Soil Moisture Modulates Rhizosphere Microbiota and Suppresses Root Rot in Organic Panax ginseng Cultivated Under Pine Forests

Soil Moisture Modulates Rhizosphere Microbiota and Suppresses Root Rot in Organic Panax ginseng Cultivated Under Pine Forests | 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 Soil Moisture Modulates Rhizosphere Microbiota and Suppresses Root Rot in Organic Panax ginseng Cultivated Under Pine Forests Huiling Wang, Rongshuang Sha, Qiongying Kang, Tao Zhou, Zhenxing Wang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8659215/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 Aims The integration of agroforestry and understory cultivation offers a sustainable approach to producing high-value medicinal plants with ecological conservation. However, optimal irrigation management for organic Panax ginseng grown under pine forests (OPPF) remains poorly studied, particularly regarding its influence on root rot disease and rhizosphere microbial communities. Methods This study evaluated four soil moisture treatments—PTh1 (55–60% field capacity, FC), PTh2 (70–75% FC), PTh3 (80–85% FC), and PTh4 (95–100% FC)—on the growth, quality, and root rot incidence of OPPF. We analyzed agronomic traits, secondary metabolites, soil properties, enzymatic activities, and rhizosphere microbial composition via high-throughput sequencing and qPCR, and validated microbial isolates functionally through pot experiments. Results Moderate soil moisture (PTh2) significantly improved plant biomass, seedling survival, and the accumulation of flavonoids, polysaccharides, and ginsenosides. In contrast, high soil moisture (PTh4) increased the incidence of root rot and the abundance of pathogens such as Fusarium solani and Ilyonectria mors-panacis . Soil properties and enzyme activities were markedly influenced by moisture levels, with PTh2 maintaining a higher pH and beneficial nutrient profile. Rhizosphere microbiome analysis revealed that PTh2 enriched potential biocontrol agents, including Trichoderma , Penicillium , Bacillus , and Streptomyces , while reducing pathogenic taxa. Six antagonistic strains were isolated, with Penicillium ortum (F1) showing the strongest suppression of root rot and promotion of plant growth in pot trials. Conclusions Maintaining soil moisture at 70–75% FC optimizes the growth and health of OPPF by fostering beneficial microbial communities and inhibiting soil-borne pathogens. These findings provide science-based irrigation strategies to support the sustainable cultivation of organic ginseng in forest-based agroecosystems. Agroforestry Panax ginseng soil moisture root rot rhizosphere microbiome disease suppression sustainable agriculture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Panax ginseng ( Panax ginseng C.A. Meyer) is a valuable medicinal herb widely recognized for its health-promoting properties, such as anti-diabetic and anti-fatigue effects (Kim et al. 2023 ). The primary cultivation methods include conventional farmland cultivation (cultivated ginseng) and understory cultivation (wild-simulated ginseng) (Bao et al. 2020 ). However, ginseng production faces significant constraints due to continuous cropping obstacles and the overuse of chemical pesticides, which contribute to a decline in crop quality. These unsustainable practices represent a common challenge in the cultivation of many medicinal plants. Extensive studies on conventional cultivation have demonstrated that continuous cropping leads to soil microbial dysbiosis and aggravates soil-borne diseases, with root rot caused by Fusarium and Ilyonectria spp. being a key factor limiting sustainable production (Xia et al. 2016 ). In response, researchers both domestically and internationally have attempted various measures such as soil disinfection, crop rotation, and soil amendments, yet these approaches often show limited efficacy or introduce new environmental risks. In recent years, the ecologically friendly understory cultivation model has gained attention. Research indicates that this system can leverage forest biodiversity to suppress foliar diseases (e.g., Alternaria panax ) and improve medicinal quality (He et al. 2022 ). However, existing studies have primarily focused on the effects of the forest environment on growth and foliar diseases, with systematic research on soil moisture management—a critical agronomic practice in this system—remaining notably scarce. Soil moisture not only directly affects plant physiology but also plays a key role in shaping rhizosphere microbial community structure and function, thereby influencing the incidence of soil-borne diseases (Patel et al. 2021 ;Berg et al. 2016 ). Although substantial knowledge exists on water management for grain crops and some commercial tree species, targeted studies on precise soil moisture regulation for understory medicinal plants, particularly ginseng, are virtually absent. Moreover, in organic P. ginseng cultivation under pine forests (OPPF) systems where rain shelters are used to control leaf diseases, the alteration of natural rainfall patterns adds complexity to soil moisture management, making the development of science-based irrigation strategies both necessary and underexplored. To address these gaps, this study focuses on the OPPF system and is the first to systematically investigate the integrated effects of different soil moisture levels (55–60%, 70–75%, 80–85%, and 95–100% of field capacity) on ginseng growth, quality, root rot incidence, and rhizosphere microecology. We hypothesize that soil moisture influences root rot occurrence by altering the rhizosphere microbial community structure and that potentially exploitable biocontrol microbial resources may exist within this microbiome. Therefore, the objectives of this study are: (1) to identify the optimal soil moisture range for enhancing OPPF biomass and quality while reducing root rot incidence, and (2) to elucidate how soil moisture drives changes in rhizosphere microbial diversity—particularly in pathogenic and antagonistic populations—and its mechanistic association with root rot disease. The findings will provide a direct theoretical basis and technical support for establishing ecologically based water management protocols in OPPF systems, offering significant academic value and practical potential. Materials and methods Experimental design and treatments The experiment was conducted in June 2023 at an organic Panax ginseng under pine forest (OPPF) test base located at the Lanheyu Forestry Center (41°09′56.98″ N, 124°26′20.70″ E) in Liaoning, China. The site experiences a mid-temperate monsoon humid climate, with a mean annual temperature of 7.5°C and annual precipitation of 1017.5 mm. Initial soil properties included pH 5.12, alkaline hydrolysis nitrogen (AHN) 441.83 mg/kg, electrical conductivity (EC) 281.63 µS/cm, and a field capacity (FC) of 48%. The starting soil moisture level was 68.52% FC. A total of 240 pots (upper diameter: 184 mm; height: 164 mm; bottom diameter: 132 mm) were used, each filled with 1090 g of soil and planted with three three-year-old P. ginseng seedlings. Pots were assigned to four soil moisture treatments: PTh1 (55–60% FC), PTh2 (70–75% FC), PTh3 (80–85% FC), and PTh4 (95–100% FC). Soil moisture was maintained by weekly pot weighing and water adjustment. All pots were placed on artificial ridges under a pine canopy to simulate natural OPPF conditions. Measurement of agronomic traits and chlorophyll content After three months of moisture treatment, plants were harvested and transported to the laboratory. Soil was carefully removed from roots, and samples were air-dried before measuring fresh weight. Leaf length, leaf width, and fibrous root length were recorded, and leaf area was calculated based on leaf dimensions (Wang et al. 2016 ). Chlorophyll content was measured using a SPAD-502 handheld meter (Konica Minolta, Japan). Per treatment, 96 plants were randomly selected and grouped into six biological replicates for average fresh weight calculation. Analysis of saponin, flavonoid, and polysaccharide quality, and total polysaccharides Saponin content (including ginsenosides Rd, Re, Rc, Rg1, Rf, Rb1, Rb3, Rb2, Rg2, Rh1, Rh2, and Rg3) in ginseng roots was quantified using a modified UPLC method adapted from Li et al. (Li et al. 2013 ). Separation was performed on a Nexera X2 system (Shimadzu, Japan) equipped with a DAD detector and a Poroshell 120 EC-C18 column (Agilent; 4 µm, 100 × 2.1 mm). The mobile phase consisted of acetonitrile (solvent A) and 0.1% aqueous solution (solvent B) under gradient conditions (Supplementary Table S1 ). The injection volume was 10 µL, flow rate 1.0 mL/min, column temperature 30°C, and detection wavelength 203 nm. Identification and quantification were performed using ginsenoside standards (Supplementary Fig. S1 ). Flavonoid, polysaccharide, and total polysaccharide contents were determined using commercial assay kits (Michy Bio, China). Root rot assessment and pathogen isolation At harvest, 144 plants per treatment (6 replicates) were evaluated for root rot severity using the rating scale established by Li et al. (Li et al. 2020 ). Disease incidence and index were calculated as described by Yang et al. (Yang et al. 2022 ). Fungal pathogens were isolated from symptomatic root tissues following Li et al. (Li et al. 2020 ). Genomic DNA was extracted using a 2×T5 Direct PCR kit (TSINGKE, China). The ITS region was amplified with primers ITS1/ITS4 (Hawa et al. 2017 ) and sequenced. Phylogenetic analysis was conducted in MEGA 7.0. Pathogenicity of isolates was confirmed using the method of Luo et al. (Luo et al. 2020 ). Soil physicochemical properties and enzyme activities Soil samples were collected and analyzed for physicochemical parameters according to Guo et al. (Guo et al. 2022 ). Activities of soil polyphenol oxidase (S-PPO), dehydrogenase (S-DHA), sucrase (S-SC), and cellulase (S-CL) were measured using specific assay kits (Michy Bio, China). DNA extraction, sequencing, and bioinformatic Total genomic DNA was extracted from soil samples, and amplification and library preparation were conducted following Edgar et al. (Edgar, 2013 ). High-throughput sequencing of bacterial 16S rRNA and fungal ITS regions was performed on the Illumina platform. Bioinformatic processing included quality filtering, OTU clustering, and taxonomic assignment using standard pipelines. Quantification of pathogenic fungi and antagonistic via qPCR DNA from soil samples was reverse-transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Quantitative real-time PCR (qPCR) was performed with strain-specific primers (Table S2 )(Géry et al. 2021 ;Ye et al. 2019 ༛Gao et al. 2020 ༛Li et al. 2012 ༛Saravanakumar et al. 2017 ༛Reiter et al. 2011 ). Each 30 µL reaction contained 10 µL qPCR Mix, 0.5 µL each of forward and reverse primers (10 µM), 2 µL template DNA, and nuclease-free water. Amplification conditions followed Géry et al.(2021). Isolation of culturable rhizosphere and functions validation Rhizosphere microorganisms were isolated and purified as described by Guo et al. ( 2022 ). Functional validation was performed using pot assays under sterile soil conditions based on Fang et al. ( 2024 ). Three-year-old ginseng seedlings were planted in pots inoculated with 50 mL of a suspension containing pathogenic fungi (10⁶ CFU/mL) and candidate antagonistic bacteria (OD 600 = 0.5). Control groups received pathogen suspension only or sterile water. Each treatment included three replicates of five pots. Plants were grown in a greenhouse at 25 ± 2°C for 60 days, after which fresh weight and root rot incidence were recorded. Statistical analysis All data were analyzed using PASW Statistics 18 (SPSS Inc. USA). One-way ANOVA followed by Tukey’s HSD test ( p < 0.05) was used to determine significant differences among treatments. Data normality and homogeneity of variance were verified beforehand. Correlation analyses were conducted using Spearman’s rank correlation method (Fang et al. 2024 ). Results Optimal soil moisture level (PTh2) enhances growth and quality in OPPF-cultivated P. ginseng Significant differences in agronomic traits and seedling survival rates were observed among P. ginseng plants subjected to varying soil moisture levels after three months of treatment (Fig. 1 A–D). Plants under the PTh2 regime (70–75% FC) exhibited markedly greater fresh weight, fibrous root length, and chlorophyll content compared to those in PTh1, PTh3, and PTh4 (Fig. 1 B). These results were consistent with field trial data from 2024 (Supplementary Table S3). Analysis of secondary metabolites revealed that PTh2 also led to significantly higher flavonoid and total carbohydrate contents in fibrous roots relative to other treatments (Fig. 1 C). Total polysaccharide content was notably elevated in PTh2 compared to the remaining moisture regimes. Although total saponin levels in roots increased with rising soil moisture (Fig. 1 C), individual ginsenosides responded differently across tissue types and treatments. In fibrous roots, significant variations were detected in the contents of Rd, Rb1, Rb3, Rh2, and Rg3. In taproots, Rd, Rc, Rg1, Rf, Rb1, Rb3, Rb2, Rg2, Rh1, Rh2, and Rg3 levels differed significantly among treatments, with Rg2, Rb1, and Rh1 increasing with soil moisture. Notably, PTh3 resulted in higher Rc content in roots and elevated Rd in fibrous roots compared to other treatments (Supplementary Fig. S2 ). Overall, soil moisture strongly influenced ginseng growth and quality, particularly flavonoid and saponin accumulation (Supplementary Fig.S3). Seedling survival was significantly higher in PTh2 than in PTh1 and PTh4 (Fig. 1 D). Both root rot incidence and disease index were significantly lower in PTh1 and PTh2 than in PTh4, and a clear positive correlation was observed between soil moisture level and root rot severity (Fig. 1 D). These findings were corroborated by 2024 field data (Supplementary Fig.S4), confirming the critical role of soil moisture in disease development under OPPF conditions. Soil properties and enzyme activities are influenced by soil moisture regimes Soil moisture levels significantly influenced various soil physicochemical properties and enzymatic activities (Fig. 2 ). Soil pH was significantly higher under the PTh2 treatment compared to PTh1, PTh3, and PTh4 (Fig. 2 A). In contrast, soil electrical conductivity was markedly elevated in PTh3 relative to the other moisture regimes (Fig. 2 B). Soil alkaline hydrolysis nitrogen (AHN), dehydrogenase (S-DHA), sucrase (S-SC), and cellulase (S-CL) activities were highest under the PTh1 treatment, showing significant differences compared to PTh2, PTh3, and PTh4 (Fig. 2 C–G). Redundancy analysis (RDA) further confirmed that soil properties and enzyme activities varied systematically with soil moisture levels (Fig. 2 H). Specifically, soil conductivity exhibited a negative correlation with S-DHA and S-SC activities, but was positively associated with moisture content in PTh3 and PTh4. Rhizosphere microbial community structure shifts with soil moisture Principal coordinate analysis (PCoA) and ANOSIM revealed significant differences in bacterial (R = 0.5467, p = 0.001) and fungal (R = 0.4779, p = 0.001) community structures among the four soil moisture treatments (Supplementary Fig.S5). Although alpha diversity indices (Chao1, Shannon, and observed species) did not differ significantly across treatments (Supplementary Fig.S4), bacterial diversity was generally higher in PTh2 and PTh3, while fungal diversity was notably elevated under the PTh2 regime. Soil moisture alters the relative abundance of key microbial taxa At the phylum level, Ascomycota dominated the fungal community under all soil moisture conditions, accompanied by Basidiomycota , Mortierellomycota , Olpidiomycota , Mucoromycota , Glomeromycota , Rozellomycota , Chytridiomycota , and Zoopagomycota (Fig. 3 A). The relative abundance of Ascomycota was higher in PTh1 and PTh4 compared to PTh2, whereas Basidiomycota and Mortierellomycota were more abundant in PTh2 and PTh3. Among bacteria, nine dominant phyla were identified: Proteobacteria , Acidobacteria , Verrucomicrobiota , Chloroflexi , Actinobacteria , Myxococcota , Firmicutes , and Bacteroidota (Fig. 3 B). Proteobacteria and Acidobacteria were the most abundant (each > 1% relative abundance), with Acidobacteria increasing and Actinobacteria decreasing as soil moisture rose. At the genus level, fungi such as Humiicola , Trichoderma , Saitozyma , Pseudogymnoascus , and Mortierella were more abundant under lower soil moisture but declined with increasing moisture (Fig. 3 C). In contrast, known pathogenic genera including Ilyonectria , Cylindrocarpon , Fusarium , Neonectria , Stagonosporopsis , and Chaetomium were most prevalent in PTh3. Among bacteria, Pseudomonas increased with soil moisture, while Arenimonas , Collimonas , Burkholderia-Caballeronia-Paraburkholderia (B-C-P), and Bacillus decreased in abundance under higher moisture conditions (Fig. 3 D). Correlation analysis and abundance of potential beneficial microbiomes Analysis of relationships between root rot incidence and microbial communities focused on taxa with relative abundances exceeding 0.1% (Fig. 4 A). Within fungal communities, disease index (DI), incidence of rot disease (IRD), and moisture gradient (MG) showed positive correlations with Fusarium , but negative correlations with Trichoderma , Chaetosphaeria , Chloridium , Mortierella , and Penicillium . Among bacterial communities, DI, IRD, and MG correlated positively with Aquicella , but negatively with Arenimonas and the Burkholderia-Caballeronia-Paraburkholderia (B-C-P) complex. Further analysis revealed significant correlations between dominant fungal and bacterial genera and key soil properties (Fig. 4 B, C). Soil electrical conductivity (EC) correlated positively with Fusarium and Ilyonectria , but negatively with Talaromyces . In contrast, alkaline hydrolysis nitrogen (AHN), dehydrogenase (S-DHA), and cellulase (S-CL) activities were positively associated with Talaromyces and negatively associated with Fusarium and Ilyonectria . Soil pH and polyphenol oxidase (S-PPO) activity showed negative correlations with Fusarium , while soil moisture correlated positively with Crossiella and negatively with Novosphingobium , B-C-P complex, Bradyrhizobium , and Arenimonas . AHN, S-DHA, and S-CL exhibited opposite correlation patterns with these bacterial genera. Quantitative PCR (qPCR) analysis of rhizosphere soil indicated that the copy numbers of the root rot pathogens Fusarium solani and Ilyonectria mors-panacis were significantly elevated under the PTh4 treatment. Conversely, antagonistic microbial populations were more abundant under both PTh1 and PTh4 conditions. Functional validation of culturable microbial strains Pathogens R3 and R6, isolated from symptomatic roots, were identified as Ilyonectria mors-panacis and Fusarium solani , respectively. Pathogenicity tests confirmed that both strains induced root rot symptoms in OPPF plants, whereas control plants remained healthy. Using a dual-culture assay, three bacterial strains (L11, A1, G1) and three fungal strains (F1, F2, F3) were identified that inhibited the growth of R6 and R3 by more than 30%. Based on BLAST analysis, the bacterial strains were identified as Bacillus mycoides (G1), Bacillus velezensis (L11), and Streptomyces drozdowiczii (A1), and the fungal strains as Penicillium ortum (F1), Talaromyces amestolkiae (F2), and Trichoderma koningii (F3). Pot experiments demonstrated that all six strains promoted ginseng growth and suppressed root rot. Among them, strain F1 ( Penicillium ortum ) exhibited the strongest disease suppression and most significantly enhanced plant biomass. Discussion Despite the potential of agroforestry systems to alleviate land-use pressures, research on the cultivation of Chinese medicinal plants—particularly organic ginseng—under forest canopy remains limited. Few studies have addressed soil moisture management specifically for organic Panax ginseng grown in understory environments. Our findings indicate that both drought (PTh1) and waterlogging (PTh4) impaired the healthy growth of understory ginseng, whereas moderate soil moisture (PTh2; 70–75% FC) promoted plant growth and increased the accumulation of polysaccharides, flavonoids, and saponins. PTh2 also resulted in superior biomass production and lower root rot incidence compared to the high-moisture PTh4 treatment. Furthermore, this study provides novel insights into how soil moisture influences rhizosphere microbial community structure and identifies several antagonistic microorganisms, offering practical strategies for irrigation management in forest-based ginseng cultivation systems. Soil moisture modulates growth and metabolite accumulation in P. ginseng Plant phenotypic traits are strongly influenced by environmental cues, which in turn affect biomass and secondary metabolism (Loretta, 2014 ). Soil moisture plays a critical role in root development and overall plant health, particularly in medicinal species such as Glycyrrhiza uralensis , where drought stress significantly curtails growth (Yang et al. 2022 ;Sun et al. 2017 ). Conversely, excess moisture can also inhibit growth after an initial increase (Li et al. 2015 ). In our study, biomass peaked under moderate soil moisture (PTh2) and declined under both water-deficit and waterlogged conditions, consistent with reports that extreme moisture levels negatively affect ginseng biomass accumulation(Liu et al. 2016 ). Notably, flavonoid and total carbohydrate contents were highest under PTh2, whereas polysaccharide and total saponin levels responded differently across treatments. Total flavonoids—commonly used as a quality marker—decreased significantly under severe water loss (Yang et al. 2020 ;Zhang, 2012 ), while polysaccharides increased under moderate stress. Total saponin content was positively correlated with soil moisture, rising significantly under high-water conditions (Puente-Garza et al. 2017 ). Individual ginsenosides (e.g., Rg1, Rf, Re, Rg3, Rb2, Rd, and Rh2) were lowest under optimal moisture and elevated under stress, underscoring how environmental conditions shape the phytochemical profile of medicinal plants (Zheng et al. 2021 ༛Lee and Mudge, 2014 ). These patterns align with observations in P. notoginseng , where saponin content varied under water stress(Zheng et al. 2021 ), highlighting species-specific metabolic plasticity. Suboptimal soil moisture alters rhizosphere microbiome and promotes root rot Soil moisture strongly influences the rhizosphere micro-environment and the prevalence of soil-borne diseases. In this study, higher soil moisture correlated with increased incidence and severity of ginseng root rot, consistent with earlier reports (Yang et al. 2022 ;Guo et al. 2022 ). Changes in moisture regimes also affected key soil properties such as pH and electrical conductivity, which in turn influenced pathogen behavior (Stover, 2011 ). We found negative correlations between root rot incidence and soil pH and alkaline hydrolysis nitrogen, whereas electrical conductivity showed a positive correlation with disease. Sustained high moisture (PTh4; 95–100% FC) favored anaerobic microorganisms and known pathogens such as Fusarium and Ilyonectria , in line with studies showing that waterlogged conditions reduce microbial diversity and disrupt community balance (Harman et al. 2021 ). Such shifts likely suppressed the activity of beneficial enzymes and microbes, exacerbating disease incidence (Guo et al. 2022 ). Maintaining soil moisture at 60–75% FC helped minimize root rot, corroborating field observations (Yang et al. 2022 ;Guo et al. 2022 ). Future studies should employ metagenomic and transcriptomic tools to unravel plant–microbe interactions under dynamic moisture conditions (Shi et al. 2019 ), offering deeper mechanistic insights into moisture-mediated disease regulation. Moisture-driven microbial community shifts influence disease resistance in OPPF High-throughput sequencing revealed that soil moisture significantly shaped the composition of bacterial and fungal communities in the ginseng rhizosphere. Dominant bacterial phyla included Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi, with genera such as Streptomyces and Bacillus —known bio-control agents—showing higher abundance under moderate moisture (Kobayashi et al. 2015 ;Moretti et al. 2021 ༛Tran, 2021 ). Pathogenic genera like Aquicella were positively correlated with root rot incidence. Fungal communities were dominated by Ascomycota, Basidiomycota, and Mortierellomycota (Liu et al. 2020 ). Known pathogens including Fusarium and Ilyonectria were more abundant in high-moisture treatments, whereas antagonistic fungi such as Trichoderma and Penicillium were enriched under drier conditions (Zhang, 2012 ;Ozimek and Hanaka, 2020 ༛Tian et al. 2021 ). These patterns support the concept that plants under stress can recruit beneficial microbes for protection, as observed in P. notoginseng with Arthrobacter (Fang et al. 2024 ). Interestingly, qPCR results indicated higher abundance of antagonistic strains under both low and high moisture, suggesting complex, context-dependent microbial interactions (Lou et al. 2018 ༛Tettamanti Boshier et al. 2020 ). Conclusions and implications This study demonstrates that maintaining soil moisture at 70–75% FC optimizes the growth, quality, and health of organic P. ginseng under pine forests. We identified key microbial taxa and several culturable strains (e.g., Bacillus velezensis , Trichoderma koningii , and Penicillium ortum ) with high biocontrol potential against root rot pathogens. These findings underscore the importance of irrigation management in balancing rhizosphere microbiota and suppressing disease in agroforestry systems. Future research would focus on the dynamic monitoring of microbial succession under fluctuating soil moisture, mechanistic studies on plant–microbe interactions using multi-omics approaches, field validation of optimal irrigation regimes and biocontrol agents, and economic and sustainability assessments of moisture management practices in ginseng agroforestry. Such efforts will advance our capacity to implement ecologically sound and productive cultivation systems for high-value medicinal plants. Declarations The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. Funding This work was supported by the China Agriculture Research System of MOF and MARA (CARS − 21), Yunnan Agricultural Joint Special Project Key Projects (202301 BD070001–138), Youth Talents Special Project of Yunnan Province, “Xingdian Talents Support Program (YNWR-QNBJ-2020194). Author contributions Huiling Wang and Xiahong He led the project. Huiling Wang and Rongshuang Sha handled field experiments and microbiom data analysis, with support from Wensong Sun and Lifu Sun in Liaoning Province. Tao Zhou, Qiongying Kang and Dan Li used HPLC-MS for data analysis. Huiling Wang, Zhenxing Wang and Baoyu Shen conducted microbial validation in pot experiments. Huiling Wang and Rongshuang Sha drafted the manuscript, which was revised by Rui Shi, Shu He and Xiahong He. 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Plant Sci 12:755733. https://doi.org/10.3389/fpls.2021.755733 Wang SJ, Jiang XL, Zhang GF, Yao YS, Xu YH, Zhao Y, Li G (2016) Establish of forecasting model of leaves aera on Panax ginseng . J Northeast Agric Sci 41(6):109–112. https://doi.org/10.16423/j.cnki.1003-8701.2016.06.024 Xia P, Guo H, Zhao H, Jiao J, Deyholos MK, Yan X, Liu Y, Liang Z (2016) Optimal fertilizer application for Panax notoginseng and effect of soil water on root rot disease and saponin contents. J Ginseng Res 40(1):38–46. https://doi.org/10.1016/j.jgr.2015.04.003 Yang K, Wang H, Luo L, Zhu S, Huang H, Wei Z, Zhu Y, Guo L, He X (2022) Effects of different soil moisture on the growth, quality, and root rot disease of organic Panax notoginseng cultivated under pine forests. J Environ Manage 329:117069. https://doi.org/10.1016/j.jenvman.2022.117069 Yang LL, Yang L, Yang X, Zhang T, Lan YM, Zhao Y, Han M, Yang LM (2020) Drought stress induces biosynthesis of flavonoids in leaves and saikosaponins in roots of Bupleurum Chinese DC. Phytochemistry 177:112434. https://doi.org/10.1016/j.phytochem.2020.112434 Ye XZ, Yang ZH, Zhang QH, Song Z, Chen QZ (2019) Molecular detection of Fusarium solani , the pathogen of Eucalyptus wilt disease. J Environ 39(6):629–635. https://doi.org/10.13324/j.cnki.jfcf.2019.06.010 Zhang UL (2012) Growth, physiological characteristics, and total flavonoid content of Glechoma longituba in response to water stress. J Med Plants Res 6(6):1015–1024. https://doi.org/10.5897/JMPR11.758 Zheng Y, Xia P, Zhao H, Zheng JF, Chai WG, Liang ZS, Yan KJ (2021) Suitable soil moisture contents for water use efficiency and saponins accumulation in Panax notoginseng . Chin Herb Med 13(2):267–273. https://doi.org/10.1016/j.chmed.2020.10.002 Supplementary Files GraphicalAbstract.docx Highlight.docx declarationStatement.docx Linefigure.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8659215","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594207386,"identity":"e59f00a1-754b-430d-8e0f-55f03ad85666","order_by":0,"name":"Huiling Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYDACCebGBwwMciCmAbFaGJuBSo1J09ImQZoWg9uNbZU/2wwSG9ibt0kw1NwhQsudg223eUFaeI6VSTAce0ZYi9mNxLbbjNv+JDZI5JhJMDYcJk5L4c9tQFvk35CghYEXpEWCh0gt9jcSm6V5/xkYt/GkFVskHCNCi+SM5IMff5wxkO1nP7zxxocaIrTAARuISCBBwygYBaNgFIwCPAAAcvE512fbMQ8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7768-8753","institution":"Southwest Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Huiling","middleName":"","lastName":"Wang","suffix":""},{"id":594207387,"identity":"05bbee1a-798e-410c-accd-1d3d106cb907","order_by":1,"name":"Rongshuang Sha","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rongshuang","middleName":"","lastName":"Sha","suffix":""},{"id":594207388,"identity":"ee331068-86e5-4a15-b593-aee534d49baa","order_by":2,"name":"Qiongying Kang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qiongying","middleName":"","lastName":"Kang","suffix":""},{"id":594207389,"identity":"dc26309e-049d-41c9-b268-757efe714c3d","order_by":3,"name":"Tao Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Zhou","suffix":""},{"id":594207390,"identity":"09b8aaf4-a029-4464-a070-e72c427f825c","order_by":4,"name":"Zhenxing Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhenxing","middleName":"","lastName":"Wang","suffix":""},{"id":594207391,"identity":"9eb10144-acfb-4512-b525-42eb97b4177c","order_by":5,"name":"Rui Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Shi","suffix":""},{"id":594207392,"identity":"5f623d7d-7f22-4f9a-8fcb-298525dbb4d2","order_by":6,"name":"Dan Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Li","suffix":""},{"id":594207393,"identity":"30b57f1b-d028-4b66-9ce7-ddd22884a488","order_by":7,"name":"Shu He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"","lastName":"He","suffix":""},{"id":594207394,"identity":"cd291563-e652-4e6d-91e5-78a29adf411b","order_by":8,"name":"Siling Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Siling","middleName":"","lastName":"Zhang","suffix":""},{"id":594207395,"identity":"a49e6a81-0c09-464d-bb17-ba9889a1daa2","order_by":9,"name":"Wensong Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wensong","middleName":"","lastName":"Sun","suffix":""},{"id":594207396,"identity":"6149ef07-cdc8-4f95-ab25-4d6178ecf1c7","order_by":10,"name":"Baoyu Shen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Baoyu","middleName":"","lastName":"Shen","suffix":""},{"id":594207397,"identity":"9f7d1ee9-50e6-433b-bb0f-255b66f67dd0","order_by":11,"name":"Lifu Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lifu","middleName":"","lastName":"Sun","suffix":""},{"id":594207398,"identity":"05dc5171-182f-4daf-a63b-850fbd15ce46","order_by":12,"name":"Xiahong He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiahong","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2026-01-21 11:43:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8659215/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8659215/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103306886,"identity":"b719db2c-b291-4f02-b857-68557fe6f7ad","added_by":"auto","created_at":"2026-02-24 09:17:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4257065,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different soil moisture treatments on the agronomic traits and quality index of OPPF. (A) Growth status of OPPF. (B) Agronomic traits of OPPF. (C) Quality index of OPPF. (D) Seedling survival rate and root rot disease for different soil moisture contents (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/9323b171d6edf2377f09e36c.png"},{"id":103506335,"identity":"de8d3d94-9b5a-444f-b74e-62c726fc8230","added_by":"auto","created_at":"2026-02-26 13:35:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":602921,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in soil properties and enzyme activities under varying soil moisture contents: (A–C) Soil properties; (D–G) Enzyme activities; (H) RDA of soil moisture, properties, and enzyme activities. Different letters above plots indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/1845677e44a10f0c55ddc46a.png"},{"id":103306893,"identity":"20634fba-3e6c-45ee-bd6d-b4709a54fdcb","added_by":"auto","created_at":"2026-02-24 09:17:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1962317,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance maps are generated at the phylum and genus taxonomic levels.\u003c/p\u003e\n\u003cp\u003e(A) Relative abundance of fungi; (B) relative abundance of bacteria; (C) heatmap of top 35 fungi genera; (D) heatmap of top 35 bacteria genera.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/9545b99268f7ddaae05a016c.png"},{"id":103506068,"identity":"c576fdeb-8991-43de-a46e-4a82c91fb264","added_by":"auto","created_at":"2026-02-26 13:33:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2676026,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between environmental factors, root rot disease, and rhizosphere microbial communities analyzed using Spearman’s correlation coefficient. (A) Correlations between root rot, soil moisture, and rhizosphere microbes, as well as the relationship between soil factors and the top 10 fungal genera; (B) correlations between soil factors and the top 10 fungal genera; (C) correlations between soil factors and the top 10 bacterial genera; (D) relative abundances of \u003cem\u003eAquicella\u003c/em\u003e, \u003cem\u003eIlyonectria\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003eCON: soil conductivity; AHN: alkali-hydrolyzed nitrogen; S-DHA: soil dehydrogenase; S-CL: soil cellulase; S-SC: soil sucrase; S-PPO: soil polyphenol oxidase; MG: moisture gradient; DI: disease index; IRD: incidence of root rot disease.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/4e1efc6f68b1f3e3b43608d7.png"},{"id":103306889,"identity":"3ef39804-ce5d-4132-8b47-005e96ef18e4","added_by":"auto","created_at":"2026-02-24 09:17:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":781590,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR copy number of pathogens and antagonists in rhizosphere soil of OPPF.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/7157bd739f8ff20af5abdbfd.png"},{"id":103306894,"identity":"f56392d9-6fb2-469f-b063-41ee77d87427","added_by":"auto","created_at":"2026-02-24 09:17:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4875313,"visible":true,"origin":"","legend":"\u003cp\u003eAntagonistic Effect of Rhizosphere Soil Strains. (A) Colony morphology and conidia of pathogens R3 and R6; (B) Phylogenetic tree based on internal transcribed spacer (ITS1) sequences (pathogens on the left, antagonists on the right); (C) Antagonistic effects of rhizosphere strains on R3 and R6; (D) Inhibition rates of rhizosphere strains against R3 and R6.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/31213d2c26ca0d3a1202091b.png"},{"id":106728969,"identity":"01c8a396-f0c5-4283-bb4c-0d0d265f4798","added_by":"auto","created_at":"2026-04-12 18:47:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16399495,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/7452c138-735d-4542-b15d-5930136c0e98.pdf"},{"id":103306892,"identity":"7a406203-a7d1-4cc7-a8b0-eda76ca4cee5","added_by":"auto","created_at":"2026-02-24 09:17:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1663774,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/bad72c50751f5c5b2cbc10fa.docx"},{"id":103306891,"identity":"a0007208-44bd-44c5-a92e-9f7a7e8db676","added_by":"auto","created_at":"2026-02-24 09:17:49","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19662,"visible":true,"origin":"","legend":"","description":"","filename":"Highlight.docx","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/042fc66ce58bea07a4cf4cd6.docx"},{"id":103306887,"identity":"baf14439-abf6-46d1-b3a3-c96c547b017d","added_by":"auto","created_at":"2026-02-24 09:17:48","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13499,"visible":true,"origin":"","legend":"","description":"","filename":"declarationStatement.docx","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/1b63b4011ad1c98e4fcb7319.docx"},{"id":103505800,"identity":"bad7920a-decd-4c5e-851f-956a7a297110","added_by":"auto","created_at":"2026-02-26 13:33:05","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2935314,"visible":true,"origin":"","legend":"","description":"","filename":"Linefigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8659215/v1/4262189d3111739c1d35da60.docx"}],"financialInterests":"","formattedTitle":"Soil Moisture Modulates Rhizosphere Microbiota and Suppresses Root Rot in Organic Panax ginseng Cultivated Under Pine Forests","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePanax\u003c/em\u003e ginseng (\u003cem\u003ePanax ginseng\u003c/em\u003e C.A. Meyer) is a valuable medicinal herb widely recognized for its health-promoting properties, such as anti-diabetic and anti-fatigue effects (Kim et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The primary cultivation methods include conventional farmland cultivation (cultivated ginseng) and understory cultivation (wild-simulated ginseng) (Bao et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, ginseng production faces significant constraints due to continuous cropping obstacles and the overuse of chemical pesticides, which contribute to a decline in crop quality. These unsustainable practices represent a common challenge in the cultivation of many medicinal plants.\u003c/p\u003e \u003cp\u003eExtensive studies on conventional cultivation have demonstrated that continuous cropping leads to soil microbial dysbiosis and aggravates soil-borne diseases, with root rot caused by \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eIlyonectria\u003c/em\u003e spp. being a key factor limiting sustainable production (Xia et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In response, researchers both domestically and internationally have attempted various measures such as soil disinfection, crop rotation, and soil amendments, yet these approaches often show limited efficacy or introduce new environmental risks. In recent years, the ecologically friendly understory cultivation model has gained attention. Research indicates that this system can leverage forest biodiversity to suppress foliar diseases (e.g., \u003cem\u003eAlternaria panax\u003c/em\u003e) and improve medicinal quality (He et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, existing studies have primarily focused on the effects of the forest environment on growth and foliar diseases, with systematic research on soil moisture management\u0026mdash;a critical agronomic practice in this system\u0026mdash;remaining notably scarce. Soil moisture not only directly affects plant physiology but also plays a key role in shaping rhizosphere microbial community structure and function, thereby influencing the incidence of soil-borne diseases (Patel et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e;Berg et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although substantial knowledge exists on water management for grain crops and some commercial tree species, targeted studies on precise soil moisture regulation for understory medicinal plants, particularly ginseng, are virtually absent. Moreover, in organic \u003cem\u003eP. ginseng\u003c/em\u003e cultivation under pine forests (OPPF) systems where rain shelters are used to control leaf diseases, the alteration of natural rainfall patterns adds complexity to soil moisture management, making the development of science-based irrigation strategies both necessary and underexplored.\u003c/p\u003e \u003cp\u003eTo address these gaps, this study focuses on the OPPF system and is the first to systematically investigate the integrated effects of different soil moisture levels (55\u0026ndash;60%, 70\u0026ndash;75%, 80\u0026ndash;85%, and 95\u0026ndash;100% of field capacity) on ginseng growth, quality, root rot incidence, and rhizosphere microecology. We hypothesize that soil moisture influences root rot occurrence by altering the rhizosphere microbial community structure and that potentially exploitable biocontrol microbial resources may exist within this microbiome. Therefore, the objectives of this study are: (1) to identify the optimal soil moisture range for enhancing OPPF biomass and quality while reducing root rot incidence, and (2) to elucidate how soil moisture drives changes in rhizosphere microbial diversity\u0026mdash;particularly in pathogenic and antagonistic populations\u0026mdash;and its mechanistic association with root rot disease. The findings will provide a direct theoretical basis and technical support for establishing ecologically based water management protocols in OPPF systems, offering significant academic value and practical potential.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and treatments\u003c/h2\u003e \u003cp\u003eThe experiment was conducted in June 2023 at an organic \u003cem\u003ePanax ginseng\u003c/em\u003e under pine forest (OPPF) test base located at the Lanheyu Forestry Center (41\u0026deg;09\u0026prime;56.98\u0026Prime; N, 124\u0026deg;26\u0026prime;20.70\u0026Prime; E) in Liaoning, China. The site experiences a mid-temperate monsoon humid climate, with a mean annual temperature of 7.5\u0026deg;C and annual precipitation of 1017.5 mm. Initial soil properties included pH 5.12, alkaline hydrolysis nitrogen (AHN) 441.83 mg/kg, electrical conductivity (EC) 281.63 \u0026micro;S/cm, and a field capacity (FC) of 48%. The starting soil moisture level was 68.52% FC.\u003c/p\u003e \u003cp\u003eA total of 240 pots (upper diameter: 184 mm; height: 164 mm; bottom diameter: 132 mm) were used, each filled with 1090 g of soil and planted with three three-year-old \u003cem\u003eP. ginseng\u003c/em\u003e seedlings. Pots were assigned to four soil moisture treatments: PTh1 (55\u0026ndash;60% FC), PTh2 (70\u0026ndash;75% FC), PTh3 (80\u0026ndash;85% FC), and PTh4 (95\u0026ndash;100% FC). Soil moisture was maintained by weekly pot weighing and water adjustment. All pots were placed on artificial ridges under a pine canopy to simulate natural OPPF conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of agronomic traits and chlorophyll content\u003c/h3\u003e\n\u003cp\u003eAfter three months of moisture treatment, plants were harvested and transported to the laboratory. Soil was carefully removed from roots, and samples were air-dried before measuring fresh weight. Leaf length, leaf width, and fibrous root length were recorded, and leaf area was calculated based on leaf dimensions (Wang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Chlorophyll content was measured using a SPAD-502 handheld meter (Konica Minolta, Japan). Per treatment, 96 plants were randomly selected and grouped into six biological replicates for average fresh weight calculation.\u003c/p\u003e\n\u003ch3\u003eAnalysis of saponin, flavonoid, and polysaccharide quality, and total polysaccharides\u003c/h3\u003e\n\u003cp\u003eSaponin content (including ginsenosides Rd, Re, Rc, Rg1, Rf, Rb1, Rb3, Rb2, Rg2, Rh1, Rh2, and Rg3) in ginseng roots was quantified using a modified UPLC method adapted from Li et al. (Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Separation was performed on a Nexera X2 system (Shimadzu, Japan) equipped with a DAD detector and a Poroshell 120 EC-C18 column (Agilent; 4 \u0026micro;m, 100 \u0026times; 2.1 mm). The mobile phase consisted of acetonitrile (solvent A) and 0.1% aqueous solution (solvent B) under gradient conditions (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The injection volume was 10 \u0026micro;L, flow rate 1.0 mL/min, column temperature 30\u0026deg;C, and detection wavelength 203 nm. Identification and quantification were performed using ginsenoside standards (Supplementary Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Flavonoid, polysaccharide, and total polysaccharide contents were determined using commercial assay kits (Michy Bio, China).\u003c/p\u003e\n\u003ch3\u003eRoot rot assessment and pathogen isolation\u003c/h3\u003e\n\u003cp\u003eAt harvest, 144 plants per treatment (6 replicates) were evaluated for root rot severity using the rating scale established by Li et al. (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Disease incidence and index were calculated as described by Yang et al. (Yang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fungal pathogens were isolated from symptomatic root tissues following Li et al. (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Genomic DNA was extracted using a 2\u0026times;T5 Direct PCR kit (TSINGKE, China). The ITS region was amplified with primers ITS1/ITS4 (Hawa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and sequenced. Phylogenetic analysis was conducted in MEGA 7.0. Pathogenicity of isolates was confirmed using the method of Luo et al. (Luo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSoil physicochemical properties and enzyme activities\u003c/h3\u003e\n\u003cp\u003eSoil samples were collected and analyzed for physicochemical parameters according to Guo et al. (Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Activities of soil polyphenol oxidase (S-PPO), dehydrogenase (S-DHA), sucrase (S-SC), and cellulase (S-CL) were measured using specific assay kits (Michy Bio, China).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction, sequencing, and bioinformatic\u003c/h2\u003e \u003cp\u003eTotal genomic DNA was extracted from soil samples, and amplification and library preparation were conducted following Edgar et al. (Edgar, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). High-throughput sequencing of bacterial 16S rRNA and fungal ITS regions was performed on the Illumina platform. Bioinformatic processing included quality filtering, OTU clustering, and taxonomic assignment using standard pipelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantification of pathogenic fungi and antagonistic via qPCR\u003c/h3\u003e\n\u003cp\u003eDNA from soil samples was reverse-transcribed using a PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Quantitative real-time PCR (qPCR) was performed with strain-specific primers (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)(G\u0026eacute;ry et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e;Ye et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e༛Gao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e༛Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e༛Saravanakumar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e༛Reiter et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Each 30 \u0026micro;L reaction contained 10 \u0026micro;L qPCR Mix, 0.5 \u0026micro;L each of forward and reverse primers (10 \u0026micro;M), 2 \u0026micro;L template DNA, and nuclease-free water. Amplification conditions followed G\u0026eacute;ry et al.(2021).\u003c/p\u003e\n\u003ch3\u003eIsolation of culturable rhizosphere and functions validation\u003c/h3\u003e\n\u003cp\u003eRhizosphere microorganisms were isolated and purified as described by Guo et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Functional validation was performed using pot assays under sterile soil conditions based on Fang et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Three-year-old ginseng seedlings were planted in pots inoculated with 50 mL of a suspension containing pathogenic fungi (10⁶ CFU/mL) and candidate antagonistic bacteria (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5). Control groups received pathogen suspension only or sterile water. Each treatment included three replicates of five pots. Plants were grown in a greenhouse at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 60 days, after which fresh weight and root rot incidence were recorded.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using PASW Statistics 18 (SPSS Inc. USA). One-way ANOVA followed by Tukey\u0026rsquo;s HSD test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was used to determine significant differences among treatments. Data normality and homogeneity of variance were verified beforehand. Correlation analyses were conducted using Spearman\u0026rsquo;s rank correlation method (Fang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOptimal soil moisture level (PTh2) enhances growth and quality in OPPF-cultivated \u003cem\u003eP. ginseng\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSignificant differences in agronomic traits and seedling survival rates were observed among \u003cem\u003eP. ginseng\u003c/em\u003e plants subjected to varying soil moisture levels after three months of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D). Plants under the PTh2 regime (70\u0026ndash;75% FC) exhibited markedly greater fresh weight, fibrous root length, and chlorophyll content compared to those in PTh1, PTh3, and PTh4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results were consistent with field trial data from 2024 (Supplementary Table S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of secondary metabolites revealed that PTh2 also led to significantly higher flavonoid and total carbohydrate contents in fibrous roots relative to other treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Total polysaccharide content was notably elevated in PTh2 compared to the remaining moisture regimes. Although total saponin levels in roots increased with rising soil moisture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), individual ginsenosides responded differently across tissue types and treatments. In fibrous roots, significant variations were detected in the contents of Rd, Rb1, Rb3, Rh2, and Rg3. In taproots, Rd, Rc, Rg1, Rf, Rb1, Rb3, Rb2, Rg2, Rh1, Rh2, and Rg3 levels differed significantly among treatments, with Rg2, Rb1, and Rh1 increasing with soil moisture. Notably, PTh3 resulted in higher Rc content in roots and elevated Rd in fibrous roots compared to other treatments (Supplementary Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Overall, soil moisture strongly influenced ginseng growth and quality, particularly flavonoid and saponin accumulation (Supplementary Fig.S3).\u003c/p\u003e \u003cp\u003eSeedling survival was significantly higher in PTh2 than in PTh1 and PTh4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Both root rot incidence and disease index were significantly lower in PTh1 and PTh2 than in PTh4, and a clear positive correlation was observed between soil moisture level and root rot severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These findings were corroborated by 2024 field data (Supplementary Fig.S4), confirming the critical role of soil moisture in disease development under OPPF conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSoil properties and enzyme activities are influenced by soil moisture regimes\u003c/h2\u003e \u003cp\u003eSoil moisture levels significantly influenced various soil physicochemical properties and enzymatic activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Soil pH was significantly higher under the PTh2 treatment compared to PTh1, PTh3, and PTh4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, soil electrical conductivity was markedly elevated in PTh3 relative to the other moisture regimes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil alkaline hydrolysis nitrogen (AHN), dehydrogenase (S-DHA), sucrase (S-SC), and cellulase (S-CL) activities were highest under the PTh1 treatment, showing significant differences compared to PTh2, PTh3, and PTh4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;G). Redundancy analysis (RDA) further confirmed that soil properties and enzyme activities varied systematically with soil moisture levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Specifically, soil conductivity exhibited a negative correlation with S-DHA and S-SC activities, but was positively associated with moisture content in PTh3 and PTh4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRhizosphere microbial community structure shifts with soil moisture\u003c/h2\u003e \u003cp\u003ePrincipal coordinate analysis (PCoA) and ANOSIM revealed significant differences in bacterial (R\u0026thinsp;=\u0026thinsp;0.5467, p\u0026thinsp;=\u0026thinsp;0.001) and fungal (R\u0026thinsp;=\u0026thinsp;0.4779, p\u0026thinsp;=\u0026thinsp;0.001) community structures among the four soil moisture treatments (Supplementary Fig.S5). Although alpha diversity indices (Chao1, Shannon, and observed species) did not differ significantly across treatments (Supplementary Fig.S4), bacterial diversity was generally higher in PTh2 and PTh3, while fungal diversity was notably elevated under the PTh2 regime.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSoil moisture alters the relative abundance of key microbial taxa\u003c/h2\u003e \u003cp\u003eAt the phylum level, \u003cem\u003eAscomycota\u003c/em\u003e dominated the fungal community under all soil moisture conditions, accompanied by \u003cem\u003eBasidiomycota\u003c/em\u003e, \u003cem\u003eMortierellomycota\u003c/em\u003e, \u003cem\u003eOlpidiomycota\u003c/em\u003e, \u003cem\u003eMucoromycota\u003c/em\u003e, \u003cem\u003eGlomeromycota\u003c/em\u003e, \u003cem\u003eRozellomycota\u003c/em\u003e, \u003cem\u003eChytridiomycota\u003c/em\u003e, and \u003cem\u003eZoopagomycota\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The relative abundance of \u003cem\u003eAscomycota\u003c/em\u003e was higher in PTh1 and PTh4 compared to PTh2, whereas \u003cem\u003eBasidiomycota\u003c/em\u003e and \u003cem\u003eMortierellomycota\u003c/em\u003e were more abundant in PTh2 and PTh3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong bacteria, nine dominant phyla were identified: \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eAcidobacteria\u003c/em\u003e, \u003cem\u003eVerrucomicrobiota\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eMyxococcota\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eBacteroidota\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eAcidobacteria\u003c/em\u003e were the most abundant (each \u0026gt;\u0026thinsp;1% relative abundance), with \u003cem\u003eAcidobacteria\u003c/em\u003e increasing and \u003cem\u003eActinobacteria\u003c/em\u003e decreasing as soil moisture rose.\u003c/p\u003e \u003cp\u003eAt the genus level, fungi such as \u003cem\u003eHumiicola\u003c/em\u003e, \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eSaitozyma\u003c/em\u003e, \u003cem\u003ePseudogymnoascus\u003c/em\u003e, and \u003cem\u003eMortierella\u003c/em\u003e were more abundant under lower soil moisture but declined with increasing moisture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In contrast, known pathogenic genera including \u003cem\u003eIlyonectria\u003c/em\u003e, \u003cem\u003eCylindrocarpon\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eNeonectria\u003c/em\u003e, \u003cem\u003eStagonosporopsis\u003c/em\u003e, and \u003cem\u003eChaetomium\u003c/em\u003e were most prevalent in PTh3. Among bacteria, \u003cem\u003ePseudomonas\u003c/em\u003e increased with soil moisture, while \u003cem\u003eArenimonas\u003c/em\u003e, \u003cem\u003eCollimonas\u003c/em\u003e, \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (B-C-P), and \u003cem\u003eBacillus\u003c/em\u003e decreased in abundance under higher moisture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation analysis and abundance of potential beneficial microbiomes\u003c/h2\u003e \u003cp\u003eAnalysis of relationships between root rot incidence and microbial communities focused on taxa with relative abundances exceeding 0.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Within fungal communities, disease index (DI), incidence of rot disease (IRD), and moisture gradient (MG) showed positive correlations with \u003cem\u003eFusarium\u003c/em\u003e, but negative correlations with \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eChaetosphaeria\u003c/em\u003e, \u003cem\u003eChloridium\u003c/em\u003e, \u003cem\u003eMortierella\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e. Among bacterial communities, DI, IRD, and MG correlated positively with \u003cem\u003eAquicella\u003c/em\u003e, but negatively with \u003cem\u003eArenimonas\u003c/em\u003e and the \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (B-C-P) complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis revealed significant correlations between dominant fungal and bacterial genera and key soil properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Soil electrical conductivity (EC) correlated positively with \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eIlyonectria\u003c/em\u003e, but negatively with \u003cem\u003eTalaromyces\u003c/em\u003e. In contrast, alkaline hydrolysis nitrogen (AHN), dehydrogenase (S-DHA), and cellulase (S-CL) activities were positively associated with \u003cem\u003eTalaromyces\u003c/em\u003e and negatively associated with \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eIlyonectria\u003c/em\u003e. Soil pH and polyphenol oxidase (S-PPO) activity showed negative correlations with \u003cem\u003eFusarium\u003c/em\u003e, while soil moisture correlated positively with \u003cem\u003eCrossiella\u003c/em\u003e and negatively with \u003cem\u003eNovosphingobium\u003c/em\u003e, B-C-P complex, \u003cem\u003eBradyrhizobium\u003c/em\u003e, and \u003cem\u003eArenimonas\u003c/em\u003e. AHN, S-DHA, and S-CL exhibited opposite correlation patterns with these bacterial genera.\u003c/p\u003e \u003cp\u003eQuantitative PCR (qPCR) analysis of rhizosphere soil indicated that the copy numbers of the root rot pathogens \u003cem\u003eFusarium solani\u003c/em\u003e and \u003cem\u003eIlyonectria mors-panacis\u003c/em\u003e were significantly elevated under the PTh4 treatment. Conversely, antagonistic microbial populations were more abundant under both PTh1 and PTh4 conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFunctional validation of culturable microbial strains\u003c/h2\u003e \u003cp\u003ePathogens R3 and R6, isolated from symptomatic roots, were identified as \u003cem\u003eIlyonectria mors-panacis\u003c/em\u003e and \u003cem\u003eFusarium solani\u003c/em\u003e, respectively. Pathogenicity tests confirmed that both strains induced root rot symptoms in OPPF plants, whereas control plants remained healthy.\u003c/p\u003e \u003cp\u003eUsing a dual-culture assay, three bacterial strains (L11, A1, G1) and three fungal strains (F1, F2, F3) were identified that inhibited the growth of R6 and R3 by more than 30%. Based on BLAST analysis, the bacterial strains were identified as \u003cem\u003eBacillus mycoides\u003c/em\u003e (G1), \u003cem\u003eBacillus velezensis\u003c/em\u003e (L11), and \u003cem\u003eStreptomyces drozdowiczii\u003c/em\u003e (A1), and the fungal strains as \u003cem\u003ePenicillium ortum\u003c/em\u003e (F1), \u003cem\u003eTalaromyces amestolkiae\u003c/em\u003e (F2), and \u003cem\u003eTrichoderma koningii\u003c/em\u003e (F3).\u003c/p\u003e \u003cp\u003ePot experiments demonstrated that all six strains promoted ginseng growth and suppressed root rot. Among them, strain F1 (\u003cem\u003ePenicillium ortum\u003c/em\u003e) exhibited the strongest disease suppression and most significantly enhanced plant biomass.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite the potential of agroforestry systems to alleviate land-use pressures, research on the cultivation of Chinese medicinal plants\u0026mdash;particularly organic ginseng\u0026mdash;under forest canopy remains limited. Few studies have addressed soil moisture management specifically for organic Panax ginseng grown in understory environments. Our findings indicate that both drought (PTh1) and waterlogging (PTh4) impaired the healthy growth of understory ginseng, whereas moderate soil moisture (PTh2; 70\u0026ndash;75% FC) promoted plant growth and increased the accumulation of polysaccharides, flavonoids, and saponins. PTh2 also resulted in superior biomass production and lower root rot incidence compared to the high-moisture PTh4 treatment. Furthermore, this study provides novel insights into how soil moisture influences rhizosphere microbial community structure and identifies several antagonistic microorganisms, offering practical strategies for irrigation management in forest-based ginseng cultivation systems.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSoil moisture modulates growth and metabolite accumulation in \u003cem\u003eP. ginseng\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePlant phenotypic traits are strongly influenced by environmental cues, which in turn affect biomass and secondary metabolism (Loretta, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Soil moisture plays a critical role in root development and overall plant health, particularly in medicinal species such as \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e, where drought stress significantly curtails growth (Yang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Sun et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Conversely, excess moisture can also inhibit growth after an initial increase (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In our study, biomass peaked under moderate soil moisture (PTh2) and declined under both water-deficit and waterlogged conditions, consistent with reports that extreme moisture levels negatively affect ginseng biomass accumulation(Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, flavonoid and total carbohydrate contents were highest under PTh2, whereas polysaccharide and total saponin levels responded differently across treatments. Total flavonoids\u0026mdash;commonly used as a quality marker\u0026mdash;decreased significantly under severe water loss (Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e;Zhang, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), while polysaccharides increased under moderate stress. Total saponin content was positively correlated with soil moisture, rising significantly under high-water conditions (Puente-Garza et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Individual ginsenosides (e.g., Rg1, Rf, Re, Rg3, Rb2, Rd, and Rh2) were lowest under optimal moisture and elevated under stress, underscoring how environmental conditions shape the phytochemical profile of medicinal plants (Zheng et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e༛Lee and Mudge, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These patterns align with observations in \u003cem\u003eP. notoginseng\u003c/em\u003e, where saponin content varied under water stress(Zheng et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), highlighting species-specific metabolic plasticity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSuboptimal soil moisture alters rhizosphere microbiome and promotes root rot\u003c/h2\u003e \u003cp\u003eSoil moisture strongly influences the rhizosphere micro-environment and the prevalence of soil-borne diseases. In this study, higher soil moisture correlated with increased incidence and severity of ginseng root rot, consistent with earlier reports (Yang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Changes in moisture regimes also affected key soil properties such as pH and electrical conductivity, which in turn influenced pathogen behavior (Stover, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). We found negative correlations between root rot incidence and soil pH and alkaline hydrolysis nitrogen, whereas electrical conductivity showed a positive correlation with disease.\u003c/p\u003e \u003cp\u003eSustained high moisture (PTh4; 95\u0026ndash;100% FC) favored anaerobic microorganisms and known pathogens such as \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eIlyonectria\u003c/em\u003e, in line with studies showing that waterlogged conditions reduce microbial diversity and disrupt community balance (Harman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Such shifts likely suppressed the activity of beneficial enzymes and microbes, exacerbating disease incidence (Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Maintaining soil moisture at 60\u0026ndash;75% FC helped minimize root rot, corroborating field observations (Yang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Future studies should employ metagenomic and transcriptomic tools to unravel plant\u0026ndash;microbe interactions under dynamic moisture conditions (Shi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), offering deeper mechanistic insights into moisture-mediated disease regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMoisture-driven microbial community shifts influence disease resistance in OPPF\u003c/h2\u003e \u003cp\u003eHigh-throughput sequencing revealed that soil moisture significantly shaped the composition of bacterial and fungal communities in the ginseng rhizosphere. Dominant bacterial phyla included Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi, with genera such as \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e\u0026mdash;known bio-control agents\u0026mdash;showing higher abundance under moderate moisture (Kobayashi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e;Moretti et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e༛Tran, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Pathogenic genera like \u003cem\u003eAquicella\u003c/em\u003e were positively correlated with root rot incidence.\u003c/p\u003e \u003cp\u003eFungal communities were dominated by Ascomycota, Basidiomycota, and Mortierellomycota (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Known pathogens including Fusarium and Ilyonectria were more abundant in high-moisture treatments, whereas antagonistic fungi such as \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e were enriched under drier conditions (Zhang, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e;Ozimek and Hanaka, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e༛Tian et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These patterns support the concept that plants under stress can recruit beneficial microbes for protection, as observed in \u003cem\u003eP. notoginseng\u003c/em\u003e with \u003cem\u003eArthrobacter\u003c/em\u003e (Fang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Interestingly, qPCR results indicated higher abundance of antagonistic strains under both low and high moisture, suggesting complex, context-dependent microbial interactions (Lou et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e༛Tettamanti Boshier et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConclusions and implications\u003c/p\u003e \u003cp\u003eThis study demonstrates that maintaining soil moisture at 70\u0026ndash;75% FC optimizes the growth, quality, and health of organic \u003cem\u003eP. ginseng\u003c/em\u003e under pine forests. We identified key microbial taxa and several culturable strains (e.g., \u003cem\u003eBacillus velezensis\u003c/em\u003e, \u003cem\u003eTrichoderma koningii\u003c/em\u003e, and \u003cem\u003ePenicillium ortum\u003c/em\u003e) with high biocontrol potential against root rot pathogens. These findings underscore the importance of irrigation management in balancing rhizosphere microbiota and suppressing disease in agroforestry systems. Future research would focus on the dynamic monitoring of microbial succession under fluctuating soil moisture, mechanistic studies on plant\u0026ndash;microbe interactions using multi-omics approaches, field validation of optimal irrigation regimes and biocontrol agents, and economic and sustainability assessments of moisture management practices in ginseng agroforestry. Such efforts will advance our capacity to implement ecologically sound and productive cultivation systems for high-value medicinal plants.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the China Agriculture Research System of MOF and MARA (CARS\u0026thinsp;\u0026minus;\u0026thinsp;21), Yunnan Agricultural Joint Special Project Key Projects (202301 BD070001\u0026ndash;138), Youth Talents Special Project of Yunnan Province, \u0026ldquo;Xingdian Talents Support Program (YNWR-QNBJ-2020194).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eHuiling Wang and Xiahong He led the project. Huiling Wang and Rongshuang Sha handled field experiments and microbiom data analysis, with support from Wensong Sun and Lifu Sun in Liaoning Province. Tao Zhou, Qiongying Kang and Dan Li used HPLC-MS for data analysis. Huiling Wang, Zhenxing Wang and Baoyu Shen conducted microbial validation in pot experiments. Huiling Wang and Rongshuang Sha drafted the manuscript, which was revised by Rui Shi, Shu He and Xiahong He. All authors contributed and approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available on request. All microbial genomic sequence data have been submitted to the National Center for Biotechnology Information (NCBI) database, with the accession number [PRJNA1031259].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBao Y, Qi B, Huang W, Liu B, Li Y (2020) The fungal community in non-rhizosphere soil of \u003cem\u003ePanax ginseng\u003c/em\u003e are driven by different cultivation modes and increased cultivation periods. PeerJ 8:e9930. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.9930\u003c/span\u003e\u003cspan address=\"10.7717/peerj.9930\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerg G, Rybakova D, Grube M, K\u0026ouml;berl M (2016) The plant microbiome explored: implications for experimental botany. 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Chin Herb Med 13(2):267\u0026ndash;273. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chmed.2020.10.002\u003c/span\u003e\u003cspan address=\"10.1016/j.chmed.2020.10.002\" 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":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Agroforestry, Panax ginseng, soil moisture, root rot, rhizosphere microbiome, disease suppression, sustainable agriculture","lastPublishedDoi":"10.21203/rs.3.rs-8659215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8659215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eThe integration of agroforestry and understory cultivation offers a sustainable approach to producing high-value medicinal plants with ecological conservation. However, optimal irrigation management for organic \u003cem\u003ePanax ginseng\u003c/em\u003e grown under pine forests (OPPF) remains poorly studied, particularly regarding its influence on root rot disease and rhizosphere microbial communities.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study evaluated four soil moisture treatments\u0026mdash;PTh1 (55\u0026ndash;60% field capacity, FC), PTh2 (70\u0026ndash;75% FC), PTh3 (80\u0026ndash;85% FC), and PTh4 (95\u0026ndash;100% FC)\u0026mdash;on the growth, quality, and root rot incidence of OPPF. We analyzed agronomic traits, secondary metabolites, soil properties, enzymatic activities, and rhizosphere microbial composition via high-throughput sequencing and qPCR, and validated microbial isolates functionally through pot experiments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eModerate soil moisture (PTh2) significantly improved plant biomass, seedling survival, and the accumulation of flavonoids, polysaccharides, and ginsenosides. In contrast, high soil moisture (PTh4) increased the incidence of root rot and the abundance of pathogens such as \u003cem\u003eFusarium solani\u003c/em\u003e and \u003cem\u003eIlyonectria mors-panacis\u003c/em\u003e. Soil properties and enzyme activities were markedly influenced by moisture levels, with PTh2 maintaining a higher pH and beneficial nutrient profile. Rhizosphere microbiome analysis revealed that PTh2 enriched potential biocontrol agents, including \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eStreptomyces\u003c/em\u003e, while reducing pathogenic taxa. Six antagonistic strains were isolated, with \u003cem\u003ePenicillium ortum\u003c/em\u003e (F1) showing the strongest suppression of root rot and promotion of plant growth in pot trials.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMaintaining soil moisture at 70\u0026ndash;75% FC optimizes the growth and health of OPPF by fostering beneficial microbial communities and inhibiting soil-borne pathogens. These findings provide science-based irrigation strategies to support the sustainable cultivation of organic ginseng in forest-based agroecosystems.\u003c/p\u003e","manuscriptTitle":"Soil Moisture Modulates Rhizosphere Microbiota and Suppresses Root Rot in Organic Panax ginseng Cultivated Under Pine Forests","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 09:17:43","doi":"10.21203/rs.3.rs-8659215/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"408ce5b0-5db6-47b9-be37-6bd44835639a","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-10T13:15:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 09:17:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8659215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8659215","identity":"rs-8659215","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}