Key Microorganism Alleviate Continuous Cropping Obstacles of Polygonatum

Key Microorganism Alleviate Continuous Cropping Obstacles of Polygonatum | 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 Key Microorganism Alleviate Continuous Cropping Obstacles of Polygonatum Chenghua Luo, Hua Yang, Zhifa Zhang, Yaping Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9201673/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background and Aims Polygonatum odoratum , a widely cultivated and valuable medicinal herb in China, is severely restricted by continuous cropping obstacles (CCOs). Traditional mitigation strategies including crop rotation and chemical control are either inefficient or environmentally unfriendly, lacking sustainable and eco-friendly solutions. This study aimed to analyze the microbiome of continuous cropping P. odoratum , identify key functional microbial taxa, and construct a synthetic microbial community (SynCom) for relieving CCOs. Methods Rhizosphere soil, root endophytic tissues and rhizome samples of P. odoratum with different planting years were collected from three major producing areas, followed by multi-omics analysis to screen core beneficial microbes. A targeted SynCom was assembled, and pot and field experiments were conducted to verify its control effect on CCOs. Results Continuous cropping significantly depleted soil nutrients, reduced soil phosphatase and nitrite reductase activities by 64.73% and 34.87%, respectively. The Chao1 index of rhizosphere bacterial α-diversity decreased by 8.41%, and the relative abundance of the key functional genus Streptomyces dropped sharply by 83.70%. The assembled SynCom effectively alleviated CCOs by restoring rhizosphere microbial homeostasis, improving soil nutrient availability, suppressing rhizome rot, and enhancing the yield and medicinal quality of P. odoratum . Conclusions The loss of Streptomyces is the core microbial factor triggering P. odoratum CCOs. Supplementing core functional microbiota via SynCom mitigates CCOs through synergistic effects of pathogen antagonism, microbial community restoration and soil fertility improvement. This study provides a novel paradigm for relieving CCOs in medicinal plants. Continuous Cropping Obstacles Synthetic Microbial Community (SynCom) Polygonatum odoratum Plant Growth-Promoting Bacteria Rhizosphere Microbiome Figures Figure 1 Figure 3 Figure 4 Figure 5 Introduction Continuous cropping obstacles (CCOs) in medicinal plants have emerged as a critical constraint to the sustainable development of the industry. These obstacles typically manifest as reduced yield, deteriorated quality, aggravated soil-borne diseases, and replanting failure, and are commonly observed across a wide range of medicinal species(Wu et al. 2015 ; Tan et al. 2017 ; Alami et al. 2021 ; Chen et al. 2021 ; Gu et al. 2022 ), often resulting in significant economic losses(Wacal et al. 2019 ). Conventional management practices such as crop rotation and chemical control are often unsuitable for large-scale cultivation and may entail environmental risks and pesticide-residue concerns(Zhou et al. 2019 ; Becker et al. 2023 ; Wang et al. 2023 ). Therefore, elucidating the mechanisms underlying continuous cropping obstacles and developing green, sustainable mitigation strategies are of paramount importance. CCOs primarily arise from the interplay of deteriorated soil physicochemical properties, accumulation of allelopathic compounds, and dysbiosis of the rhizosphere microbial community. Continuous cropping leads to soil acidification, nutrient imbalance, and secondary salinization(Alami et al. 2021 ). Meanwhile, root-secreted allelochemicals—such as organic acids, phenolic acids, and flavonoids—can directly inhibit plant growth or indirectly compromise plant health by altering the rhizosphere microenvironment(Liu Ping 2018; Sasse et al. 2018 ; Alami et al. 2021 ; Bao et al. 2022 ; Hao et al. 2022 ). Of particular importance is the disruption of rhizosphere microbial communities continuous cropping generally reduces microbial diversity, enriches pathogenic taxa, and diminishes beneficial bacteria(Fei et al. 2021 ; Feng et al. 2022 ; Su et al. 2023 ). Rhizosphere beneficial bacteria are considered as a "second genome" for plants, enhancing nutrient acquisition, stress tolerance and pathogen resistanc. Plant growth-promoting rhizobacteria (PGPR) improve nutrient availability via phosphate solubilization, nitrogen fixation, or phytohormone production (e.g., IAA and zeatin)(Yu et al. 2021 ; Zheng et al. 2024 ). Certain PGPR strains also antagonize pathogens through antibiotic synthesis for instance, Bacillus amyloliquefaciens suppresses Ralstonia solanacearum in tomato(Zhou et al. 2022 ). Other microbes enhance plant resilience to abiotic stress through ACC deaminase activity, which modulates ethylene levels, or through extended hyphal networks (e.g., arbuscular mycorrhizal fungi) that improve water and nutrient uptake(Chandwani& Amaresan 2022 ; Chen et al. 2023 ). Nevertheless, the key microorganisms that play vital roles in continuous cropping and their specific functional changes remain poorly understood. Polygonatum odoratum , a perennial medicinal herb highly susceptible to CCOs, exhibits severe root rot and significant declines in both yield and quality under continuous cropping. These effects have become a primary cause for the contraction of its cultivation area in major production regions(Ni et al. 2021 ). Studies showed that the main disease of obstacle was Fusarium root rot, and continuous cropping of P. odoratum leading to a significant decrease in soil nitrogen and phosphorus nutrition, and its own photosynthesis was weakened and growth was blocked(Wang et al. 2023 ; Xu et al. 2023 ). Elucidating the mechanisms underlying CCOs and developing biological solutions are therefore of considerable practical importance. In this study, the key microorganisms causing the CCOs were identified through microbial diversity and metagenomic analysis. Afterword, a SynCom composed of functionally defined strains was constructed by combining microbial separation and culture, and its effectiveness in alleviating the CCOs was verified through pot and field trials, providing a feasible microbial community regulation strategy for the green and sustainable cultivation of P. odoratum . Materials and Methods Sample collection, soil physicochemical property and enzyme activity measurement Three representative P. odoratum production area ShaoYang (SY: 111°38′25″E, 27°16′07″N), GuiYang (GY: 112°39′28″E, 26°04′55″N), CiLi (CL: 111°07′12″E, 29°25′12″N) in Hunan Province, China, were selected as sampling locations. Each location included P. odoratum cropped in new field for first-planting 1 year (Y1), 3 years (Y3) and cropped in continuous cropping field for 3 years (Y6) with 3 replicates (n = 3). First, three plants with similar growth status were randomly select and there were more than 1m apart within a plot. After removing dead leaves and debris from the ground, P. odoratum were dug out with depth 20-30cm soil cube. After removed top 5 cm of soil and loosely attached large soil clumps, we collected 200g of soil from 10cm below the surface and 5 cm away from the root system for physical and chemical property analysis. The physical and chemical properties of the soil and soil enzyme activity (Supplementary Materials) were measured from soil samples according to Castillo et al.(Castillo& Regan 2025 ; Liu et al. 2025 ). Then, 20 g rhizosphere soil sample were collected by brushing the soil attach to the roots. Finally, we cleaned the underground parts of the plants, and used scissors toting off 10g of fresh, clean roots growing on the rhizome as the roots sample. All samples were transferred to the laboratory on dry ice, each replicate sample was analyzed separately for the omics data described in the text. Rhizosphere soil nontarget metabolomics and root targeted metabolomics analysis Rhizosphere soil and plant root samples were first lyophilization treated. Metabolites were extracted and re-purified. Final supernatants and quality control (QC) pooled samples were analyzed. Untargeted metabolomics and widely targeted metabolomics were performed according to literature(Yue et al. 2023 ; Wang et al. 2024 ), with minor modifications (Supplementary Materials). High-throughput DNA extraction, amplification, and sequencing Total genomic DNA of the rhizosphere soil and roots sample was extracted by using the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.). The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F: 5'-ACTCCTACGGGAGGCAGCA-3' and 806R: 5'- GGACTACHVGGGTWTCTAAT-3'. The its2 gene of fungi were amplified with primer pairs its2F: 5'-GCATCGATGAAGAACGCAGC-3'; its2R: 5'-TCCTCCGCTTATTGATATGC-3'; Both the forward and reverse primers were tailed with sample-specific Illumina index sequences to allow for deep sequencing. The purified PCR products was paired-end sequenced (2×250) on an Illumina novaseq6000 (Beijing Biomarker Technologies Co., Ltd., Beijing, China). Raw data were assembled and quality-filtered according to Caporaso et al., and chimeric sequences were removed using the UCHIME tool in USEARCH. The sequences matching the mitochondria and chloroplast were also removed, and the remaining effective sequences were clustered into operational taxonomic units (OTUs) at 97% similarity(Caporaso et al. 2010 ; Zhang et al. 2021 ). Metagenomic sequencing workflow and data analysis Rhizosphere soil DNA was sequenced on an Illumina platform following the manufacturer’s standard protocol. Raw reads were quality-filtered using Fastp (v0.23.2) to obtain clean reads(Chen et al. 2018 ). Host-derived reads were removed by alignment to the host genome with Bowtie2 (v2.4.5)(Langmead& Salzberg 2012 ). Metagenome assembly was performed using MEGAHIT (v1.2.9), retaining contigs longer than 300 bp(Li et al. 2015 ). Coding regions were predicted with MetaGeneMark (v3.26) under default parameters(Zhu et al. 2010 ). Redundant genes were clustered using MMseqs2 (v12-113e3) at 95% sequence identity and 90% coverage thresholds(Mirdita et al. 2019 ). Non-redundant protein sequences were annotated against the NR database using BLASTp (E-value ≤ 1e − 5). Downstream analyses, including alpha diversity, principal coordinate analysis (PCoA), intergroup taxonomic differences, KEGG functional profiling (P < 0.05), spearman correlation-based microbial interaction network at the genus level (correlation coefficient threshold = 0.1, P < 0.05) and analysis of microbial nitrogen and phosphorus cycling were conducted using the BMKCloud platform ( www.biocloud.net ). Microbial Isolation and SynCom assembly The P. odoratum roots and rhizosphere soil in continuous cropping field for 3 years(Y6) of Cili was used as the separation material, and the rhizosphere bacteria and plant endophyte were isolated according to Haiyambo et al.(Mittapalli et al. 2014 ; Haiyambo et al. 2015 ) with minor modifications (Supplementary Materials). 16S rDNA of bacteria and its2 of fungi were amplified and sequenced. Sequence alignment was performed on the NCBI website. The identified bacteria were subjected to a series of plant growth-promoting trait experiments and in vitro co-culture experiments with phenolic acids and other compounds (Supplementary Materials) following the methods described in the literature(R. et al. 1982; Haiyambo et al. 2015 ; Liu et al. 2017 ; Cortazar-Murillo et al. 2023 ; Ansari et al. 2025 ). Subsequently, strains with excellent plant growth-promoting characteristics were selected to construct SynCom; the preparation method of SynCom was referenced from Li Z et al.(Li et al. 2021 ), and the medium formulation was modified according to the strains (Supplementary Materials). Control effect of SynCom on CCOs Pot experiment was conducted using 60 one-year-old P. odoratum seedlings from local farmers in Cili, Hunan Province, and 20 seedlings in planted in three 50×50 cm square pots at 10 cm spacing per group. There were three treatment groups (A, B, and C). Group A was the disease control group, treated only with the pathogenic bacteria. Group B was the mental group, treated with both the pathogenic bacteria and SynCom. Group C was the blank control group, using distilled water instead of the pathogenic bacteria and SynCom. To simulate continuous cropping stress, 0.5 cm wounds were made on the surface of P. odoratum rhizomes using a sharp needle, followed by irrigation with 500 mL of Fusarium solani spore suspension (1×10⁷ CFU/mL) per pot. Detailed methods are provided in the Supplementary Materials. A concurrent field trial was established in September 2024 in Cili, Hunan Province, using a P. odoratum field with one prior cropping season. Planting material, selected from the previous crop, consisted of rhizomes approximately 13 cm in length with intact buds. Field methods are described in detail in the Supplementary Materials. Statistical analyses The differential abundances of soil chemical properties were screened out using analysis of variance (ANOVA) and Tukey test for soil chemical properties (significance level of P value < 0.05). KEGG pathway 3 metabolic pathways of Streptomyces among different groups were compared and plotted by their ratio to the total value. Histogram, Heatmap, and violin chart were visualized by the GraphPad Prism 9.5.0 and embellished by Adobe Illustrator 2022. Results Plant and soil physicochemical properties Compared with the first-year plantings, continuous cropping significantly advanced plant withering ( Fig. 1 a ). The rhizomes of continuous cropping plants were notably smaller and exhibited clear disease symptoms, including softening, rot, and reddish-brown surface spots ( Fig. 1 b , c ). After continuous cropping, the soil total nitrogen content and the relative contents of nitrate nitrogen and available phosphorus all significantly decreased, with total nitrogen deceasing by 27.41%, and the relative contents of nitrate nitrogen and available phosphorus decreasing by 78.26% and 34.69%. In addition, the activities of phosphatase and nitrite reductase decreased by 64.73% and 34.87% ( Fig. 1 d , e ). Rhizosphere soil nontarget metabolomics results indicated that the rhizosphere soil of P. odoratum contained various potential allelopathic substances, including phenolic acid and flavonoids. However, the contents of these substances did not change significantly under continuous cropping, while volcano plot analysis revealed that α-linolenyl alcohol increased significantly with continuous cropping ( Fig. 1 f, Fig. S1a ). Fig. 1 Note: a: Plant performance of the first planting and CCP in same time . b: Disease phenotype of the CCP . c: Comparison of the rhizomers of the first planting and CCP . d: Changes in the contents of total nitrogen and total phosphorus in the soil. e: Heat map of soil physical and chemical properties and enzyme activity, from top to bottom, the order is alkali hydrolyzable nitrogen (AhN), nitrate nitrogen (N), ammonium nitrogen (AN), nitrite nitrogen (NN), soluble organic nitrogen (SN), available phosphorus (SP), nitrite reductase (Nir), phosphatase (PP), soluble organic matter (SOM), humic acid (Hu). The nitrogen and phosphorus components were compared by their ratio to total nitrogen and total phosphorus. f. Volcano map of rhizosphere soil metabolites. Microbial diversity and community composition Diversity analysis showed that continuous cropping significantly reduced the α-diversity of rhizosphere bacteria and fungi at the genus level, as indicated by a decrease in the Chao1 index by 8.41% and 59.02% in bacteria and fungi, respectively while the diversity of root endophyte remained unchanged ( Fig. 2 a , b , Table S1 ). β-diversity analysis (PCoA) further confirmed a clear separation between the rhizosphere microbial communities of continuously cropped and first-year planted P. odoratum ( Fig. 2 c ). Taxonomic analysis revealed that the relative abundances of rhizosphere Actinobacteria , Curvularia , and Firmicutes were significantly reduced, the Actinobacteria and Firmicutes in continuous cropping were significantly reduced by 69.67% and 50.41%, with Firmicutes also declining in root communities ( Fig. 2 d , e ). Notably, several bacterial genera with relative abundance > 0.001, including Streptomyces (83.70%), Gaiella , Frankia , Jatrophihabitans and Bacillus showed substantial depletion under continuous cropping ( Fig. 2 f ). Additionally, the abundance of arbuscular mycorrhizal fungi (AMF) decreased in continuously cropped soils, whereas several other fungal taxa (e.g., Fusarium ) increased. ( Fig. 2 d ) . Fig.2 Note: a: Changes in the Chao1 index of alpha diversity of rhizosphere bacteria. b: Changes in the Chao1 index of alpha diversity of rhizosphere fungi. c: PCoA analysis of rhizosphere microorganisms in different years. d: Histogram of fungal composition in rhizosphere and root(genus level). e: Histogram of bacteria composition in rhizosphere and root(genus level). f: The content of rhizosphere microorganisms decreased significantly (relative abundance greater than 0.001). Microbial interaction networks and microbial functions According to the microbial interaction network at the genus level of the first cropping healthy P. odoratum and the continuous cropping P. odoratum ( Fig. 3 ), we was found that from the relationship weight of the network nodes (the number of connections) and the relative abundance (Size value), the microbial interaction network of the first cropping P. odoratum showed significant core dominance of Streptomyces and Frankia . The network weights of the two were both 11, which was most connected group among all nodes, and the relative abundances were 0.00855 and 0.00260, respectively, with the corresponding Size of 7.259 and 4.897. Interspecific interaction showed that the two genera were strongly positively correlated with a variety microorganisms in the Actinobacteria (such as Actinomadura , Kribbella , Micromonospora , etc.) (coefficient ≥ 0.9667, p < 0.001). With the increase of the number of cropping, the microbial interaction network of P. odoratum changed significantly. The network dominance of Streptomyces and Frankia was greatly weakened, and both were respectively reduced from 11 to 8, and the relative abundance also decreased significantly, and the positively correlated cluster formed in the Actinobacteria integrated. Instead, the Acidipila and Silvibacterium genera of the Acidobacteria became the new core nodes of the network, with a weight of 8 and a Size value as high as 8.850 and 7.440, respectively. Fig. 3 Note: a: Network map of rhizosphere microbial interaction of the first cropping. b. Network map of rhizosphere microbial interaction of the continuous cropping. Metagenomic analysis revealed declines in metabolic pathways associated with sugar utilization, bacterial flagella assembly, and the synthesis of various secondary metabolites, antibiotics, and immune-related compounds ( Fig. S 2 ). Metagenomic analysis of immune-related pathways indicated that the reduction in antibiotic synthesis and other defense-associated functions was primarily linked to a decline in Streptomyces abundance ( Fig. 4a ). Analysis of soil nitrogen cycling processes revealed significant reductions in nitrification and dissimilatory nitrate reduction ( Fig. 4b ). Furthermore, key genes involved in bacterial phosphorus solubilization ( gdh and pqqE ) were significantly down regulated ( Fig. 4c ). Importantly, these nitrogen- and phosphorus-cycling genes were predominantly associated with microbial genera that were reduced under continuous cropping, particularly Streptomyces ( Fig. 4d – f ). Fig. 4 Note: a: Heatmap of major metabolic pathways in Streptomyces . b: Heat map of soil nitrogen cycle pathway changes. c: Changes in the relative abundance of genes related to bacterial phosphorus solubilization. d: Heatmap of the abundance of related genera in the nitrification process. e: Heatmap of bacterial abundance related to dissimilatory nitrate reduction process. f: Source species abundance of phosphate solubilizing genes. Application effects of SynCom 16 Streptomyces strains and 33 other plant-growth-promoting bacteria were obtained from P. odoratum roots or rhizosphere. Based on in vitro assay for growth-promoting traits and pathogen antagonism ( Fig. S 4 ), one Streptomyces strain, two Bacillus strains and one Serratia strain were selected to construct a SynCom with complementary plant-beneficial functions. In both controlled pot and field experiments, plant health promotion, microecological balance restoration, and soil nutrient remediation occurred significant improvement. In pot experiments, SynCom treatment reduced rhizome disease incidence by 62.50% ( Fig. 5 a ) and promoted wound healing. Under field conditions, treated plots exhibited 2.5-fold more sprouting points than controls, and newly formed rhizomes developed more extensive root systems ( Fig. 5 b , Fig. S4 ). Importantly, beyond increasing biomass, SynCom significantly improved rhizome quality, as evidenced by elevated levels of total sugars and key bioactive compounds including salicylic acid and asperosaponin ( Fig. 5c ). Regarding soil nutrient remediation, pot experiments showed that SynCom treatment significantly increased soil available phosphorus, nitrate nitrogen, and humic acid contents compared to the control group ( Fig. S3 ). This growth-promoting effect was further validated under field conditions, where SynCom application significantly enhanced soil available phosphorus, alkali-hydrolyzable nitrogen, and nitrate nitrogen by 43%, 12%, and 58%, respectively ( Fig. 5 d ). At the level of microecological balance restoration, the SynCom not only successfully colonized the rhizosphere but also reshaped the rhizosphere microbial community structure. In both pot and field environments, the abundances of the introduced Bacillus , Streptomyces , and Serratia strains were significantly increased by 18.44-fold, 8.50-fold, and 4.81-fold, respectively, under field conditions ( Fig. 5 e ), which concomitantly enriched beneficial phylum including Firmicutes and Actinobacteria , with relative abundance increases of 64.85% and 15.46%, respectively, in pot experiments ( Fig. 5 f, g ). Concurrently, the abundance of pathogenic Fusarium species was effectively suppressed, while potentially beneficial fungi such as Aspergillus welwitschae were enriched ( Fig. 5 h ). Fig. 5 Note: a. Comparison of artificial wound healing of rhizomes in different treatment groups. b. Statistics of rhizomes status in different treatment groups. c. Differences in component content of rhizomes among different treatment groups. d. Comparison of soil nutrient content among different treatment groups. e. Heat map of relative abundance differences between introduced bacteria in different treatment groups. f. The difference heatmap of relative abundance of microorganisms (phylum level) in different treatment pots. g. Comparison of microbial composition and structure between the field experimental group and the initial sample group (phylum level). h. Heat map of relative abundance differences between fungi in different treatment groups. Discusson The rational design and application of SynCom represent a promising strategy for mitigating CCOs by restoring rhizosphere microecological functions. In this study, we demonstrated that a streamlined SynCom, composed of three functionally complementary beneficial strains ( Streptomyces ., Bacillus , and Serratia ) consistently alleviated CCOs in P. odoratum across both controlled pot and field conditions. Our results provide evidence that targeted supplementation of core functional microbes can simultaneously improve soil nutrient availability, reshape rhizosphere microbial communities, and enhance plant health and quality, offering a viable approach for sustainable cultivation of medicinal plants. The drivers of CCOs in P. odoratum Phenolic acids have long been considered key allelochemicals driving continuous cropping obstacles (CCOs) through their inhibitory effects on beneficial microbes(Li et al. 2014 ; Wang et al. 2023 ; Xu et al. 2025 ). However, our findings challenge this established paradigm in P. odoratum . Although phenolic acids were detectable in the rhizosphere, they exhibited no significant increase in CCOs. Consistently, the beneficial taxa that declined under continuous cropping—including Streptomyces and Bacillus —were not susceptible to phenolic acids suppression ( Fig. S1 b ). Instead, metabolite–microbe correlation analysis and validation experiments identified α-linolenyl alcohol specifically inhibiting Streptomyces and Bacillus (Fig. 1 f, Fig. S5 ). These results demonstrate that the drivers of CCOs are species-specific and cannot be generalized across cropping systems. Our study expands the current understanding of CCO mechanisms by revealing that fatty acid-derived compounds, rather than phenolic acids, can serve as primary triggers of rhizosphere microecological imbalance in certain medicinal plants. SynCom restores rhizosphere functions and plant health under continuous cropping. Building on the mechanistic insight that LA-driven decline of core beneficial taxa underlies CCOs in P. odoratum , we rationally designed a streamlined SynCom comprising Streptomyces , Bacillus , and Serratia . Across both pot and field experiments, SynCom application consistently improved soil nutrient availability (Fig. 5 d). This aligns with previous studies demonstrating that PGPR-containing consortia enhance nutrient mobilization through phosphate solubilization and nitrogen cycling(Li et al. 2026 ). Notably, the significant enrichment of Firmicutes and Actinobacteria (Fig. 5 f, g), alongside suppression of pathogenic Fusarium and enrichment of beneficial fungi such as Aspergillus welwitschae (Fig. 5 h), indicates that the SynCom functions as a microbiome modulator rather than merely a bioinoculant. This observation is consistent with emerging evidence that keystone taxa can drive community-wide shifts through metabolite-mediated interactions or modulation of plant immunity(Pieterse et al. 2014 ; Zhou et al. 2022 ). The successful recruitment of additional beneficial taxa suggests that the SynCom may help re-establish a resilient, disease-suppressive rhizosphere microbiome—a key goal in CCOs management(Zhou et al. 2023 ). These rhizosphere-level improvements translated directly into enhanced plant performance,including the reduced disease incidence and the improved rhizome quality with elevated bioactive compounds including salicylic acid and asperosaponin (Fig. 5 c). The increase in salicylic acid content is particularly noteworthy, as this phytohormone is central to systemic acquired resistance; similar priming effects have been reported for beneficial Bacillus and Streptomyces strains in other plant systems(Vergnes et al. 2020 ; Gogoi et al. 2025 ). Moreover, the improved accumulation of asperosaponin—a key medicinal compound in P. odoratum —suggests that SynCom-mediated rhizosphere restoration may indirectly modulate secondary metabolite biosynthesis, consistent with reports that beneficial microbes can trigger metabolic pathways(Zhong et al. 2022 ; Su et al. 2023 ). Our results demonstrate that targeted restoration of missing core functions, rather than broad-spectrum microbial supplementation, offers an efficient strategy for managing CCOs. Conclusion This study uncovers the mechanistic basis of continuous cropping obstacles in P. odoratum and presents a targeted solution. We reveal that α-linolenyl alcohol, rather than traditionally implicated phenolic acids, acts as the key allelochemical driving the decline of core beneficial taxa, particularly Streptomyces . Based on this insight, a SynCom was designed to restore these missing functions. Pot and field validations confirmed that this SynCom effectively rehabilitates rhizosphere microecology, suppresses disease, and improves both yield and medicinal quality. Our work establishes a paradigm for mechanism-guided development of precision microbial consortia, offering a sustainable approach to overcoming CCOs in medicinal plant cultivation. Declarations Funding Funding by Science and Technology Projects in Guangzhou Competing interest The authors declare no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Author Contributions Chenghua Luo: conceptualization, data curation, formal analysis, visualization, writing the original draft. Hua Yang: investigation, methodology. Zhifa Zhang: data curation, methodology. Yaping Chen: conceptualization, funding acquisition, manuscript review, and editing. All authors substantially contributed to the subsequent drafts. 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Microbiome 11(1). 10.1186/s40168-023-01513-1 Zhang J, Liu Y-X, Guo X, Qin Y, Garrido-Oter R, Schulze-Lefert P, Bai Y (2021) High-throughput cultivation and identification of bacteria from the plant root microbiota. Nat Protoc 16(2):988–1012. 10.1038/s41596-020-00444-7 Zheng Y, Cao X, Zhou Y, Ma S, Wang Y, Li Z, Zhao D, Yang Y, Zhang H, Meng C, Xie Z, Sui X, Xu K, Li Y, Zhang C-S (2024) Purines enrich root-associated Pseudomonas and improve wild soybean growth under salt stress. Nat Commun 15(1). 10.1038/s41467-024-47773-9 Zhong C, Chen C, Gao X, Tan C, Bai H, Ning K (2022) Multi-omics profiling reveals comprehensive microbe–plant–metabolite regulation patterns for medicinal plant Glycyrrhiza uralensis Fisch. Plant Biotechnol J 20(10):1874–1887. 10.1111/pbi.13868 Zhou X, Li C, Liu L, Zhao J, Zhang J, Cai Z, Huang X (2019) Control of Fusarium wilt of lisianthus by reassembling the microbial community in infested soil through reductive soil disinfestation. Microbiol Res 220:1–11. 10.1016/j.micres.2018.12.001 Zhou X, Wang J, Liu F, Liang J, Zhao P, Tsui CKM, Cai L (2022) Cross-kingdom synthetic microbiota supports tomato suppression of Fusarium wilt disease. Nat Commun 13(1). 10.1038/s41467-022-35452-6 Zhou Y, Yang Z, Liu J, Li X, Wang X, Dai C, Zhang T, Carrión VJ, Wei Z, Cao F, Delgado-Baquerizo M, Li X (2023) Crop rotation and native microbiome inoculation restore soil capacity to suppress a root disease. Nat Commun 14(1). 10.1038/s41467-023-43926-4 Zhu W, Lomsadze A, Borodovsky M (2010) Ab initio gene identification in metagenomic sequences. Nucleic Acids Res 38(12):e132–e132. 10.1093/nar/gkq275 Supplementary Files Supplementmaterial.docx Supplementary.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 06 May, 2026 Editor invited by journal 02 Apr, 2026 Editor assigned by journal 02 Apr, 2026 First submitted to journal 31 Mar, 2026 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-9201673","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635318893,"identity":"556c444c-41ea-4661-9f9c-6cb72eeec6be","order_by":0,"name":"Chenghua Luo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chenghua","middleName":"","lastName":"Luo","suffix":""},{"id":635318894,"identity":"b7b42523-1c11-4c6e-b8e1-5256b10225fb","order_by":1,"name":"Hua Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Yang","suffix":""},{"id":635318895,"identity":"533636d0-d824-4236-bc1c-0326cf7a4851","order_by":2,"name":"Zhifa Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhifa","middleName":"","lastName":"Zhang","suffix":""},{"id":635318896,"identity":"80155d22-2fb1-412d-8cab-2861d797d77c","order_by":3,"name":"Yaping Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDACCTBpA2MQryWNdC2HSdDCP7v52GPeHeft+We3X2D4UcMgb07QkjvH0o15z9xOnHHnTAFjzzEGw50NBLQYSOSYSfO23U4AMhIYeBsYEgwOENSS/w2o5Zw9SAvjX+K05LABtRxg3CCRfoCZKFskbqSZSc49kwzyC8NhmWMShhsIaeGfkfxM4u0OO1CIPXz4psZGnqAtIMAEdA8Q8IAUExk7jD/BWtgfEKd8FIyCUTAKRhwAAAIsPopysTrCAAAAAElFTkSuQmCC","orcid":"","institution":"UCAS: University of the Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-03-23 14:12:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9201673/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9201673/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109335970,"identity":"e9546a55-c4f9-41cf-96f0-f3f1abdd5590","added_by":"auto","created_at":"2026-05-15 17:10:54","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e a: Plant performance of the first planting and CCP in same time\u003cem\u003e.\u003c/em\u003e b: Disease phenotype of the CCP\u003cem\u003e.\u003c/em\u003e c: Comparison of the rhizomers of the first planting and CCP\u003cem\u003e. \u003c/em\u003ed: Changes in the contents of total nitrogen and total phosphorus in the soil. e: Heat map of soil physical and chemical properties and enzyme activity, from top to bottom, the order is alkali hydrolyzable nitrogen (AhN), nitrate nitrogen (N), ammonium nitrogen(AN), nitrite nitrogen (NN), soluble organic nitrogen (SN), available phosphorus(SP), nitrite reductase (Nir), phosphatase(PP), soluble organic matter (SOM), humic acid(Hu). The nitrogen and phosphorus components were compared by their ratio to total nitrogen and total phosphorus. f. Volcano map of rhizosphere soil metabolites.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/ee34ab8c39f860f33b92941a.jpeg"},{"id":109335974,"identity":"ac0fee26-a8df-4315-9601-bfcff85bdd5d","added_by":"auto","created_at":"2026-05-15 17:10:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225242,"visible":true,"origin":"","legend":"\u003cp\u003eNote: a: Network map of rhizosphere microbial interaction of the first cropping. b. Network map of rhizosphere microbial interaction of the continuous cropping.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/c77bbceb0581381f1223a733.jpeg"},{"id":109335971,"identity":"3272eead-1eb8-493e-b074-c2635158d098","added_by":"auto","created_at":"2026-05-15 17:10:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e a: Heatmap of major metabolic pathways in \u003cem\u003eStreptomyces.\u003c/em\u003e b: Heat map of soil nitrogen cycle pathway changes. c: Changes in the relative abundance of genes related to bacterial phosphorus solubilization. d: Heatmap of the abundance of related genera in the nitrification process. e: Heatmap of bacterial abundance related to dissimilatory nitrate reduction process. f: Source species abundance of phosphate solubilizing genes.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/14f94d52ff5d98d814a3c8a7.jpeg"},{"id":109335975,"identity":"8b946897-4993-4a6e-af61-c8ddb01ccbdc","added_by":"auto","created_at":"2026-05-15 17:10:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003ea. Comparison of artificial wound healing of rhizomes in different treatment groups. b. Statistics of rhizomes status in different treatment groups. c. Differences in component content of rhizomes among different treatment groups. d. Comparison of soil nutrient content among different treatment groups. e. Heat map of relative abundance differences between introduced bacteria in different treatment groups. f. The difference heatmap of relative abundance of microorganisms (phylum level) in different treatment pots. g. Comparison of microbial composition and structure between the field experimental group and the initial sample group (phylum level). h. Heat map of relative abundance differences between fungi in different treatment groups.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/18321b1532d26c768b8d6a92.jpeg"},{"id":109405571,"identity":"661159c2-ce41-4734-8a80-516c883ae3f4","added_by":"auto","created_at":"2026-05-17 13:19:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1063843,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/374d6dd2-19ef-402b-b10e-c541f5dc4fdf.pdf"},{"id":109405494,"identity":"29d726a6-7ae5-45f2-acf6-fabc1eafdc74","added_by":"auto","created_at":"2026-05-17 13:18:29","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":23977,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/acefa51e700a145b7aca2f04.docx"},{"id":109335973,"identity":"b22d6c6f-f495-40c7-ba23-9fcd61c13c0e","added_by":"auto","created_at":"2026-05-15 17:10:54","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":707730,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9201673/v1/7e72267d275684b2852eabcd.docx"}],"financialInterests":"","formattedTitle":"Key Microorganism Alleviate Continuous Cropping Obstacles of Polygonatum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eContinuous cropping obstacles (CCOs) in medicinal plants have emerged as a critical constraint to the sustainable development of the industry. These obstacles typically manifest as reduced yield, deteriorated quality, aggravated soil-borne diseases, and replanting failure, and are commonly observed across a wide range of medicinal species(Wu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Alami et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), often resulting in significant economic losses(Wacal et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Conventional management practices such as crop rotation and chemical control are often unsuitable for large-scale cultivation and may entail environmental risks and pesticide-residue concerns(Zhou et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Becker et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, elucidating the mechanisms underlying continuous cropping obstacles and developing green, sustainable mitigation strategies are of paramount importance.\u003c/p\u003e \u003cp\u003eCCOs primarily arise from the interplay of deteriorated soil physicochemical properties, accumulation of allelopathic compounds, and dysbiosis of the rhizosphere microbial community. Continuous cropping leads to soil acidification, nutrient imbalance, and secondary salinization(Alami et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Meanwhile, root-secreted allelochemicals\u0026mdash;such as organic acids, phenolic acids, and flavonoids\u0026mdash;can directly inhibit plant growth or indirectly compromise plant health by altering the rhizosphere microenvironment(Liu Ping 2018; Sasse et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Alami et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bao et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Of particular importance is the disruption of rhizosphere microbial communities continuous cropping generally reduces microbial diversity, enriches pathogenic taxa, and diminishes beneficial bacteria(Fei et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Rhizosphere beneficial bacteria are considered as a \"second genome\" for plants, enhancing nutrient acquisition, stress tolerance and pathogen resistanc. Plant growth-promoting rhizobacteria (PGPR) improve nutrient availability via phosphate solubilization, nitrogen fixation, or phytohormone production (e.g., IAA and zeatin)(Yu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Certain PGPR strains also antagonize pathogens through antibiotic synthesis for instance, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e suppresses \u003cem\u003eRalstonia solanacearum\u003c/em\u003e in tomato(Zhou et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Other microbes enhance plant resilience to abiotic stress through ACC deaminase activity, which modulates ethylene levels, or through extended hyphal networks (e.g., arbuscular mycorrhizal fungi) that improve water and nutrient uptake(Chandwani\u0026amp; Amaresan \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nevertheless, the key microorganisms that play vital roles in continuous cropping and their specific functional changes remain poorly understood.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePolygonatum odoratum\u003c/em\u003e, a perennial medicinal herb highly susceptible to CCOs, exhibits severe root rot and significant declines in both yield and quality under continuous cropping. These effects have become a primary cause for the contraction of its cultivation area in major production regions(Ni et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studies showed that the main disease of obstacle was \u003cem\u003eFusarium\u003c/em\u003e root rot, and continuous cropping of \u003cem\u003eP. odoratum\u003c/em\u003e leading to a significant decrease in soil nitrogen and phosphorus nutrition, and its own photosynthesis was weakened and growth was blocked(Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Elucidating the mechanisms underlying CCOs and developing biological solutions are therefore of considerable practical importance.\u003c/p\u003e \u003cp\u003eIn this study, the key microorganisms causing the CCOs were identified through microbial diversity and metagenomic analysis. Afterword, a SynCom composed of functionally defined strains was constructed by combining microbial separation and culture, and its effectiveness in alleviating the CCOs was verified through pot and field trials, providing a feasible microbial community regulation strategy for the green and sustainable cultivation of \u003cem\u003eP. odoratum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection, soil physicochemical property and enzyme activity measurement\u003c/h2\u003e \u003cp\u003eThree representative \u003cem\u003eP. odoratum\u003c/em\u003e production area ShaoYang (SY: 111\u0026deg;38\u0026prime;25\u0026Prime;E, 27\u0026deg;16\u0026prime;07\u0026Prime;N), GuiYang (GY: 112\u0026deg;39\u0026prime;28\u0026Prime;E, 26\u0026deg;04\u0026prime;55\u0026Prime;N), CiLi (CL: 111\u0026deg;07\u0026prime;12\u0026Prime;E, 29\u0026deg;25\u0026prime;12\u0026Prime;N) in Hunan Province, China, were selected as sampling locations. Each location included \u003cem\u003eP. odoratum\u003c/em\u003e cropped in new field for first-planting 1 year (Y1), 3 years (Y3) and cropped in continuous cropping field for 3 years (Y6) with 3 replicates (n\u0026thinsp;=\u0026thinsp;3). First, three plants with similar growth status were randomly select and there were more than 1m apart within a plot. After removing dead leaves and debris from the ground, \u003cem\u003eP. odoratum\u003c/em\u003e were dug out with depth 20-30cm soil cube. After removed top 5 cm of soil and loosely attached large soil clumps, we collected 200g of soil from 10cm below the surface and 5 cm away from the root system for physical and chemical property analysis. The physical and chemical properties of the soil and soil enzyme activity (Supplementary Materials) were measured from soil samples according to Castillo et al.(Castillo\u0026amp; Regan \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Then, 20 g rhizosphere soil sample were collected by brushing the soil attach to the roots. Finally, we cleaned the underground parts of the plants, and used scissors toting off 10g of fresh, clean roots growing on the rhizome as the roots sample. All samples were transferred to the laboratory on dry ice, each replicate sample was analyzed separately for the omics data described in the text.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRhizosphere soil nontarget metabolomics and root targeted metabolomics analysis\u003c/h3\u003e\n\u003cp\u003eRhizosphere soil and plant root samples were first lyophilization treated. Metabolites were extracted and re-purified. Final supernatants and quality control (QC) pooled samples were analyzed. Untargeted metabolomics and widely targeted metabolomics were performed according to literature(Yue et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with minor modifications (Supplementary Materials).\u003c/p\u003e\n\u003ch3\u003eHigh-throughput DNA extraction, amplification, and sequencing\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA of the rhizosphere soil and roots sample was extracted by using the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.). The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F: 5'-ACTCCTACGGGAGGCAGCA-3' and 806R: 5'- GGACTACHVGGGTWTCTAAT-3'. The its2 gene of fungi were amplified with primer pairs its2F: 5'-GCATCGATGAAGAACGCAGC-3'; its2R: 5'-TCCTCCGCTTATTGATATGC-3'; Both the forward and reverse primers were tailed with sample-specific Illumina index sequences to allow for deep sequencing. The purified PCR products was paired-end sequenced (2\u0026times;250) on an Illumina novaseq6000 (Beijing Biomarker Technologies Co., Ltd., Beijing, China). Raw data were assembled and quality-filtered according to Caporaso et al., and chimeric sequences were removed using the UCHIME tool in USEARCH. The sequences matching the mitochondria and chloroplast were also removed, and the remaining effective sequences were clustered into operational taxonomic units (OTUs) at 97% similarity(Caporaso et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMetagenomic sequencing workflow and data analysis\u003c/h3\u003e\n\u003cp\u003eRhizosphere soil DNA was sequenced on an Illumina platform following the manufacturer\u0026rsquo;s standard protocol. Raw reads were quality-filtered using Fastp (v0.23.2) to obtain clean reads(Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Host-derived reads were removed by alignment to the host genome with Bowtie2 (v2.4.5)(Langmead\u0026amp; Salzberg \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Metagenome assembly was performed using MEGAHIT (v1.2.9), retaining contigs longer than 300 bp(Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Coding regions were predicted with MetaGeneMark (v3.26) under default parameters(Zhu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Redundant genes were clustered using MMseqs2 (v12-113e3) at 95% sequence identity and 90% coverage thresholds(Mirdita et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Non-redundant protein sequences were annotated against the NR database using BLASTp (E-value\u0026thinsp;\u0026le;\u0026thinsp;1e\u0026thinsp;\u0026minus;\u0026thinsp;5). Downstream analyses, including alpha diversity, principal coordinate analysis (PCoA), intergroup taxonomic differences, KEGG functional profiling (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), spearman correlation-based microbial interaction network at the genus level (correlation coefficient threshold\u0026thinsp;=\u0026thinsp;0.1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and analysis of microbial nitrogen and phosphorus cycling were conducted using the BMKCloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.biocloud.net\" target=\"_blank\"\u003ewww.biocloud.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMicrobial Isolation and SynCom assembly\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eP. odoratum\u003c/em\u003e roots and rhizosphere soil in continuous cropping field for 3 years(Y6) of Cili was used as the separation material, and the rhizosphere bacteria and plant endophyte were isolated according to Haiyambo et al.(Mittapalli et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Haiyambo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) with minor modifications (Supplementary Materials). 16S rDNA of bacteria and its2 of fungi were amplified and sequenced. Sequence alignment was performed on the NCBI website. The identified bacteria were subjected to a series of plant growth-promoting trait experiments and in vitro co-culture experiments with phenolic acids and other compounds (Supplementary Materials) following the methods described in the literature(R. et al. 1982; Haiyambo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cortazar-Murillo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ansari et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Subsequently, strains with excellent plant growth-promoting characteristics were selected to construct SynCom; the preparation method of SynCom was referenced from Li Z et al.(Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and the medium formulation was modified according to the strains (Supplementary Materials).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eControl effect of SynCom on CCOs\u003c/h2\u003e \u003cp\u003ePot experiment was conducted using 60 one-year-old \u003cem\u003eP. odoratum\u003c/em\u003e seedlings from local farmers in Cili, Hunan Province, and 20 seedlings in planted in three 50\u0026times;50 cm square pots at 10 cm spacing per group. There were three treatment groups (A, B, and C). Group A was the disease control group, treated only with the pathogenic bacteria. Group B was the mental group, treated with both the pathogenic bacteria and SynCom. Group C was the blank control group, using distilled water instead of the pathogenic bacteria and SynCom. To simulate continuous cropping stress, 0.5 cm wounds were made on the surface of \u003cem\u003eP. odoratum\u003c/em\u003e rhizomes using a sharp needle, followed by irrigation with 500 mL of \u003cem\u003eFusarium solani\u003c/em\u003e spore suspension (1\u0026times;10⁷ CFU/mL) per pot. Detailed methods are provided in the Supplementary Materials. A concurrent field trial was established in September 2024 in Cili, Hunan Province, using a \u003cem\u003eP. odoratum\u003c/em\u003e field with one prior cropping season. Planting material, selected from the previous crop, consisted of rhizomes approximately 13 cm in length with intact buds. Field methods are described in detail in the Supplementary Materials.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eThe differential abundances of soil chemical properties were screened out using analysis of variance (ANOVA) and Tukey test for soil chemical properties (significance level of P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). KEGG pathway 3 metabolic pathways of \u003cem\u003eStreptomyces\u003c/em\u003e among different groups were compared and plotted by their ratio to the total value. Histogram, Heatmap, and violin chart were visualized by the GraphPad Prism 9.5.0 and embellished by Adobe Illustrator 2022.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePlant and soil physicochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the first-year plantings, continuous cropping significantly advanced plant withering (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e). The rhizomes of continuous cropping plants were notably smaller and exhibited clear disease symptoms, including softening, rot, and reddish-brown surface spots (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e). After continuous cropping, the soil total nitrogen content and the relative contents of nitrate nitrogen and available phosphorus all significantly decreased, with total nitrogen deceasing by 27.41%, and the relative contents of nitrate nitrogen and available phosphorus decreasing by 78.26% and 34.69%.\u0026nbsp;In addition, the activities of phosphatase and nitrite reductase decreased by 64.73% and 34.87% (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e). Rhizosphere soil nontarget metabolomics results indicated that the rhizosphere soil of \u003cem\u003eP. odoratum\u003c/em\u003e contained various potential allelopathic substances, including phenolic acid and flavonoids. However, the contents of these substances did not change significantly under continuous cropping, while volcano plot analysis revealed that \u0026alpha;-linolenyl alcohol increased significantly with continuous cropping (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1\u003c/strong\u003e\u003cstrong\u003ef, Fig.\u003c/strong\u003e\u003cstrong\u003eS1a\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFig. 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e a: Plant performance of the first planting and CCP in same time\u003cem\u003e.\u003c/em\u003e b: Disease phenotype of the CCP\u003cem\u003e.\u003c/em\u003e c: Comparison of the rhizomers of the first planting and CCP\u003cem\u003e.\u0026nbsp;\u003c/em\u003ed: Changes in the contents of total nitrogen and total phosphorus in the soil.\u0026nbsp;e: Heat map of soil physical and chemical properties and enzyme activity, from top to bottom, the order is alkali hydrolyzable nitrogen\u0026nbsp;(AhN), nitrate nitrogen\u0026nbsp;(N), ammonium nitrogen\u0026nbsp;(AN), nitrite nitrogen\u0026nbsp;(NN), soluble organic nitrogen\u0026nbsp;(SN), available phosphorus\u0026nbsp;(SP), nitrite reductase\u0026nbsp;(Nir), phosphatase\u0026nbsp;(PP), soluble organic matter\u0026nbsp;(SOM), humic acid\u0026nbsp;(Hu). The nitrogen and phosphorus components were compared by their ratio to total nitrogen and total phosphorus.\u0026nbsp;f. Volcano map of rhizosphere soil metabolites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial diversity and community composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiversity analysis showed that continuous cropping significantly reduced the \u0026alpha;-diversity of rhizosphere bacteria and fungi at the genus level, as indicated by a decrease in the Chao1 index by 8.41% and 59.02% in bacteria and fungi, respectively while the diversity of root endophyte remained unchanged (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e, Table S1\u003c/strong\u003e). \u0026beta;-diversity analysis (PCoA) further confirmed a clear separation between the rhizosphere microbial communities of continuously cropped and first-year planted \u003cem\u003eP. odoratum\u003c/em\u003e (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e). Taxonomic analysis revealed that the relative abundances of rhizosphere \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eCurvularia\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e were significantly reduced, the \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e in continuous cropping were significantly reduced by 69.67% and 50.41%, with \u003cem\u003eFirmicutes\u0026nbsp;\u003c/em\u003ealso declining in root communities (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e). Notably, several bacterial genera with relative abundance \u0026gt; 0.001, including \u003cem\u003eStreptomyces\u003c/em\u003e (83.70%), \u003cem\u003eGaiella\u003c/em\u003e, \u003cem\u003eFrankia\u003c/em\u003e, \u003cem\u003eJatrophihabitans\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacillus\u003c/em\u003e showed substantial depletion under continuous cropping (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;2\u003c/strong\u003e\u003cstrong\u003ef\u003c/strong\u003e). Additionally, the abundance of arbuscular mycorrhizal fungi (AMF) decreased in continuously cropped soils, whereas several other fungal taxa (e.g., \u003cem\u003eFusarium\u003c/em\u003e) increased. (\u003cem\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFig.2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u0026nbsp;\u003c/strong\u003ea: Changes in the Chao1 index of alpha diversity of rhizosphere bacteria. b: Changes in the Chao1 index of alpha diversity of rhizosphere fungi. c: PCoA analysis of rhizosphere microorganisms in different years. d: Histogram of fungal composition in rhizosphere and root(genus level). e: Histogram of bacteria composition in rhizosphere and root(genus level). f: The content of rhizosphere microorganisms decreased significantly (relative abundance greater than 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial interaction networks and microbial functions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the microbial interaction network at the genus level of the first cropping healthy \u003cem\u003eP. odoratum\u003c/em\u003e and the continuous cropping \u003cem\u003eP. odoratum\u003c/em\u003e (\u003cstrong\u003eFig. 3\u003c/strong\u003e), we was found that from the relationship weight of the network nodes (the number of connections) and the relative abundance (Size value), the microbial interaction network of the first cropping \u003cem\u003eP. odoratum\u003c/em\u003e showed significant core dominance of \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFrankia\u003c/em\u003e. The network weights of the two were both 11, which was most connected group among all nodes, and the relative abundances were 0.00855 and 0.00260, respectively, with the corresponding Size of 7.259 and 4.897. Interspecific interaction showed that the two genera were strongly positively correlated with a variety microorganisms in the \u003cem\u003eActinobacteria\u003c/em\u003e(such as \u003cem\u003eActinomadura\u003c/em\u003e, \u003cem\u003eKribbella\u003c/em\u003e, \u003cem\u003eMicromonospora\u003c/em\u003e, etc.) (coefficient \u0026ge; 0.9667, p \u0026lt; 0.001). With the increase of the number of cropping, the microbial interaction network of \u003cem\u003eP. odoratum\u003c/em\u003e changed significantly. The network dominance of \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFrankia\u0026nbsp;\u003c/em\u003ewas greatly weakened, and both were respectively reduced from 11 to 8, and the relative abundance also decreased significantly, and the positively correlated cluster formed in the \u003cem\u003eActinobacteria\u003c/em\u003e integrated. Instead, the \u003cem\u003eAcidipila\u0026nbsp;\u003c/em\u003eand \u003cem\u003eSilvibacterium\u0026nbsp;\u003c/em\u003egenera of the \u003cem\u003eAcidobacteria\u0026nbsp;\u003c/em\u003ebecame the new core nodes of the network, with a weight of 8 and a Size value as high as 8.850 and 7.440, respectively.\u003c/p\u003e\n\u003cp\u003eFig. 3\u003c/p\u003e\n\u003cp\u003eNote: a: Network map of rhizosphere microbial interaction of the first cropping. b. Network map of rhizosphere microbial interaction of the continuous cropping.\u003c/p\u003e\n\u003cp\u003eMetagenomic analysis revealed declines in metabolic pathways associated with sugar utilization, bacterial flagella assembly, and the synthesis of various secondary metabolites, antibiotics, and immune-related compounds (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;S\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e). Metagenomic analysis of immune-related pathways indicated that the reduction in antibiotic synthesis and other defense-associated functions was primarily linked to a decline in \u003cem\u003eStreptomyces\u003c/em\u003e abundance (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e4a\u003c/strong\u003e). Analysis of soil nitrogen cycling processes revealed significant reductions in nitrification and dissimilatory nitrate reduction (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e4b\u003c/strong\u003e). Furthermore, key genes involved in bacterial phosphorus solubilization (\u003cem\u003egdh\u003c/em\u003e and \u003cem\u003epqqE\u003c/em\u003e) were significantly down regulated (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e4c\u003c/strong\u003e). Importantly, these nitrogen- and phosphorus-cycling genes were predominantly associated with microbial genera that were reduced under continuous cropping, particularly \u003cem\u003eStreptomyces\u003c/em\u003e (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e4d\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFig. 4\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e a: Heatmap of major metabolic pathways in \u003cem\u003eStreptomyces\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e b: Heat map of soil nitrogen cycle pathway changes. c: Changes in the relative abundance of genes related to bacterial phosphorus solubilization. d: Heatmap of the abundance of related genera in the nitrification process. e: Heatmap of bacterial abundance related to dissimilatory nitrate reduction process. f: Source species abundance of phosphate solubilizing genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApplication effects of SynCom\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e16 \u003cem\u003eStreptomyces\u003c/em\u003e strains and 33 other plant-growth-promoting bacteria were obtained from \u003cem\u003eP. odoratum\u003c/em\u003e roots or rhizosphere. Based on in vitro assay for growth-promoting traits and pathogen antagonism (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;S\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e), one \u003cem\u003eStreptomyces\u003c/em\u003e strain, two \u003cem\u003eBacillus\u003c/em\u003e strains and one \u003cem\u003eSerratia\u003c/em\u003e\u003cem\u003e\u0026nbsp;strain\u0026nbsp;\u003c/em\u003ewere selected to construct a SynCom with complementary plant-beneficial functions.\u003c/p\u003e\n\u003cp\u003eIn both controlled pot and field experiments, plant health promotion, microecological balance restoration, and soil nutrient remediation occurred significant improvement. In pot experiments, SynCom treatment reduced rhizome disease incidence by 62.50% (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e) and promoted wound healing. Under field conditions, treated plots exhibited 2.5-fold more sprouting points than controls, and newly formed rhizomes developed more extensive root systems (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;S4\u003c/strong\u003e). Importantly, beyond increasing biomass, SynCom significantly improved rhizome quality, as evidenced by elevated levels of total sugars and key bioactive compounds including salicylic acid and asperosaponin (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e5c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eRegarding soil nutrient remediation, pot experiments showed that SynCom treatment significantly increased soil available phosphorus, nitrate nitrogen, and humic acid contents compared to the control group (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;S3\u003c/strong\u003e). This growth-promoting effect was further validated under field conditions, where SynCom application significantly enhanced soil available phosphorus, alkali-hydrolyzable nitrogen, and nitrate nitrogen by 43%, 12%, and 58%, respectively (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAt the level of microecological balance restoration, the SynCom not only successfully colonized the rhizosphere but also reshaped the rhizosphere microbial community structure. In both pot and field environments, the abundances of the introduced \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003estrains were significantly increased by 18.44-fold, 8.50-fold, and 4.81-fold, respectively, under field conditions (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e), which concomitantly enriched beneficial phylum including \u003cem\u003eFirmicutes\u003c/em\u003eand \u003cem\u003eActinobacteria\u003c/em\u003e, with relative abundance increases of 64.85% and 15.46%, respectively, in pot experiments (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5\u003c/strong\u003e\u003cstrong\u003ef, g\u003c/strong\u003e). Concurrently, the abundance of pathogenic \u003cem\u003eFusarium\u003c/em\u003especies was effectively suppressed, while potentially beneficial fungi such as \u003cem\u003eAspergillus welwitschae\u003c/em\u003ewere enriched (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5\u003c/strong\u003e\u003cstrong\u003eh\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFig. 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u0026nbsp;\u003c/strong\u003ea. Comparison of artificial wound healing of rhizomes in different treatment groups. b. Statistics of rhizomes status in different treatment groups. c. Differences in component content of rhizomes among different treatment groups. d. Comparison of soil nutrient content among different treatment groups. e. Heat map of relative abundance differences between introduced bacteria in different treatment groups. f. The difference heatmap of relative abundance of microorganisms (phylum level) in different treatment pots. g. Comparison of microbial composition and structure between the field experimental group and the initial sample group (phylum level). h. Heat map of relative abundance differences between fungi in different treatment groups.\u003c/p\u003e"},{"header":"Discusson","content":"\u003cp\u003eThe rational design and application of SynCom represent a promising strategy for mitigating CCOs by restoring rhizosphere microecological functions. In this study, we demonstrated that a streamlined SynCom, composed of three functionally complementary beneficial strains (\u003cem\u003eStreptomyces\u003c/em\u003e., \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003e) consistently alleviated CCOs in \u003cem\u003eP. odoratum\u003c/em\u003e across both controlled pot and field conditions. Our results provide evidence that targeted supplementation of core functional microbes can simultaneously improve soil nutrient availability, reshape rhizosphere microbial communities, and enhance plant health and quality, offering a viable approach for sustainable cultivation of medicinal plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe drivers of CCOs in\u003c/b\u003e \u003cb\u003eP. odoratum\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhenolic acids have long been considered key allelochemicals driving continuous cropping obstacles (CCOs) through their inhibitory effects on beneficial microbes(Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, our findings challenge this established paradigm in \u003cem\u003eP. odoratum\u003c/em\u003e. Although phenolic acids were detectable in the rhizosphere, they exhibited no significant increase in CCOs. Consistently, the beneficial taxa that declined under continuous cropping\u0026mdash;including \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e\u0026mdash;were not susceptible to phenolic acids suppression (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e). Instead, metabolite\u0026ndash;microbe correlation analysis and validation experiments identified α-linolenyl alcohol specifically inhibiting \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cb\u003eFig. S5\u003c/b\u003e). These results demonstrate that the drivers of CCOs are species-specific and cannot be generalized across cropping systems. Our study expands the current understanding of CCO mechanisms by revealing that fatty acid-derived compounds, rather than phenolic acids, can serve as primary triggers of rhizosphere microecological imbalance in certain medicinal plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynCom restores rhizosphere functions and plant health under continuous cropping.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBuilding on the mechanistic insight that LA-driven decline of core beneficial taxa underlies CCOs in \u003cem\u003eP. odoratum\u003c/em\u003e, we rationally designed a streamlined SynCom comprising \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003e. Across both pot and field experiments, SynCom application consistently improved soil nutrient availability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). This aligns with previous studies demonstrating that PGPR-containing consortia enhance nutrient mobilization through phosphate solubilization and nitrogen cycling(Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Notably, the significant enrichment of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, g), alongside suppression of pathogenic \u003cem\u003eFusarium\u003c/em\u003e and enrichment of beneficial fungi such as \u003cem\u003eAspergillus welwitschae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), indicates that the SynCom functions as a microbiome modulator rather than merely a bioinoculant. This observation is consistent with emerging evidence that keystone taxa can drive community-wide shifts through metabolite-mediated interactions or modulation of plant immunity(Pieterse et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The successful recruitment of additional beneficial taxa suggests that the SynCom may help re-establish a resilient, disease-suppressive rhizosphere microbiome\u0026mdash;a key goal in CCOs management(Zhou et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese rhizosphere-level improvements translated directly into enhanced plant performance,including the reduced disease incidence and the improved rhizome quality with elevated bioactive compounds including salicylic acid and asperosaponin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The increase in salicylic acid content is particularly noteworthy, as this phytohormone is central to systemic acquired resistance; similar priming effects have been reported for beneficial \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eStreptomyces\u003c/em\u003e strains in other plant systems(Vergnes et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gogoi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, the improved accumulation of asperosaponin\u0026mdash;a key medicinal compound in \u003cem\u003eP. odoratum\u003c/em\u003e\u0026mdash;suggests that SynCom-mediated rhizosphere restoration may indirectly modulate secondary metabolite biosynthesis, consistent with reports that beneficial microbes can trigger metabolic pathways(Zhong et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our results demonstrate that targeted restoration of missing core functions, rather than broad-spectrum microbial supplementation, offers an efficient strategy for managing CCOs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study uncovers the mechanistic basis of continuous cropping obstacles in \u003cem\u003eP. odoratum\u003c/em\u003e and presents a targeted solution. We reveal that α-linolenyl alcohol, rather than traditionally implicated phenolic acids, acts as the key allelochemical driving the decline of core beneficial taxa, particularly \u003cem\u003eStreptomyces\u003c/em\u003e. Based on this insight, a SynCom was designed to restore these missing functions. Pot and field validations confirmed that this SynCom effectively rehabilitates rhizosphere microecology, suppresses disease, and improves both yield and medicinal quality. Our work establishes a paradigm for mechanism-guided development of precision microbial consortia, offering a sustainable approach to overcoming CCOs in medicinal plant cultivation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding by Science and Technology Projects in Guangzhou\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e\n\u003ch3\u003eAuthor Contributions\u003c/h3\u003e\n\u003cp\u003eChenghua Luo: conceptualization, data curation, formal analysis, visualization, writing the original draft. Hua Yang: investigation,\u0026nbsp;methodology. Zhifa Zhang: data curation, methodology. Yaping Chen: conceptualization, funding acquisition, manuscript review, and editing. All authors substantially contributed to the subsequent drafts. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available upon reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlami MM, Pang Q, Gong Z, Yang T, Tu D, Zhen O, Yu W, Alami MJ, Wang X (2021) Continuous Cropping Changes the Composition and Diversity of Bacterial Communities: A Meta-Analysis in Nine Different Fields with Different Plant Cultivation. 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Nucleic Acids Res 38(12):e132\u0026ndash;e132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkq275\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkq275\" 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":false,"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":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Continuous Cropping Obstacles, Synthetic Microbial Community (SynCom), Polygonatum odoratum, Plant Growth-Promoting Bacteria, Rhizosphere Microbiome","lastPublishedDoi":"10.21203/rs.3.rs-9201673/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9201673/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Aims\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePolygonatum odoratum\u003c/em\u003e, a widely cultivated and valuable medicinal herb in China, is severely restricted by continuous cropping obstacles (CCOs). Traditional mitigation strategies including crop rotation and chemical control are either inefficient or environmentally unfriendly, lacking sustainable and eco-friendly solutions. This study aimed to analyze the microbiome of continuous cropping \u003cem\u003eP. odoratum\u003c/em\u003e, identify key functional microbial taxa, and construct a synthetic microbial community (SynCom) for relieving CCOs.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRhizosphere soil, root endophytic tissues and rhizome samples of \u003cem\u003eP. odoratum\u003c/em\u003e with different planting years were collected from three major producing areas, followed by multi-omics analysis to screen core beneficial microbes. A targeted SynCom was assembled, and pot and field experiments were conducted to verify its control effect on CCOs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eContinuous cropping significantly depleted soil nutrients, reduced soil phosphatase and nitrite reductase activities by 64.73% and 34.87%, respectively. The Chao1 index of rhizosphere bacterial α-diversity decreased by 8.41%, and the relative abundance of the key functional genus \u003cem\u003eStreptomyces\u003c/em\u003e dropped sharply by 83.70%. The assembled SynCom effectively alleviated CCOs by restoring rhizosphere microbial homeostasis, improving soil nutrient availability, suppressing rhizome rot, and enhancing the yield and medicinal quality of \u003cem\u003eP. odoratum\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe loss of \u003cem\u003eStreptomyces\u003c/em\u003e is the core microbial factor triggering \u003cem\u003eP. odoratum\u003c/em\u003e CCOs. Supplementing core functional microbiota via SynCom mitigates CCOs through synergistic effects of pathogen antagonism, microbial community restoration and soil fertility improvement. This study provides a novel paradigm for relieving CCOs in medicinal plants.\u003c/p\u003e","manuscriptTitle":"Key Microorganism Alleviate Continuous Cropping Obstacles of Polygonatum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 17:10:50","doi":"10.21203/rs.3.rs-9201673/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-05-06T14:35:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T07:50:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-04-03T00:48:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T06:26:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-01T00:31:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a5c150f5-1d66-48a8-aded-c6d8e16304e0","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"","date":"2026-05-06T14:35:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T07:50:59+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T17:10:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 17:10:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9201673","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9201673","identity":"rs-9201673","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}