited conditions to an active nitrogen cycle, a pattern previously reported for Caragana korshinskii in Chinese loess dunes [ 47 ]. However, the decrease in soil moisture highlights the trade-off between plant water uptake microenvironmental amelioration, a process that may become more pronounced under future conditions of precipitation variability [ 48 ]. 4.2 Successional dynamics of bacterial and fungal communities. Principal coordinate analysis (PCoA) (Fig. 3 ) and α diversity (Table 2 ) indicators revealed a clear succession trajectory. Within 1 year planting Vitex rotundifolia L. f. bacterial richness consistently increased (XGJ, XGZ), but long-term planting (> 3 years) led to a decrease in diversity. The temporary surge in bacterial diversity may be attributed to initial plant-microbe interactions promoting the colonization of Vitex rotundifolia L. f. while the competitive exclusion effect of dominant groups in later stages (e.g., Cyanobacteria) reduced bacterial diversity (Fig. 4 A). A similar pattern has been reported in studies of the desert shrub Artemisia ordosica suggesting that ecological restoration requires dynamic monitoring of microbial community succession [ 3 ]. Studies have shown that plants take up microbes, especially bacteria, from these pre-existing soil legacies during the early stages of seedling development [ 35 ]. These proliferating bacterial microorganisms may play a more important role in promoting the growth of Vitex rotundifolia L. f. than the microbial communities present around the roots of older plants. Fungal richness declined with increasing growth duration. After three years of growth, the fungal community of Vitex rotundifolia L. f., the fungal community became dominated by competitively superior taxa, primarily saprotrophic Ascomycota (82.8% in LGJ) and mycorrhizal Basidiomycetes (Fig. 6 ). These antagonistic dynamic changes are similar to those observed in the rhizosphere of Salix psammophila during the early stabilization phase of sand dunes [ 49 ]. 4.3 Key taxonomic groups and functional communities supporting ecosystem functions Changes in the quantity and structure of microorganisms will inevitably impact the associated ecological functions, among which the nitrogen cycle is considered particularly important [ 50 ]. Microorganisms play a key role in regulating the nitrogen cycle, and changes in their abundance will affect their functions [ 50 ]. LEfSe identified Proteobacteria, Actinobacteria and Cyanobacteria, as biomarkers for the soil bacterial community of Vitex rotundifolia L. f. (Fig. 8 A). Proteobacteria is the largest bacterial phylum, involved in almost all nitrogen cycling processes, including ammonia oxidation, nitrite oxidation-reduction reactions, and nitrite reduction to nitric oxide [ 51 , 52 ]. Therefore, Proteobacteria is one of the main bacterial phyla responsible for changes in nitrogen cycling in the soil of Poyang Lake. In addition, previous studies have shown that Chloroflexi, Actinobacteria and Cyanobacteria are also involved in nitrogen cycling, and changes in the abundance of these phyla lead to changes in the dominant functions of the sand dune soil in Poyang Lake [ 53 , 54 ]. Biological soil crusts (BSC) are considered a subsystem or micro-ecosystem, covering up to 70% of the gaps between sparse vegetation in semi-arid and arid regions worldwide [ 55 , 56 ]. They perform a series of ecological functions related to soil stabilization, including carbon and nitrogen fixation and nutrient cycling [ 55 , 56 ]. Particularly noteworthy is the 122-fold increase in cyanobacterial abundance observed in the rhizosphere soil (LGJ) of Vitex rotundifolia L. f. grown for over three years, which may accelerate sand surface stabilization through the establishment of biological crusts formed by filamentous cyanobacteria (Fig. 4 F), while also promoting an increase in soil surface stability and/or water availability. Cyanobacteria and Acidobacteria belong to green bacteria, characterized by the use of chloroplasts for light energy capture [ 57 ]. Studies have found that seasonal changes and extreme drought can temporarily or periodically expose the lake bed of Poyang Lake to sunlight, leading to the transformation of sediment into soil [ 58 ]. Moreover, the abundance of cyanobacteria in the water of Poyang Lake is higher than that in the sediments [ 57 ], a phenomenon also observed in the Yangtze River [ 59 ]. Moreover, the abundance of cyanobacteria in the water of Poyang Lake is higher than that in the sediment, a phenomenon also observed in the Yangtze River. This indicates that the cyanobacteria in the sand dune soil of Poyang Lake may originate from the water of Poyang Lake, and the wet-dry-wet cycle system of Poyang Lake indirectly enhances the microbial diversity of the sand hills. Under current climate change-induced alterations in environmental parameters, ecological restoration work is particularly necessary [ 60 ]. Consequently, developing technologies to enhance ecosystem resilience represents an immediate priority [ 61 , 62 ]. The use of microorganisms in ecological restoration has been mentioned in many studies. Researchers have isolated bacteria that promote plant growth from the rhizosphere soil of Shepherdia utahensis 'Torrey' [ 63 ]. And a study reported plant growth-promoting rhizobacteria from Ceanothus velutinus [ 64 ]. The microbial communities in the sandy hills where Vitex rotundifolia L. f. grows may contain microorganisms that help the plant adapt to extreme conditions representing valuable resources for developing biostimulants or biofertilizers. Subsequent research will implement function-based isolation of rhizosphere bacteria with biological nitrogen fixation capabilities to formulate microbial amendments for combating desertification in humid regions. In fungal communities, the dominance of wood saprotrophs and ectomycorrhizal fungi in rhizosphere soil (Fig. 11 B) suggests the promotion of nutrient cycling by plant-fungal symbiotic systems. Notably, the reduction of plant pathogens in long-term cultivation areas (LGJ) (Fig. 11 B) indicates a shift towards a \"healthy\" microbial community succession. KEGG and FUNGuild analyses revealed significant enrichment in amino acid metabolism and membrane transport pathways in bacterial communities (Fig. 10 C), while the proportion of saprotrophic-symbiotic mixed trophic types (e.g., Purpureocillium) in fungal communities increased with cultivation years (Fig. 11 A). The expression of these functional genes may promote organic matter decomposition and nutrient availability, forming a positive feedback loop of \"plant-microbe-soil\". 4.4 Implications for the ecological restoration of Humid Dune Ecosystems Vitex rotundifolia L. f. promoted microbial and species diversity in the Poyang Lake sand hills by activating positive plant-soil feedback mechanisms. Within two years, the significant improvement in soil total nitrogen content and microbial functions was comparable to that reported for Ammophila arenaria [ 65 ] in temperate sand dunes, yet occurred under significantly higher rainfall conditions (> 1300 mm/year). Therefore, Vitex rotundifolia L. f. is a promising candidate plant for low-input restoration of lake and coastal dunes in subtropical China, especially when traditional engineering measures are costly. 5. Conclusions This study employed 16S rRNA and ITS sequencing technologies to investigate the impact of Vitex rotundifolia L. f. growth on the diversity, composition, and function of bacterial and fungal communities in the sand hills soil of Poyang Lake. Vitex rotundifolia L. f. enhanced rhizosphere soil total nitrogen (TN) by 17-fold and alkaline-hydrolyzable nitrogen (AN) by approximately 20%, with significantly increased urease (UA) and nitrogenase (NA) activities accelerating nutrient accumulation and soil formation processes in the sand hills. Bacterial richness initially increased with plant growth duration of Vitex rotundifolia L. f., and then decreased due to competition from dominant groups such as Cyanobacteria, while fungal richness decreased progressively. Actinobacteria and Proteobacteria were the dominant bacterial groups in the bacterial community, with the relative abundances of Cyanobacteria in the rhizosphere soil being 22.7% and 15.5%, respectively, increasing by 104-fold and 122-fold. In the fungal community, Ascomycota and Basidiomycetes were the dominant groups, with Ascomycota having the highest relative abundance. Redundancy analysis (RDA) revealed that at the genus level of bacteria, the two most influential factors were moisture content (MC) and nitrogenase activity (NA), while at the genus level of fungi, the two most influential factors were soil moisture content (MC) and total nitrogen content (TN). KEGG and FUNGuild analyses revealed significant enrichment in nitrogen fixation, amino acid metabolism and membrane transport pathways in bacterial communities, with a substantial reduction in the proportion of plant pathogens, marking the formation of a \"healthy\" microecosystem. By enhancing nitrogen availability and selecting beneficial microbial communities, Vitex rotundifolia L. f. rapidly transformed its underground environment, thereby accelerating the transformation of mobile sand dunes into stable, nutrient-rich soil. Vitex rotundifolia L. f. can serve as a low-cost, high-benefit pioneer plant to replace or supplement traditional engineering measures, providing a replicable model for ecological restoration of subtropical lakeshores and coastal sand dunes. Abbreviations TCM: Traditional Chinese Medicine PCoA: Principal coordinates analysis PDA: Redundancy analysis KEGG: Kyoto Encyclopedia of Genes and Genomes FAPROTAX: Functional annotation of prokaryotic taxa BSC: Biological soil crusts MC: moisture content TN: total nitrogen AN: Alkali-hydrolyzable nitrogen UA: urease activity NA: nitrogenase activity (NA) Declarations Data availability The 16S rRNA and ITS Sequencing datasets generated during this current study were submitted to the National Center for Biotechnology Information (NCBI) Sequence read archive (SRA) under Bioproject accession PRJNA1304043. The Soil Physicochemical Properties Data were newly generated for this study. The data are available in the following table: [Table S1]. The Microbial Community Correlation Data were newly generated for this study. The data are available in the following table: [Table S2-S5]. The Microbial Community Diversity Data were newly generated for this study. The data are available in the following supplementary file: [Table S6-S9, Supplementary Fig S1]. The Soil Microbial Biomarker Data were newly generated for this study. The data are available in the following table or supplementary file: [Table S10, S11, Supplementary Fig S1]. The KEGG Functional Prediction Data were newly generated and are available in the following table: [Table S12, S13]. The Fungal Functional Prediction Data were newly generated for this study. The data are available in the following table: [Table S14, S15]. Acknowledgements We would like to thank Kai Chen for his help during sampling. Funding The study was supported by Jiangxi Province Double Thousand Talent-Leader of Natural Science Project (jxsq2023101038), Jiangxi Province Urgently Overseas Talent Project (2022BCJ25027), The Key Research Projects in Jiangxi Province (20223BBH8007 & 20232BBG70014), Jiujiang Xinglin Key Laboratory for Traditional Chinese Medicines (S2024ZDSYS037), Natural Science Foundation of Jiujiang (S2024KXJJ0001), and The Key Research Project in Jiujiang (S2023ZDYFN796). Author information Authors and Affiliations Jiangxi Key Laboratory for Sustainable Utilization of Chinese Materia Medica Resources, Lushan Botanical Garden, Chinese Academy of Science, Jiujiang City, Jiangxi Province, 332900, PR China Haijing Xiao, Min Guo, Yiqing Luo, Yiyun Xu, Zhaoqi Xie, Chunsong Cheng Lushan Xinglin Institute for Medicinal Plants & Jiujiang Xinglin Key Laboratory for Traditional Chinese Medicines, Lushan, Jiujiang City, Jiangxi Province, 332900, PR China Haijing Xiao, Min Guo, Yiqing Luo, Yiyun Xu, Zhaoqi Xie, Chunsong Cheng Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences (Joint post-doctoral plan with Jiangxi Key Laboratory for Sustainable Utilization of Chinese Materia Medica Resources), Guangzhou City, Guangdong Province, 510650, PR China Yiqing Luo Contributions HJX analyzed and interpreted the data, and was a major contributor in writing the manuscript. MG and YYX provided and participated in the collection of experimental samples. YQL and ZQX participated in the writing of the manuscript. CSC provided overall guidance for the collection of experimental data and manuscript preparation. All authors read and approved the final manuscript. Corresponding author Correspondence to Chunsong Cheng Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. 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(B) Soil fungal microbial.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/f271a9f3d22fcdcf641eddea.png\"},{\"id\":92980073,\"identity\":\"381e22a6-bbbf-41d1-a1ea-c3ef4d7887ac\",\"added_by\":\"auto\",\"created_at\":\"2025-10-07 19:12:10\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3938983,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRedundancy analysis of the relationship between the microbial communities and soil physicochemical properties. (A) Bacterial communities at the genus levels. (B) Bacterial communities at the species levels. (A) Fungal communities at the genus levels. (B) Fungal communities at the species levels. MC, moisture content; TN, total nitrogen; AN, Alkali-hydrolyzable nitrogen; UA, urease activity; NA, nitrogenase activity.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/809ab634d8c5f5aa547f5073.png\"},{\"id\":92979581,\"identity\":\"53f3bff6-62b5-4856-821f-fe03b9a81c0c\",\"added_by\":\"auto\",\"created_at\":\"2025-10-07 19:04:10\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":8274347,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnalysis of soil bacterial microbial functions. (A) Circle chart of the proportion of functional microorganisms (KEGG level 1). (B) Bar chart of the proportion of functional microorganisms (KEGG level 1). Different lowercase letters indicate significant differences (\\u003cem\\u003ep \\u0026lt; \\u003c/em\\u003e0.05). (C) The relative abundance of the functions (KEGG level 2), where color depth indicates the relative abundance of genes.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/9de2cd0c86ae6a920b06748c.png\"},{\"id\":92980076,\"identity\":\"7c7d9696-0529-4063-9ea4-c204a31939c6\",\"added_by\":\"auto\",\"created_at\":\"2025-10-07 19:12:10\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":10232134,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnalysis of soil fungal microbial functions. (A) Circle chart of fungal trophic modes.(B) Heatmap of the first 25 metabolic pathways. (C) The relative abundances of the top 20 functional groups.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig11.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/86076aef3529604ce612e36b.png\"},{\"id\":96453416,\"identity\":\"466867a4-ae91-4199-80af-fab020034b1b\",\"added_by\":\"auto\",\"created_at\":\"2025-11-21 09:59:43\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":65558392,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/334cf4af-f1e0-430c-8597-5bed3ce78911.pdf\"},{\"id\":92980072,\"identity\":\"7fc3b33f-bc06-475e-b35d-620f0469b46a\",\"added_by\":\"auto\",\"created_at\":\"2025-10-07 19:12:10\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":4224734,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigureSupplementary.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/989d70dba58e7ed12c3021da.docx\"},{\"id\":92978948,\"identity\":\"b8c2d900-e0e1-4421-99ff-5c239015545c\",\"added_by\":\"auto\",\"created_at\":\"2025-10-07 18:56:10\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":130072,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"TableSupplementary.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7181804/v1/a29809ea69baf3d0e3529425.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Vitex rotundifolia L. f. Engineered Microbial Interaction Reverse Desertification: Nitrogen-Fixing Consortia Drive Ecosystem Restoration in Sandy Wetlands of Poyang Lake\",\"fulltext\":[{\"header\":\"1. Background\",\"content\":\"\\u003cp\\u003eDesertification in humid areas means land degradation processes occurring in humid areas resulting from various factors, including climatic variations and human activities [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. The historical development of desertification in humid areas has been closely correlated with anthropogenic activities during the past one to two centuries [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. It has occurred across multiple regions, including South Asia, Southeast Asia, the Mediterranean coast, several sub-Saharan African countries, and extensive areas of southwestern China, exerting substantial negative impacts on both environmental systems and socioeconomic conditions at local and regional scales [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. The desertified land area in humid southern China is estimated at 198,000 km\\u003csup\\u003e2\\u003c/sup\\u003e [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. It exhibits a patchy distribution across the hilly and mountainous terrain of southern China, primarily occurring in areas with weathering crusts developed from red clay, quaternary red clay, granite, and limestone formations, as well as within alluvial floodplains, fluvial sandy terraces along lower river reaches, and lacustrine shoreline environments [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. The main types of desertification in China's humid areas include: water erosion-induced desertification, characterized by eroded badlands and rocky slopes, and wind erosion-induced desertification, marked by wind-eroded lands and mobile sand dunes [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. The Poyang Lake periphery experiences severe land desertification, locally referred to as \\\"sand hills\\\", which belongs to the second desertification type (wind erosion-induced desertification) [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. The desertification area around Poyang Lake spans up to 38,900 km\\u003csup\\u003e2\\u003c/sup\\u003e, accounting for 19.65% of the desertification in China's humid areas [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. It is primarily distributed in the lakeside areas of Poyang Lake and the delta regions of the \\\"Five Rivers\\\" (the Gan, Fu, Xin, Rao, and Xiu River) [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Severe wind and sand erosion has buried farmland, destroyed arable land, exacerbated poverty among local residents, intensified human-land conflicts, caused ecological degradation, and seriously threatening regional ecological and food security [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. is a deciduous shrub or small tree of the Lamiaceae family in the \\u003cem\\u003eVitex\\u003c/em\\u003e L., and evolved from \\u003cem\\u003eVitex trifolia\\u003c/em\\u003e L. [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. It has been used as medicine for over 2,000 years, spanning the Middle Ages and the Renaissance periods [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. has been listed in the prestigious \\u003cem\\u003ePharmacopoeia Londinensis\\u003c/em\\u003e which dates back to the year 1618, and is indicated for the management of traumatic injuries and inflammatory disorders [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. In China, the mature dried fruits of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. or \\u003cem\\u003eVitex trifolia\\u003c/em\\u003e L. are known as \\\"Manjingzi\\\" [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. This herbal medicine was first documented in the \\u003cem\\u003eDivine Farmer\\u0026rsquo;s Classic of Materia Medica\\u003c/em\\u003e and is currently included in the \\u003cem\\u003ePharmacopoeia of the People's Republic of China\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. It is widely used in Traditional Chinese Medicine (TCM) for treating various conditions, including premenstrual syndrome, headache disorders, migraines, colds, and eye pain [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. thrives in beach and dune habitats, widely distributed in desert areas and coastal regions. with mainly found in in provinces such as Jiangxi, Fujian, Shandong, Anhui and Zhejiang [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. The stems develop nodal root systems that enable the formation of dense clonal clusters, extending over 10 meters from the mother plants. Both roots and stems exhibit rapid regeneration rapidly [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. demonstrates remarkable adaptability to sandy and saline-alkali environments, and possesses ecological functions such as windbreak stabilization, sand fixation, water storage, and moisture retention [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Recognized as a pioneer species for sandy ecosystem rehabilitation and coastal landscaping, it represents a multifunctional resource with integrated social, ecological, and medicinal values [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe vegetation of the sand hills in Poyang Lake has the characteristics of a subtropical desert [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. The shrub layer vegetation in Poyang Lake's sand hills ecosystem is primarily constructed by dominant and pioneer species including \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f., \\u003cem\\u003eArtemisia capillaris\\u003c/em\\u003e Thunb., and \\u003cem\\u003eLespedeza bicolor\\u003c/em\\u003e Turcz.. Among these, \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. holds a significant position on different types of sand dunes (flowing sand dunes, semi-flowing sand dunes, and fixed sand dunes) [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Our field observations reveal that \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. exhibits a patchy distribution pattern across the sand hills. The rhizosphere soil within these patches displays significantly darker coloration compared to bare sand, demonstrating this species' ability to improve edaphic conditions in dune systems. The research on \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. has primarily focused on traditional medicine, phytochemistry, and pharmacology [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]., with relatively few studies investigating its mechanisms for soil amelioration and ecological restoration. Plants can modify the nitrogen cycle in the soil, transforming inherent and unchanging componentsinto available nutrients, and altering the structure and function of the soil microbial communities. Meanwhile, soil microorganisms regulating the nitrogen cycle through decomposition of soil organic matter and plant residues [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Therefore, understanding the compositional composition and function characteristics of soil microbial communities is crucial for studying \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. growth, contributing to a deeper exploration of the microbial ecological mechanisms underlying variations in Poyang Lake's sand hills [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThis study employed 16S rRNA and ITS sequencing techniques to investigate the diversity and composition of bacterial and fungal communities in the soil of the sand hills in Poyang Lake. A comparative microbiome analysis was performed across three chronosequence stages: (1) non-\\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. areas, (2) 1-year \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. growth areas, and (3) multi-year \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. growth areas, to assess successional microbial shifts. The relationship between soil physicochemical properties (environmental factors and enzyme activities) related to the nitrogen cycle and microorganisms was studied. The functions of soil microorganisms were predicted to identify metabolic pathways related to different growth areas of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f.. This study aimed to provide insights into the ecological mechanisms of growth of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f., providing scientific data for standardized cultivation in Poyang Lake's desertified areas, and offering governance approaches for the ecological restoration of deserts in humid regions, thus achieving the goal of ecological conservation (lucid waters and lush mountains) and sustainable socio-economic benefits (golden and silver mountains).\\u003c/p\\u003e\"},{\"header\":\"2. Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e\\u003cb\\u003e2.1 Study sites description\\u003c/b\\u003e\\u003c/h2\\u003e\\u003cp\\u003eIn this study, sandy soil from the experimental area of Liaohua Town, Xingzi County, Jiujiang City, Jiangxi Province (115\\u0026deg;48\\u0026prime; to 116\\u0026deg;10\\u0026prime;E, 29\\u0026deg;8\\u0026prime; to 29\\u0026deg;36\\u0026prime;N) was used as the material. The test area is located on the left bank of the floodway connecting Poyang Lake to the Yangtze River. It belongs to the mid-subtropical humid zone, characterized by high temperatures, abundant rainfall, distinct dry and wet seasons, and an uneven distribution of seasonal precipitation throughout the year [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. The average annual temperature is 16.5\\u0026thinsp;~\\u0026thinsp;17.8℃, while the average and highest surface temperatures are 21.3℃ and 69.5℃, respectively [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. The annual evaporation is 1880 mm, and the annual precipitation ranges from 1300\\u0026thinsp;~\\u0026thinsp;1600 mm. Precipitation mainly occurs in spring and summer, with limited rainfall in autumn and winter. Cold north winds are common in winter, with wind speeds reaching up to 17 m/s, resulting in lake shrinkage and the exposure of lakebed sand [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.2 Soil sampling\\u003c/h2\\u003e\\u003cp\\u003eTwo sampling areas, L and X, were established in the experimental area, and sampling was conducted in October 2023 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Area L consisted of a \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. community with more than three years of growth. Area X was prepared in October 2022 by leveling 20 acres of sandy land and planting \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f.. Thus, Area X consists of a \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. community that has been growing for less than two years. Blank control (LCK, XCK) samples were collected from sandy areas (L area, X area) without the growth of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f.. The root-surrounding soil (LGZ, XGZ) samples were collected from soil within 5\\u0026ndash;20 cm around the roots of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f.. The rhizosphere soil samples (LGJ, XGJ) were collected by gently brushing the tightly adhered soil from the roots of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. using a sterile soft-bristled brush. Samplings were selected five-point sampling method, soil samples from five different locations were mixed and transferred into sterile plastic bags. The samples were divided into two parts, one part was air-dried for the determination of soil pH, moisture content (MC), total nitrogen (TN), Alkali-hydrolyzable nitrogen (AN), urease activity (UA) and nitrogenase activity (NA) content, and the other part was kept in sterile centrifugal tubes and frozen in -80 ℃ refrigerator for spare.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e\\u003cb\\u003e2.3 Soil physicochemical indicators and enzyme activity analysis\\u003c/b\\u003e\\u003c/h2\\u003e\\u003cp\\u003eSoil water content was determined using the drying method. pH was determined using pH 368 (resolution 0.01 pH). TN was determined using the Semi-micro kjeldahl method [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. AN was determined using the alkali diffusion method [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. UA was determined using the indophenol blue colorimetric method [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. NA was determined using the microbial nitrogenase ELISA kit [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.4 DNA extraction, amplification, and sequencing\\u003c/h2\\u003e\\u003cp\\u003eNucleic acids were extracted from samples using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA) kit. The DNA concentration was then detected using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Scientific, USA). We used an ABI GeneAmp\\u0026reg; 9700 thermal cycler for DNA fragment amplification. The 16s rRNA V3-V4 region was selected for bacterial PCR amplification with primer sequences 341F (5'-CCTACGGGGNGGCWGCAG-3'), 805R (5'-GACTACHVGGGGTATCTAATCC-3 '). The ITS rDNA ITS region was selected for PCR amplification of the fungus, and the primer sequences were ITS1F (5'-GGAAGTAAAAGTCGTAACAAGG-3'), ITS2R (5'-GCTGCGTTCTTCATCGATGC-3 '). The PCR reflection parameters were: the DNA was denatured at 98\\u0026deg;C for 5 min in a single cycle; then denatured at 98\\u0026deg;C for 30 s, annealed at 52\\u0026deg;C for 30 s, and extended at 72\\u0026deg;C for 45 s, for a total of 28 cycles; and finally, the product was kept at 72\\u0026deg;C for 5 min to make the product extension complete and stored at 12\\u0026deg;C. Finally, the amplification products were screened by 2% agarose gel electrophoresis, and the target fragments were recovered by sorted magnetic bead recovery method. Nova Seq 6000 SP Reagent Kit (500 cycles) was utilized to perform 2\\u0026times;250bp double-end sequencing to obtain raw sequencing data.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.5 Statistical analysis\\u003c/h2\\u003e\\u003cp\\u003eRaw microbiome sequencing data were spliced and optimized using Fastp v0.19.6 and FLASH v1.2.7 software. The optimized data were then processed using the DADA2 method for sequence noise reduction, resulting in the identification of amplicon sequence variants (ASVs) within the samples. The representative sequences of the ASVs were classified and annotated with QIIME2 v2022.2 software, utilizing the bacterial 16S rRNA gene Silva database and the fungal ITS UNITE database as reference sources, respectively.\\u003c/p\\u003e\\u003cp\\u003eFigs were performed applying Excel 2019, Origin 2018 and GraphPad Prism 9. One-way ANOVA was carried out using SPSS version 25.0, and the significance of differences was assessed using the Least Significant Difference method. Differences in the values with P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 (*) were considered statistically significant. LEfSe analyses were performed using an online data analysis tool (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://huttenhower.sph.harvard.edu/galaxy/\\u003c/span\\u003e\\u003cspan address=\\\"http://huttenhower.sph.harvard.edu/galaxy/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), and the LDA discriminant value of 4.0 was selected. Redundancy analysis (RDA) was used to elucidate the overall relationships between microbial communities and related factors. Functional differences among the microbial communities were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Fungal functional guild assignment for each ASV was performed using FUNGuild version 1.0.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.1 Physicochemical properties of the soil samples\\u003c/h2\\u003e\\n \\u003cp\\u003eThe growth of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. has changed the Physicochemical properties of the sand hills in Poyang Lake (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, Table \\u003cspan class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). Soil water content was significantly lower in plots grown with Vi\\u003cem\\u003etex rotundifolia\\u003c/em\\u003e L. f. than without growing in plots (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The root-surrounding soil and rhizosphere soil exhibited decreasing water content with increasing growth duration. Soils planted with\\u0026nbsp;\\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. showed significantly higher values of pH, TN, AN, and UA compared to non-planted control soils. Furthermore, these parameters in root-surrounding and rhizosphere soils showed increases with extended growth duration.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003ctable id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eSoil physicochemical properties of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. with different stand ages and locations.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eIndex\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLGZ\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLGJ\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eXGZ\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eXGJ\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLCK\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eXCK\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMoisture\\u003c/p\\u003e\\n \\u003cp\\u003eContent (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.21\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15bc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.57\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.23c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.29\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.27b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.92\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.30bc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.22a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.27a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003epH\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.17ab\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.38a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21bc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.72\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.20a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.54\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.24c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTN (g/kg)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.05b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.65\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.77\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.84\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.27\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02d\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.29\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01d\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAN (mg/kg)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e53.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.96a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e53.57\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.73a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e48.73\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.93b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e53.91\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.00a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e45.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.51c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e43.68\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.12c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eUA (U/g)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e255.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.11c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e462.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;15.67a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e240.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;7.17c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e299.22\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.78b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e214.79\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.13d\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e191.87\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.20e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNA (U/g)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.49\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03d\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.68\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.04b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.40\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01d\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003ctfoot\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"7\\\"\\u003eNote: Different letters in the same row indicate significant differences among different treatments (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05).\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tfoot\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.2 Diversity of the microbial community\\u003c/h2\\u003e\\n \\u003cp\\u003eMicrobiome sequencing of soil samples from areas L and X yielded a total of 24,172 bacterial ASVs and 6,456 fungal ASVs, with average sequence lengths of 221 bp and 220 bp, respectively. Shared species analysis was performed for common bacterial and fungal species among the six sampling sites (LGZ, LGJ, XGZ, XGJ, LCK, XCK), and a petal diagram was generated to visualize the results (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The proportions of species in bacteria and fungi were 78.9% and 21.1%, respectively, and shared bacterial and fungal species among the six sampling sites accounted for 0.22% and 1.17%, respectively. In the bacterial community, the number of ASVs shared and unique in area X (XGZ, XGJ) is the highest among all groups (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). In the fungal community, the highest number of ASVs shared with other groups was found in area X (XGZ, XGJ), while the highest number of ASVs unique in LCK (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB).\\u003c/p\\u003e\\n \\u003cp\\u003eThe Chao and Shannon indices were used to assess the alpha diversity of the microbial community, with the former reflecting species richness and the latter reflecting species diversity (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, Table S6-S9). In the bacterial community, both indices of the X area exhibited highest values than those of the L area (XCK\\u0026thinsp;\\u0026gt;\\u0026thinsp;LCK, XGJ\\u0026thinsp;\\u0026gt;\\u0026thinsp;LGJ, XGZ\\u0026thinsp;\\u0026gt;\\u0026thinsp;LGZ), particularly in the Shannon index, which displayed significant differences across all regions (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Regarding the fungal community, both indices of the L area exhibited highest values than those of the X area (LCK\\u0026thinsp;\\u0026gt;\\u0026thinsp;XCK, LGJ\\u0026thinsp;\\u0026gt;\\u0026thinsp;XGJ, LGZ\\u0026thinsp;\\u0026gt;\\u0026thinsp;XGZ). However, there were no significant differences in the Shannon and Chao indices of fungal communities among the different groups. These findings indicate that growth\\u0026nbsp;\\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. increased increased the species richness and diversity of bacterial and fungal communities (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), but as the growth time increased, species richness and diversity decreased.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003ctable id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eAlpha diversity of soil microorganisms\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eGroup\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eBacteria\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eFungi\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eChao1 index\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eShannon index\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eChao1 index\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eShannon index\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLGZ\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2965.96\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;78.59a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.79\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.25ab\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e565.06\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;91.70bc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.04\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15ab\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLGJ\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1836\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;83.69b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.04\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19bc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e488.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;55.64c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.44\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.27ab\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eXGZ\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3345.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;997.30a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10.28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.58a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e699.23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;66.46ab\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.53\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.44a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eXGJ\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3476.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;611.60a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10.16\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.22a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e624.59\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;136.04abc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.55b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLCK\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1297.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;30.56b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.79\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21d\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e766.17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;108.42a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.56\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.28ab\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eXCK\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1697.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;69.84c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8.80\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e437.04\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;42.88c\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.42\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.70b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003ctfoot\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"5\\\"\\u003eNote: The values in the table are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation. Different lowercase letters following the values in the same column indicate a significant difference (\\u003cem\\u003ep\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.05).\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tfoot\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003ePCOA of the 16S rRNA sequencing data revealed that in the bacterial community 40.03% of the variation accounted for by PCoA1 and PCoA2 (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), while PCOA of the ITS rDNA sequencing revealed that 28.99% of the variation accounted for by PCoA1 and PCoA2 in the fungal community composition (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). According to the 16S rRNA data, LGJ was separated from XCK on the X-axis, and LGJ was separated from the other groups on the Y-axis. Based on ITS gene sequencing, LGJ was separated from XCK and LCK on the X-axis, LCK showed the widest span on the y-axis. These findings support the hypothesis that regional differences in soil microbial communities may be associated with the growth of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f..\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.3 Differences in the microbial relative abundance and community structure\\u003c/h2\\u003e\\n \\u003cp\\u003eThe relative abundances of soil bacterial communities were compared at the phylum level among the LGZ, LGJ, XGZ, XGJ, LCK and XCK samples (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Table \\u003cspan class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e). The top 10 taxa accounted for 93.2% of all species, with Actinobacteria, Proteobacteria, Acidobacteriota, and Cyanobacteria being the most abundant phylum (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The relative abundances of Cyanobacteria were 22.7% and 15.5% in XGJ and LGJ, respectively, representing 104-fold and 122-fold increases compared to XCK and LCK (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF). The relative abundances of Actinobacteriota (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB) and Proteobacteria (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC) were consistently higher in root-surrounding soil and rhizosphere soil compared to the control group. Overall, the relative abundance of dominant microbial populations (Actinobacteria, Proteobacteria, and Cyanobacteria) increased by 125% in the root-surrounding soil (LGZ, XGZ) and 141% in the rhizoplane soil (LGJ, XGJ). In addition, the composition of the bacterial communities was measured at different taxonomic levels (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, Table S3). At the genus level, the relative abundances of \\u003cem\\u003eChloroplast\\u003c/em\\u003e, \\u003cem\\u003eAcidothermus\\u003c/em\\u003e, and \\u003cem\\u003eMycobacterium\\u003c/em\\u003e were significantly higher in root-surrounding soil (LGZ, XGZ) and rhizosphere soils (LGJ, XGJ) compared to the control group (LCK, XCK) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). The relative abundance of \\u003cem\\u003eAcidothermus\\u003c/em\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD) and \\u003cem\\u003eMycobacterium\\u003c/em\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eF) increased with increasing growth duration.\\u003c/p\\u003e\\n \\u003cp\\u003eThe relative abundances of soil fungal communities were compared at the phylum level among the LGZ, LGJ, XGZ, XGJ, LCK and XCK samples (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e, Table S4). The top 10 taxa accounted for 81.2% of all species, with Ascomycota, Basidiomycota, Mucoromycota, and Chytridiomycota being the most abundant phylum (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). The relative abundance of Ascomycota was the highest among all six groups, with the highest abundance at 82.8% in LGJ (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). The relative abundance of Basidiomycota was highest at 43.5% in XGJ, representing 4.93-fold and 2.18-fold increases compared to XCK and LCK respectively, with significant differences observed across all groups (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC). The relative abundance of Mucoromycota was highest in root-surrounding soil, followed by rhizosphere soil (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD). The relative abundance of Mucoromycota increased with increasing cultivation duration, with LGZ showing significant differences compared to all other groups (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD). The composition of the fungal communities was measured at different taxonomic levels (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e, Table S5). At the genus level, \\u003cem\\u003ePurpureocillium\\u003c/em\\u003e and \\u003cem\\u003eGongronella\\u003c/em\\u003e were most abundant in root-surrounding soil (LGZ, XGZ), followed by rhizosphere soil (LGJ and XGJ) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA). The relative abundance of \\u003cem\\u003ePurpureocillium\\u003c/em\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eD) and \\u003cem\\u003eGongronella\\u003c/em\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eE) increased with increasing growth duration.\\u003c/p\\u003e\\n \\u003cp\\u003eBiomarkers were compared among the plots (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). LEfSe is an algorithm used for identifying high-dimensional biomarkers from multiple taxa. In the cladogram of soil bacterial communities (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA, Table S10), LGJ contains the highest number of specific biomarkers, primarily composed of Actinobacteriota, Planctomycetota, and Acidobacteriota. Among these, WD2101_soil_group and Isosphaeraceae are involved in carbon fixation and cycling. The specific biomarkers in LGZ and XGJ include Rhizobiales, Chthoniobacterales, and Burkholderiaceae, which are related to nitrogen fixation. There are significantly more specific biomarkers in LCK than in XCK, mainly composed of Chloroflexi, Firmicutes, and Proteobacteria. In the cladogram of soil fungal communities (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eB, Table S11), specific biomarkers mainly comprised Ascomycota, which are most abundant in root-surrounding soil (LGZ, XGZ).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4 Correlation between soil physicochemical properties and microorganisms\\u003c/h2\\u003e\\n \\u003cp\\u003eThe redundancy analysis (RDA) plot showed the overall relationships between genus populations, environmental factors, and enzyme activities (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e). The first two axes of the RDA explained 47.45% and 12.88% of the total bacteria variation (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eA). The top two influential factors were moisture content (MC) and nitrogenase activity (NA) for microbial taxa at the bacterial genus level and moisture content (MC) and pH for microbial taxa at the bacterial species level (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eB). Among the top ten genera in terms of relative abundance (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eA), \\u003cem\\u003eIMCC26256\\u003c/em\\u003e and \\u003cem\\u003eSubgroup_2\\u003c/em\\u003e were positively correlated with MC, and \\u003cem\\u003eBrybacter\\u003c/em\\u003e, \\u003cem\\u003eMycobacterium\\u003c/em\\u003e and \\u003cem\\u003eConexibacter\\u003c/em\\u003e were positively correlated with total nitrogen (TN). \\u003cem\\u003eThermonosporaceae_bacterium\\u003c/em\\u003e and \\u003cem\\u003eAcidibacter_ferrireducens\\u003c/em\\u003e abundance was positively correlated with urease activity (UA). \\u003cem\\u003eActinobacterium_BGR\\u003c/em\\u003e abundance was positively correlated with nitrogenase activity (NA) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eB). For fungal genus (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eC), RDA1 accounted for 18.58%, while RDA2 accounted for 10.93% of the total variations. Soil moisture content (MC) was the highest determinant factor followed by total nitrogen (TN) at the fungal genus level and species level (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eD). Among the top ten genera in terms of relative abundance (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eC), \\u003cem\\u003eCalcarisporiella\\u003c/em\\u003e, \\u003cem\\u003eAspergillus\\u003c/em\\u003e and \\u003cem\\u003eTalaromyces\\u003c/em\\u003e were positively correlated with MC, and \\u003cem\\u003ePenicillium\\u003c/em\\u003e, \\u003cem\\u003ePurpureocillium\\u003c/em\\u003e and \\u003cem\\u003eAgaricus\\u003c/em\\u003e were positively correlated with total nitrogen (TN). \\u003cem\\u003eAcremonium_pinkertoniae\\u003c/em\\u003e and \\u003cem\\u003ePurpureocillium_lilacinum\\u003c/em\\u003e abundance was positively correlated with urease activity (UA) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eD).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4 Functional predictions of the microorganisms\\u003c/h2\\u003e\\n \\u003cp\\u003eBased on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the abundance of functional genes is predicted. Functional predictions of the bacterial communities were performed using PICRUSt2, and FAPROTAX. Comparisons of our data with the KEGG pathway database suggested that metabolism was the main function of soil bacterial, accounting for 70.8% in all genes, followed by genetic information processing, environmental information processing, cellular processes, human diseases, and organismal systems (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eA, Table S12). Furthermore, the experimental groups (LGJ, LGZ, XGJ, XGZ) exhibited significant differences compared to control group (LCK) in metabolism, environmental information processing, genetic information processing, and human diseases (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eB, Table S13). Relatively abundant genes were found to be associated with metabolism of cofactors and vitamins and Metabolism of other amino acids in the L area (LGJ, LGZ); while genes associated with the lipid metabolism, carbohydrate metabolism, and metabolism of terpenoids and polyketides were more prevalent in the X area (XGJ, XGZ) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eC). Functional genes with statistical differences groups (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), primarily concentrated between the experimental groups (LGJ, LGZ, XGJ) and the control group (LCK). Notably, 19 identical metabolic genes exhibited significant differences compared to LCK (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), with highly significant differences (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) in the following pathways: metabolism of other amino acids, transport and catabolism, translation, replication and repair, nucleotide metabolism, and membrane transport (Supplementary Fig \\u003cspan class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). The metabolic and ecological functions of the bacteria were predicted using FAPROTAX software. The results (Supplementary Fig \\u003cspan class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e) showed that chemoheterotrophy and aerobic chemoheterotrophs were the most abundant in each sample. The abundance of chloroplasts were mainly concentrated in the rhizosphere soil (LGJ, XGJ), while cellulolysis was primarily concentrated in the L region (LGJ, LGZ). Animal parasites or symbionts, aromatic compound degradation, and human pathogens were all significantly enriched in LCK.\\u003c/p\\u003e\\n \\u003cp\\u003eFunctional Guild (FUNGuild) is a tool used for the classification and analysis of fungal communities in a microecological guild. The fungal ASVs into distinct ecological locations after assigning them to various nutritional categories (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003e). These ASVs belong to nine trophic modes (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eA, Table S14): saprotroph (54.31%), pathotroph-saprotroph-symbiotroph (22.16%), pathotroph-symbiotroph (9.04%), pathotroph (8.48%), pathotroph-saprotroph (2.38%), pathotroph-saprotroph-symbiotroph (2.13%), symbiotroph (1.19%), saprotroph-symbiotroph (0.31%), and saprotroph-pathotroph-symbiotroph (0.01%). The relative abundance of pathotroph-symbiotroph was highest in the rhizosphere soil (LGJ, XGJ), showing statistically significant differences compared to all other groups (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). A total of 73 KEGG metabolic pathways were annotated, with 52 having relative abundances greater than 0.01%, including pathways related to wood saprotroph, plant pathogen, fungal parasite, ectomycorrhizal and endophyte. The top 25 most abundant metabolic pathways, the results show: Wood Saprotroph relative abundances were higher in XGJ than in other groups, Ectomycorrhizal fungi were most abundant in XGZ, whereas LCK contained the highest abundances of both plant pathogens and dung saprotrophs (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eB, Table S15). The top 20 functional groups in abundance were displayed, with other groups combined into \\u0026apos;Others\\u0026apos;(Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eC). Among the 20 functional groups of fungi, 3 were saprotroph, including undefined Saprotroph, wood saprotroph and dung saprotroph, totaling 50.53%. Only two were pathotroph, including plant pathogen and animal pathogen, which accounted for 7.24%. Moreover, there were ten mixed trophic types of fungi, totaling 29.40%.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003e\\u003cb\\u003e4.1 The growth of\\u003c/b\\u003e \\u003cb\\u003eVitex rotundifolia\\u003c/b\\u003e \\u003cb\\u003eL. f. accelerates the soil formation process and promotes nitrogen accumulation.\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eConsistent with our first hypothesis, the growth of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. significantly increased soil pH, total nitrogen (TN), and alkaline-hydrolyzable nitrogen (AN) compared to bare sandy (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), which is consistent with the previously reported mechanisms where pioneer plants improve soil nutrients through root exudates and litter inputs [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. In root zones (LGJ) with more than 3 years of growth, TN increased by 17-fold, confirming the role of nodal rooting systems in capturing fine particles and enhancing organic matter deposition [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Elevated urease and nitrogenase activities further indicate a shift from nitrogen-limited conditions to an active nitrogen cycle, a pattern previously reported for \\u003cem\\u003eCaragana korshinskii\\u003c/em\\u003e in Chinese loess dunes [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. However, the decrease in soil moisture highlights the trade-off between plant water uptake microenvironmental amelioration, a process that may become more pronounced under future conditions of precipitation variability [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e4.2 Successional dynamics of bacterial and fungal communities.\\u003c/h2\\u003e\\u003cp\\u003ePrincipal coordinate analysis (PCoA) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) and α diversity (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e) indicators revealed a clear succession trajectory. Within 1 year planting \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. bacterial richness consistently increased (XGJ, XGZ), but long-term planting (\\u0026gt;\\u0026thinsp;3 years) led to a decrease in diversity. The temporary surge in bacterial diversity may be attributed to initial plant-microbe interactions promoting the colonization of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. while the competitive exclusion effect of dominant groups in later stages (e.g., Cyanobacteria) reduced bacterial diversity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). A similar pattern has been reported in studies of the desert shrub Artemisia ordosica suggesting that ecological restoration requires dynamic monitoring of microbial community succession [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Studies have shown that plants take up microbes, especially bacteria, from these pre-existing soil legacies during the early stages of seedling development [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. These proliferating bacterial microorganisms may play a more important role in promoting the growth of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. than the microbial communities present around the roots of older plants. Fungal richness declined with increasing growth duration. After three years of growth, the fungal community of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f., the fungal community became dominated by competitively superior taxa, primarily saprotrophic Ascomycota (82.8% in LGJ) and mycorrhizal Basidiomycetes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). These antagonistic dynamic changes are similar to those observed in the rhizosphere of Salix psammophila during the early stabilization phase of sand dunes [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e4.3 Key taxonomic groups and functional communities supporting ecosystem functions\\u003c/h2\\u003e\\u003cp\\u003eChanges in the quantity and structure of microorganisms will inevitably impact the associated ecological functions, among which the nitrogen cycle is considered particularly important [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. Microorganisms play a key role in regulating the nitrogen cycle, and changes in their abundance will affect their functions [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. LEfSe identified Proteobacteria, Actinobacteria and Cyanobacteria, as biomarkers for the soil bacterial community of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA). Proteobacteria is the largest bacterial phylum, involved in almost all nitrogen cycling processes, including ammonia oxidation, nitrite oxidation-reduction reactions, and nitrite reduction to nitric oxide [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. Therefore, Proteobacteria is one of the main bacterial phyla responsible for changes in nitrogen cycling in the soil of Poyang Lake. In addition, previous studies have shown that Chloroflexi, Actinobacteria and Cyanobacteria are also involved in nitrogen cycling, and changes in the abundance of these phyla lead to changes in the dominant functions of the sand dune soil in Poyang Lake [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eBiological soil crusts (BSC) are considered a subsystem or micro-ecosystem, covering up to 70% of the gaps between sparse vegetation in semi-arid and arid regions worldwide [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. They perform a series of ecological functions related to soil stabilization, including carbon and nitrogen fixation and nutrient cycling [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. Particularly noteworthy is the 122-fold increase in cyanobacterial abundance observed in the rhizosphere soil (LGJ) of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. grown for over three years, which may accelerate sand surface stabilization through the establishment of biological crusts formed by filamentous cyanobacteria (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF), while also promoting an increase in soil surface stability and/or water availability.\\u003c/p\\u003e\\u003cp\\u003eCyanobacteria and Acidobacteria belong to green bacteria, characterized by the use of chloroplasts for light energy capture [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. Studies have found that seasonal changes and extreme drought can temporarily or periodically expose the lake bed of Poyang Lake to sunlight, leading to the transformation of sediment into soil [\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e]. Moreover, the abundance of cyanobacteria in the water of Poyang Lake is higher than that in the sediments [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e], a phenomenon also observed in the Yangtze River [\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e]. Moreover, the abundance of cyanobacteria in the water of Poyang Lake is higher than that in the sediment, a phenomenon also observed in the Yangtze River. This indicates that the cyanobacteria in the sand dune soil of Poyang Lake may originate from the water of Poyang Lake, and the wet-dry-wet cycle system of Poyang Lake indirectly enhances the microbial diversity of the sand hills.\\u003c/p\\u003e\\u003cp\\u003eUnder current climate change-induced alterations in environmental parameters, ecological restoration work is particularly necessary [\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e]. Consequently, developing technologies to enhance ecosystem resilience represents an immediate priority [\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e]. The use of microorganisms in ecological restoration has been mentioned in many studies. Researchers have isolated bacteria that promote plant growth from the rhizosphere soil of \\u003cem\\u003eShepherdia utahensis\\u003c/em\\u003e 'Torrey' [\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e]. And a study reported plant growth-promoting rhizobacteria from \\u003cem\\u003eCeanothus velutinus\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e]. The microbial communities in the sandy hills where \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. grows may contain microorganisms that help the plant adapt to extreme conditions representing valuable resources for developing biostimulants or biofertilizers. Subsequent research will implement function-based isolation of rhizosphere bacteria with biological nitrogen fixation capabilities to formulate microbial amendments for combating desertification in humid regions.\\u003c/p\\u003e\\u003cp\\u003eIn fungal communities, the dominance of wood saprotrophs and ectomycorrhizal fungi in rhizosphere soil (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eB) suggests the promotion of nutrient cycling by plant-fungal symbiotic systems. Notably, the reduction of plant pathogens in long-term cultivation areas (LGJ) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eB) indicates a shift towards a \\\"healthy\\\" microbial community succession. KEGG and FUNGuild analyses revealed significant enrichment in amino acid metabolism and membrane transport pathways in bacterial communities (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eC), while the proportion of saprotrophic-symbiotic mixed trophic types (e.g., Purpureocillium) in fungal communities increased with cultivation years (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eA). The expression of these functional genes may promote organic matter decomposition and nutrient availability, forming a positive feedback loop of \\\"plant-microbe-soil\\\".\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e4.4 Implications for the ecological restoration of Humid Dune Ecosystems\\u003c/h2\\u003e\\u003cp\\u003e\\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. promoted microbial and species diversity in the Poyang Lake sand hills by activating positive plant-soil feedback mechanisms. Within two years, the significant improvement in soil total nitrogen content and microbial functions was comparable to that reported for \\u003cem\\u003eAmmophila arenaria\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e] in temperate sand dunes, yet occurred under significantly higher rainfall conditions (\\u0026gt;\\u0026thinsp;1300 mm/year). Therefore, \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. is a promising candidate plant for low-input restoration of lake and coastal dunes in subtropical China, especially when traditional engineering measures are costly.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"5. Conclusions\",\"content\":\"\\u003cp\\u003eThis study employed 16S rRNA and ITS sequencing technologies to investigate the impact of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. growth on the diversity, composition, and function of bacterial and fungal communities in the sand hills soil of Poyang Lake. \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. enhanced rhizosphere soil total nitrogen (TN) by 17-fold and alkaline-hydrolyzable nitrogen (AN) by approximately 20%, with significantly increased urease (UA) and nitrogenase (NA) activities accelerating nutrient accumulation and soil formation processes in the sand hills. Bacterial richness initially increased with plant growth duration of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f., and then decreased due to competition from dominant groups such as Cyanobacteria, while fungal richness decreased progressively. Actinobacteria and Proteobacteria were the dominant bacterial groups in the bacterial community, with the relative abundances of Cyanobacteria in the rhizosphere soil being 22.7% and 15.5%, respectively, increasing by 104-fold and 122-fold. In the fungal community, Ascomycota and Basidiomycetes were the dominant groups, with Ascomycota having the highest relative abundance. Redundancy analysis (RDA) revealed that at the genus level of bacteria, the two most influential factors were moisture content (MC) and nitrogenase activity (NA), while at the genus level of fungi, the two most influential factors were soil moisture content (MC) and total nitrogen content (TN). KEGG and FUNGuild analyses revealed significant enrichment in nitrogen fixation, amino acid metabolism and membrane transport pathways in bacterial communities, with a substantial reduction in the proportion of plant pathogens, marking the formation of a \\\"healthy\\\" microecosystem. By enhancing nitrogen availability and selecting beneficial microbial communities, \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. rapidly transformed its underground environment, thereby accelerating the transformation of mobile sand dunes into stable, nutrient-rich soil. \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. can serve as a low-cost, high-benefit pioneer plant to replace or supplement traditional engineering measures, providing a replicable model for ecological restoration of subtropical lakeshores and coastal sand dunes.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eTCM:\\u003c/em\\u003e\\u003c/strong\\u003eTraditional Chinese Medicine\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003ePCoA:\\u003c/em\\u003e\\u003c/strong\\u003e Principal coordinates analysis\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003ePDA:\\u003c/em\\u003e\\u003c/strong\\u003e Redundancy analysis\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eKEGG:\\u003c/em\\u003e\\u003c/strong\\u003e Kyoto Encyclopedia of Genes and Genomes\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eFAPROTAX:\\u003c/em\\u003e\\u003c/strong\\u003e Functional annotation of prokaryotic taxa\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eBSC:\\u003c/em\\u003e\\u003c/strong\\u003e Biological soil crusts\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eMC:\\u003c/em\\u003e\\u003c/strong\\u003e moisture content\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eTN:\\u003c/em\\u003e\\u003c/strong\\u003e total nitrogen\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAN:\\u003c/em\\u003e\\u003c/strong\\u003e Alkali-hydrolyzable nitrogen\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eUA:\\u003c/em\\u003e\\u003c/strong\\u003e urease activity\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eNA:\\u003c/em\\u003e\\u003c/strong\\u003e nitrogenase activity (NA)\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe 16S rRNA and ITS Sequencing datasets generated during this current study were submitted to the National Center for Biotechnology Information (NCBI) Sequence read archive (SRA) under Bioproject accession PRJNA1304043.\\u003c/p\\u003e\\n\\u003cp\\u003eThe Soil Physicochemical Properties Data were newly generated for this study. The data are available in the following table: [Table S1].\\u003c/p\\u003e\\n\\u003cp\\u003eThe Microbial Community Correlation Data were newly generated for this study. The data are available in the following table: [Table S2-S5].\\u003c/p\\u003e\\n\\u003cp\\u003eThe Microbial Community Diversity Data were newly generated for this study. The data are available in the following supplementary file: [Table S6-S9, Supplementary Fig S1].\\u003c/p\\u003e\\n\\u003cp\\u003eThe Soil Microbial Biomarker Data were newly generated for this study. The data are available in the following table or supplementary file: [Table S10, S11, Supplementary Fig S1].\\u003c/p\\u003e\\n\\u003cp\\u003eThe KEGG Functional Prediction Data were newly generated and are available in the following table: [Table S12, S13].\\u003c/p\\u003e\\n\\u003cp\\u003eThe Fungal Functional Prediction Data were newly generated for this study. The data are available in the following table: [Table S14, S15].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe would like to thank Kai Chen for his help during sampling.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe study was supported by Jiangxi Province Double Thousand Talent-Leader of Natural Science Project (jxsq2023101038), Jiangxi Province Urgently Overseas Talent Project (2022BCJ25027), The Key Research Projects in Jiangxi Province (20223BBH8007 \\u0026amp; 20232BBG70014), Jiujiang Xinglin Key Laboratory for Traditional Chinese Medicines (S2024ZDSYS037), Natural Science Foundation of Jiujiang (S2024KXJJ0001), and The Key Research Project in Jiujiang (S2023ZDYFN796).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors and Affiliations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eJiangxi Key Laboratory for Sustainable Utilization of Chinese Materia Medica Resources, Lushan Botanical Garden, Chinese Academy of Science, Jiujiang City, Jiangxi Province, 332900, PR China\\u003c/p\\u003e\\n\\u003cp\\u003eHaijing Xiao, Min Guo, Yiqing Luo, Yiyun Xu, Zhaoqi Xie, Chunsong Cheng\\u003c/p\\u003e\\n\\u003cp\\u003eLushan Xinglin Institute for Medicinal Plants \\u0026amp; Jiujiang Xinglin Key Laboratory for Traditional Chinese Medicines, Lushan, Jiujiang City, Jiangxi Province, 332900, PR China\\u003c/p\\u003e\\n\\u003cp\\u003eHaijing Xiao, Min Guo, Yiqing Luo, Yiyun Xu, Zhaoqi Xie, Chunsong Cheng\\u003c/p\\u003e\\n\\u003cp\\u003eGuangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences (Joint post-doctoral plan with Jiangxi Key Laboratory for Sustainable Utilization of Chinese Materia Medica Resources), Guangzhou City, Guangdong Province, 510650, PR China\\u003c/p\\u003e\\n\\u003cp\\u003eYiqing Luo\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eContributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHJX analyzed and interpreted the data, and was a major contributor in writing the manuscript. MG and YYX provided and participated in the collection of experimental samples. YQL and ZQX participated in the writing of the manuscript. CSC provided overall guidance for the collection of experimental data and manuscript preparation. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCorresponding author\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCorrespondence to Chunsong Cheng\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePublisher\\u0026apos;s Note\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBelow is the link to the electronic supplementary material.\\u003c/p\\u003e\\n\\u003cp\\u003eFigure Supplementary\\u003c/p\\u003e\\n\\u003cp\\u003eTable Supplementary\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRights and permissions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eOpen Access\\u003c/strong\\u003e This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eCui SH. Desertification in humid area. Quaternary Sci. 1998;18(2):173\\u0026ndash;81.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZhu ZD, Concept. Cause and control of Desertification in China. Quaternary Sci. 1998;18(2):145\\u0026ndash;55.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFeng Q, Ma H, Jiang X, Wang X, Cao S. 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Mycorrhiza. 2024;34(3):159\\u0026ndash;71. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s00572-024-01144-w\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s00572-024-01144-w\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e\\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\":\"info@researchsquare.com\",\"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\":\"Vitex rotundifolia L. f., microbiome analysis, function prediction, Poyang Lake, sand hills\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7181804/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7181804/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground:\\u003c/strong\\u003e The sandy areas of Poyang Lake are a typical region of wind erosion desertification in humid areas, and their ecological restoration urgently requires efficient, low-cost plant and microbial co-management measures. \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f., a local dominant pioneer shrub, has both medicinal and ecological value, but its mechanism of influence on sandy soil microorganisms remains unclear. To systematically assess how \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. drives sand dune ecological restoration by altering soil physical and chemical properties and microbial community structure.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults:\\u003c/strong\\u003e The total nitrogen (TN) and alkali-hydrolyzable nitrogen (AN) in the rhizosphere soil of \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003e L. f. grown for more than 3 years were 17-fold and 1.2-fold higher than those of bare sand, respectively. The activities of urease (UA) and nitrogenase (NA) significantly increased, while the moisture content slightly decreased. The bacterial α-diversity first increased and then slightly decreased, while fungal diversity decreased with increasing growth years. Cyanobacteria in the rhizosphere soil surged by 122-fold, while Actinobacteria and Proteobacteria increased by 1.3-fold and 1.4-fold, respectively. Fungi were dominated by Ascomycota and Basidiomycota. Functional prediction showed that nitrogen fixation, amino acid metabolism, and membrane transport pathways were significantly enriched. The proportion of wood-decaying fungi and ectomycorrhizal fungi increased, while pathogenic bacteria significantly decreased.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion:\\u003c/strong\\u003e \\u003cem\\u003eVitex rotundifolia\\u003c/em\\u003eL. f. can significantly accelerate soil formation and ecological restoration of wet sand dunes in Poyang Lake by promoting nitrogen accumulation, constructing beneficial microbial communities, and forming biological crusts, providing a new strategy for low-cost and large-scale restoration of sandy land in humid areas.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Vitex rotundifolia L. f. Engineered Microbial Interaction Reverse Desertification: Nitrogen-Fixing Consortia Drive Ecosystem Restoration in Sandy Wetlands of Poyang Lake\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-10-07 18:56:05\",\"doi\":\"10.21203/rs.3.rs-7181804/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"e4b9ab88-5473-45f8-b664-b44a13db5125\",\"owner\":[],\"postedDate\":\"October 7th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-11-24T01:53:10+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-10-07 18:56:05\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7181804\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7181804\",\"identity\":\"rs-7181804\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}