Enterobacter asburiae S13 enhances the promoting effect of polyaspartic acid on potato growth by improving rhizosphere nutrient availability and reshaping microbial community

Enterobacter asburiae S13 enhances the promoting effect of polyaspartic acid on potato growth by improving rhizosphere nutrient availability and reshaping microbial community | 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 Enterobacter asburiae S13 enhances the promoting effect of polyaspartic acid on potato growth by improving rhizosphere nutrient availability and reshaping microbial community Xin Zhou, Haiyan Ma, Chao Luo, Hafsa Nazir Cheema, Ruilin Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6917360/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 Polyaspartic acid (PASP), as an environmentally friendly fertilizer synergist, has been widely applied in agricultural production. However, the effects of combined application of PASP and microbial inoculants have not been fully investigated. Through integrating pot experiments, soil biochemical property analysis, and microbiome sequencing, this study revealed that inoculation with Enterobacter asburiae S13 improved the plant height (70.83%), stem diameter (38.43%), root length (41.12%), and root-shoot biomass (50.00–45.83%) of potato seedlings under PASP application. Meanwhile, it simultaneously enhanced the contents of ammonium nitrogen (40.00%), nitrate nitrogen (57.70%), available potassium (47.56%), and urease activity in rhizosphere soil. 16S rRNA sequencing showed that Enterobacter asburiae S13 addition enriched beneficial microbial communities (e.g., Paucibacter , Massilia ) and suppressed potential competitive taxa (e.g., Duganella , Pedobacter ). Redundancy analysis (RDA) indicated that available potassium and ammonium nitrogen were the core factors driving microbial community structure changes. These results elucidated the causal relationship between rhizosphere nutrient dynamics and microbial community reshaping under the combined application of PGPR and PASP, providing theoretical and technical support for sustainable fertilization strategies in agriculture. Potato Plant Growth-Promoting Rhizobacteria Polyaspartic Acid Soil Nutrients Microbial Community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Potato ( Solanum tuberosum L.), the fourth-largest staple crop globally, with a cultivation area of approximately 18.9 million hectares (He et al., 2024 ), plays a crucial role in ensuring food security and sustainable agricultural development. In China, efforts to enhance potato yield have historically relied on intensive fertilizer and pesticide applications, which exacerbate environmental issues such as soil degradation, declining fertility, and non-point source pollution (Zhong et al., 2018 ). Addressing these challenges requires eco-friendly strategies to optimize nutrient use and improve soil health. Polyaspartic acid (PASP), a biodegradable amino acid-based polymer naturally found in snails and mollusks, can also be synthesized through the thermal polycondensation of aspartic acid (Obst & Steinbüchel, 2004 ). As a fertilizer synergist, PASP has shown considerable potential in enhancing crop growth and yield by chelating rhizosphere nutrients (Machingura et al., 2024 ; Liu et al., 2022 ), stimulating microbial activity (Liu et al., 2019 ), and regulating plant metabolism via its degradation product, aspartic acid (Machingura et al., 2024 ). Notably, PASP application has been reported to improve nitrogen assimilation pathways in potatoes, promoting growth under low-nitrogen conditions (Cheema et al., 2024 ). However, the synergistic effects of PASP with microbial inoculants, particularly plant growth-promoting rhizobacteria (PGPR), remain unexplored. PGPR are root-colonizing microorganisms that promote plant growth through various mechanisms (Yang et al., 2022 ), including nutrient solubilization and modulation of soil-plant-microbe interactions ( Sughra et al., 2021 ; Luo et al., 2025 ). For example, phosphate- and silicate-solubilizing strains enhance the availability of essential elements in the rhizosphere (Arjun et al., 2020 ), while nitrogen-fixing bacteria improve plant nitrogen assimilation efficiency (Li et al., 2023 ). PGPR that colonize mineral surfaces proliferate by forming biofilms composed of extracellular polymers, such as cellulose, proteins, and galactose, while releasing metabolites, including organic acids, proteins, and enzymes (Pankaj et al., 2020 ; Maurya et al., 2024). The dynamic chemical reactions within these biofilms create favorable conditions for PGPR to assimilate inorganic nutrients, facilitating mineral weathering and enhancing plant nutrient absorption (Pankaj et al., 2020 ). The restructuring of the rhizosphere microbial community is primarily driven by changes in the composition and concentration of soil exudates. Exogenously applied PGPR influence interspecies interactions through their secreted products (Zhao et al., 2024 ) and can also induce host plants to alter the composition and quantity of root exudates (Sun et al., 2024 ). This modulation recruits complementary microbial taxa, collectively promoting plant growth. The combined use of growth-promoting agents has emerged as an effective strategy to enhance crop resilience and productivity (A.Rahou et al., 2022 ). For instance, the combination of biochar and nano-silicon improves drought resistance in wheat ( Triticum aestivum L.) (Zulfiqar et al., 2024 ), while microbial fertilizers integrated with biochar enhance soil phosphorus and potassium fertility, promoting tomato ( Solanum lycopersicum L.) growth (Yang et al., 2022 ). Despite these advancements, the effects of PGPR inoculation on rhizosphere nutrient dynamics, microbial community structure, and plant performance—particularly under PASP-supplemented cultivation systems—remain underexplored. A pot experiment was conducted to assess the effects of PGPR inoculation combined with PASP application on potato growth, rhizosphere nutrient content, and microbial community composition. We hypothesized that PGPR inoculation would synergize with PASP to (1) increase potato biomass, (2) enhance rhizosphere nutrient availability, and (3) shift the microbial community toward beneficial taxa. This study elucidates the mechanisms underlying plant-soil-microbe interactions in the context of combined PASP-PGPR application, providing both theoretical and practical insights for developing sustainable "fertilizer synergist-microbial inoculant" strategies in agriculture. Materials and methods Experimental Site and Materials The pot experiment was conducted at the Chengdu Academy of Agricultural and Forestry Sciences, Sichuan Province, China (30°42′32″N, 103°51′49″E; altitude 540 m). Potato cultivation began in late December 2023 and concluded in May 2024, with pots maintained under open-air conditions. The potato variety used was "Chuanyu 50," known for its strong field growth potential, resistance to late blight, and high resistance to viral diseases. Seed tubers were provided by the Key Laboratory of Crop Cultivation and Farming Systems, College of Agronomy, Sichuan Agricultural University. The PGPR strain S13, identified as Enterobacter asburiae (NCBI accession number SUB14859840) through 16S rRNA sequencing, was supplied by the same laboratory. PASP (purity: analytical grade [AR], active ingredient > 99.7%) was obtained from Zhengzhou Guanda Chemical Products Co., Ltd. Pot Experiment Design The experiment was designed using a completely randomized design with four treatments: (1) untreated control (CK); (2) control with PGPR inoculation only (S1P0); (3) PASP application without PGPR inoculation (S0P1); (4) combined application of PASP and PGPR inoculation (S1P1). Each treatment consisted of 15 pots, totaling 60 pots. Vermiculite and perlite (3:1 v/v) were used as the growth substrate, filled to 4/5 of the pots (370 mm upper diameter × 240 mm height). Seed Preparation and Inoculation Seed tubers (~ 50 g each) were surface sterilized with 0.1% NaClO for 20 min, rinsed with distilled water, and air-dried. For PGPR inoculation (S1P0 and S1P1), tubers were immersed in a bacterial suspension for 30 min, while the control groups (S0P1 and CK) were received with sterile water. Through preliminary germination tests, a working concentration of 1×10 4 CFU· mL -1 was selected for the bacterial suspension (Supplemental Fig. 1) . The bacterial suspension was prepared by culturing a single colony of E. asburiae in LB broth (37°C, 170 rpm, 24 h), adjusting the concentration to 1×10 8 CFU· mL -1 (OD 600 = 0.8 ~ 1.0), and diluting 1:10,000 in sterile water (The final concentration is 1×10 4 CFU· mL -1 ). Fertilization and Cultivation A potassium sulfate compound fertilizer (N: P 2 O 5 : K 2 SO 4 = 16:6:8; total nutrients ≥ 40%) was applied at a rate of 10 g per pot. PASP (0.5 g per pot, 5% of the fertilizer mass) was dissolved in sterile water and administered through root irrigation. Routine watering and manual weeding were conducted throughout the growth period. Sample Collection and Processing Plant Samples Ten days post-emergence, nine uniformly growing plants per treatment were harvested. Three plants were used for the collection of phenotypic images. Six plants were used for growth parameter analysis, including stem diameter, root length, and dry weight determination. Stem diameter was measured at the base using vernier calipers, while root length was recorded as the distance from the root origin to the longest tip. For dry weight measurement, roots, stems, and leaves were separated, oven-dried at 105°C for 30 min, followed by drying at 80°C until a constant weight was achieved, and then weighed. Rhizosphere Soil Samples Rhizosphere soil was collected from six plants per treatment by gently removing loosely adhered soil and brushing off tightly bound soil from the roots. The samples were then divided into two subsets: one was flash-frozen in liquid nitrogen (-80°C) for microbial analysis, while the other was air-dried, ground, and sieved through a 60-mesh for biochemical assays. Soil Biochemical Analysis Ammonium nitrogen (NH 4 + -N): Extracted using 2 M KCl and quantified via indophenol blue colorimetry (UV-756 spectrophotometer, 625 nm) (Zhang et al., 2023 ). Nitrate nitrogen (NO 3 - -N): Extracted with 2 M KCl, acidified with 10% H 2 SO4, and quantified using dual-wavelength spectrophotometry (UV-756 spectrophotometer, 220 nm and 275 nm) (Zheng et al., 2024 ). Available potassium (AK): Extracted with 1 M CH 3 COONH 4 (pH 7.0) and analyzed using flame photometry (FP6400) (Zhao et al., 2022 ). Soli Urease activity (S-Ure): Measured using a commercial kit (Jiangsu Adison Biotechnology Co., Cat#ADS-F-TR001) according to the manufacturer’s instructions. Rhizosphere Microbiome Sequencing of Potato Total genomic DNA was extracted from soil samples using the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified with primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were analyzed by agarose gel electrophoresis and purified using the D6492-00 OMEGA Cycle Pure Kit (Noblebio Technology Co., Ltd., Beijing). Sequencing was performed on the Illumina Novaseq 6000 platform with paired-end (2×250 bp) reads. To obtain high-quality sequencing data, raw data were first filtered using Trimmomatic v0.33 software, followed by primer sequence identification and removal using Cutadapt 1.9.1 software. Denoising, assembly, and chimera removal were then performed using the DADA2 plugin in QIIME2 software, resulting in high-quality sequences for subsequent analysis (Bolyen et al., 2019 ; Callahan et al., 2016 ). Statistical Analysis Plant growth parameters and soil biochemical properties were analyzed using one-way ANOVA followed by Duncan's multiple range test (p < 0.05) in SPSS Statistics 26. Data visualization was performed using GraphPad Prism 9.5. For microbial community analysis, α-diversity indices (Chao1 and Shannon) were calculated using QIIME2 (v2023.2). Operational taxonomic units (OTUs) were clustered at a 97% similarity threshold using USEARCH (v10.0) in R, with low-abundance OTUs (< 0.005% of total sequences) filtered out. β-Diversity was visualized via principal coordinate analysis (PCoA), PERMANOVA and ANOSIM analyses based on Bray-Curtis dissimilarity. Functional profiling of microbial communities was inferred using PICRUSt2 against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Taxonomic annotation was performed in QIIME2 with reference to the NCBI database ( https://www.ncbi.nlm.nih.gov ). Differential abundance of taxa between groups was assessed using Metastats (p < 0.05) (Nizigiyimana et al., 2022 ). Feature taxa contributing to community differences were identified via random forest analysis using the RandomForest package (v4.6-10) in R (ntree = 500). Relationships between microbial diversity and environmental factors were evaluated through redundancy analysis (RDA) and variance partitioning analysis (VPA) implemented in the vegan package (v2.3) in R (Rui et al., 2015 ). Results Effects of PGPR Inoculation on Potato Seedling Growth To assess the effects of PGPR inoculation on the growth of potato seedlings and potato yield under PASP application, we measured growth indices and the dry weights of various plant parts. Specifically, the S1P1 treatment showed a 70.83% increase in plant height ( Fig. 1 a ) , a 38.43% increase in stem diameter ( Fig. 1 b ) , and a 41.12% increase in root length (p < 0.05) ( Fig. 1 c ) compared with the S0P1 treatment. S1P1 treatment also notably enhanced the biomass of both roots and stems (p < 0.05), with the dry weights of roots and stems increasing by 50.00% and 45.83%, respectively, relative to the S0P1 treatment ( Fig. 1 d ) . Effects of PGPR Inoculation on Rhizosphere Soil Biochemical Properties To assess the effects of PGPR inoculation under PASP application on rhizosphere soil chemistry and enzyme activity, we measured soil nutrient content and urease activity. Compared to the S0P1 treatment, the S0P1 treatment significantly enhanced NH 4 + -N ( Fig. 2 a ) , NO 3 − -N ( Fig. 2 b ) , AK ( Fig. 2 c ) , and S-Ure activity ( Fig. 2 d ) in the rhizosphere, increased NH 4 + -N, NO 3 − -N, and AK by 40.00%, 57.70%, and 47.56%, respectively, with all differences being statistically significant (p < 0.05). Effects of PGPR Inoculation on Rhizosphere Microbial Community Structure and Function To further assess the effect of PGPR on the rhizosphere soil microenvironment under PASP application, we conducted 16S rRNA sequencing for the S0P1 and S1P1 treatments. Results showed that PGPR inoculation under PASP application altered the bacterial community diversity. Specifically, PGPR inoculation under PASP application decreased α-diversity indices (Chao1 and Shannon) (p < 0.05) ( Fig. 3 a, 3 b ) . PCoA based on Bray - Curtis dissimilarity indicated a significant change in β - diversity (p = 0.001) ( Fig. 3 c ) , suggesting a reorganization of the microbial community structure. Venn analysis of OTU abundance showed that the PGPR-inoculated group under PASP application had fewer unique OTUs (3,713) compared to the PASP-alone group, with only 122 shared OTUs ( Fig. 3 d ) . However, KEGG pathway analysis did not show significant changes in microbial metabolic functions ( Fig. 3 e ) . Effects of PGPR Inoculation on Rhizosphere Microbial Composition At the phylum level, PGPR inoculation increased the relative abundances of Bacteroidota and Acidobacteriota while decreasing those of Proteobacteria , Firmicutes , and Actinobacteria ( Fig. 4 a ) . At the genus level, PGPR inoculation enriched beneficial taxa such as Paucibacter , Pseudomonas , and Massilia , while reducing the abundance of Duganella , Flavobacterium , and Pedobacter ( Fig. 4 b ) . Genus-level enrichment analysis revealed distinct microbial signatures, with several taxa showing statistically significant variations in relative abundance (p < 0.01). Specifically, the inoculated group exhibited marked increases in Turicibacter and Wolbachia , along with significant reductions Shinella , Pseudarcicella , and Marmoricola . Furthermore, taxa such as Rhodanobacter , Hirschia , and Jatrophihabitans displayed significant abundance differences (p < 0.05) ( Fig. 4 c ) . Notably, genera such as Turicibacter (a genus associated with host metabolic regulation) and Rhodanobacter (a denitrification-related taxon), whose abundances increased, along with genera like Shinella (a potential nitrogen-cycling participant) and Marmoricola (a stress-responsive genus), whose abundances decreased, were identified by Random Forest analysis as major drivers of microbial community restructuring between treatments ( Fig. 4 d ) . Correlations Between Seedling Biomass and Rhizosphere Soil Properties Correlation analysis showed that root dry weight was significantly positively correlated with AK (p < 0.05), whereas S-Ure activity exhibited strong positive correlations with NH 4 + -N (p < 0.01) and NO 3 − -N (p < 0.001) ( Fig. 5 ) . Interactions Between Soil Biochemical Properties and Microbial Diversity RDA analysis of rhizosphere microbial communities and environmental factors indicated that NH 4 + -N content had the greatest influence on microbial community structure, followed by AK content, while NO 3 − -N content and S-Ure activity made marginal contributions. Notably, bacterial communities in PGPR-inoculated treatments under PASP application exhibited positive correlations with all environmental factors, whereas non-inoculated groups showed negative correlations ( Fig. 6 a ) . VPA further quantified the contributions of individual environmental factors to microbial community variation. AK and NO 3 − -N explained 1.22% and 0.61% of the total variation, respectively. Additionally, the combined effects of NH 4 + -N, NO 3 − -N, and S-Ure activity accounted for 0.62% of the microbial community variation ( Fig. 6 b ) . At the genus level, correlation analysis between dominant taxa and environmental factors revealed that beneficial genera such as Paucibacter , Massilia , and Methylophilus were positively correlated with nutrient availability (e.g., NH 4 + -N, NO 3 − -N, AK), whereas genera including Duganella , Flavobacterium , and Pedobacter showed negative correlations. Notably, Pedobacter and Janthinobacterium exhibited significant negative correlations with AK (p < 0.05), suggesting their sensitivity to potassium dynamics in PASP-supplemented systems ( Fig. 6 c ) . Discussion PGPR Inoculation Enhances Potato Growth Through Rhizosphere Nutrient Modulation Most PGPR strains exhibit growth-promoting properties. For instance, Bacillus sp. can stimulate plant growth by producing plant hormones such as IAA and solubilizing inorganic phosphorus (Sun and Shahrajabian, 2025 ), while Paenibacillus polymyxa HL14-3 induces ABA production in cucumbers, enhancing their growth under drought stress (Qin et al., 2024 ). In this study, PGPR inoculation under PASP application increased the plant height, stem diameter, root length, and dry matter accumulation in the stems and roots of potato seedlings ( Fig. 1 ) , confirming the growth-promoting efficacy of PGPR as an inoculant. To explore the mechanism by which PGPR promotes potato growth, we measured the nutrient content in the rhizosphere soil and analyzed its correlation with dry matter accumulation in different plant parts. The results indicated that PGPR inoculation increased the levels of NH 4 + -N, NO 3 − -N, and AK in the rhizosphere soil of potato seedlings ( Fig. 2 ) , with a significant positive correlation observed between AK content and root dry matter accumulation ( Fig. 5 ) . Mechanistically, plant growth and development are strongly correlated with rhizosphere nutrient availability. For example, soil nitrogen content influences the absorption and utilization of potassium in rapeseed ( Brassica napus L.), with high nitrogen conditions alleviating the effects of low potassium stress on photosynthesis, while low nitrogen conditions require more potassium to maintain normal photosynthetic function (Li et al., 2022 ). Soil potassium content can alter root architecture, under low potassium conditions, tea plant ( Camellia sinensis L. Kuntze) roots secrete organic acids to solubilize potassium salts, whereas under high potassium conditions, they upregulate genes related to cellulose degradation, thereby increasing total root length and fine root proportion in response to soil potassium gradients (Ruan et al., 2022 ). These findings suggest that the increased biomass of potato seedlings in this study may be linked to changes in soil nitrogen and potassium content, with the increase in soil potassium content potentially triggering root response mechanisms that enhance root length and dry weight. The changes in soil biochemical properties are the result of the combined action of rhizosphere microbial communities mediated by PGPR inoculation PGPR can enhance nutrient availability in the plant rhizosphere, increasing the content of available nitrogen, potassium, and phosphorus (Razack et al., 2024 ). For example, Azoarcus sp. BH72 has been shown to increase nitrogen content in the rhizosphere of kallar grass ( Leptochloa fusca L. Kunth) under low nitrogen conditions (Igiehon and Babalola, 2018 ), while Pseudomonas sp. and Bacillus sp. can secrete oxalic acid, citric acid, and phytase to solubilize organic or inorganic phosphorus, thereby enhancing its availability to plants (Rawat et al., 2020 ). In this study, PGPR inoculation significantly increased the levels of NH 4 + -N, NO 3 − -N, AK, and S-Ure activity in the rhizosphere soil of potatoes ( Fig. 2 ) , which is consistent with previous findings. Furthermore, RDA combined with VPA revealed that changes in soil biochemical characteristics within the potato rhizosphere were predominantly driven by the synergistic effects of PGPR-inoculated rhizosphere microbial communities. The analysis indicated that AK content was the most significantly affected parameter, followed by NH 4 + -N and NO 3 − -N content ( Fig. 6 a, 6 b ) . Subsequent correlation analysis between microbial diversity indices and edaphic factors showed that these changes were associated with specific functional taxa, such as Paucibacter (a genus recognized for its metabolic versatility in carbon cycling and xenobiotic degradation), Massilia (a rhizosphere-competent genus involved in root exudate utilization and phytohormone modulation), and Methylophilus (a methylotrophic bacterium contributing to single-carbon metabolism and nitrogen mineralization) ( Fig. 6 c ) . Studies have shown that the addition of exogenous PGPR can increase the abundance of beneficial microorganisms in the microbial community, thereby promoting plant growth (Xie et al., 2025 ). For example, the use of Bacillus velezensis SQR9 increased the relative abundance of Pseudomonas sp. and Bacillus sp. in the rhizosphere of cucumber ( Cucumis sativus L.) and promoted cucumber growth in association with the native strain Pseudomonas stutzeri (Sun et al., 2021 ). Inoculation with Bacillus velezensis containing agricultural waste enhanced the biofilm formation and colonization ability of native Pseudomonas fluorescens , promoting strawberry ( Fragaria × ananassa ) growth (Wang et al., 2024 ). This suggests that changes in rhizosphere soil nutrient content are not solely attributed to a single microbial species but are likely the result of the combined action of rhizosphere microbial communities mediated by exogenous PGPR. The introduction of exogenous microorganisms can reshape the structure of the rhizosphere soil microbial community Previous studies have demonstrated that the use of microbial inoculants can not only directly affect host plants but also regulate the structure and composition of soil microbial communities, thereby synergistically promoting plant growth, disease resistance, and stress tolerance (Xie et al., 2025 ). In this study, PGPR inoculation reduced the richness and diversity of soil microbial communities under PASP application, but increased the compositional differences between samples ( Fig. 3 ) , indicating that PGPR inoculation significantly impacted the soil microbial community structure, consistent with previous findings. Although there were no significant changes in α - diversity ( Fig. 3 a, 3 b ) , the changes in β - diversity ( Fig. 3 c ) and the dynamics of key species ( Fig. 4 ) jointly validated the rhizosphere reshaping hypothesis. This aligns with the ecological tenet: Function transcends taxonomy in soil-microbe dialogues (Pankaj et al., 2020 ). The reduction in microbial community richness and diversity, alongside the increase in compositional differences between treatments, may be related to PGPR colonization activities in the potato rhizosphere. The introduction of exogenous microorganisms may stimulate the host plant to produce specific secretions that favor their colonization, enhancing their competitive advantage in nutrient and ecological niche competition (Nicolle et al., 2024 ; Segura and Ramos, 2013 ). For instance, inoculation with Pseudomonas simiae WCS417 can induce the secretion of scopoletin by Arabidopsis ( Arabidopsis thaliana L. Heynh) roots, facilitating the colonization of this strain while inhibiting the growth of pathogens and other native microbial communities (Stringlis et al., 2018 ). Additionally, PGPR inoculation increased the abundance of phyla such as Bacteroidota and Acidobacteriota in the potato rhizosphere soil, as well as the abundance of genera like Paucibacter and Massilia . Random Forest analysis revealed that the increased abundance of genera such as Turicibacter and Rhodanobacter significantly influenced the differences in microbial community composition between groups ( Fig. 4 ) . These increased microbial populations often exhibit strong growth-promoting and soil nutrient-activating abilities. For example, Massilia strains JJY03 and JJY04 can solubilize phosphate and produce IAA (Chhetri et al., 2024 ), while Rhodanobacter strains Si-c and S2-g demonstrate robust phosphate solubilization and nitrogen fixation capabilities (Woo et al., 2024 ). This suggests that PGPR inoculation promotes the recruitment of beneficial microorganisms associated with growth promotion, thereby reshaping the structure of the potato rhizosphere microbial community. Conclusion In this study, the inoculation of PGPR under PASP application led to a significant restructuring of the rhizosphere microbial community composition and architecture in potato, along with the selective enrichment of beneficial microbial taxa. The PGPR-mediated microbial consortia collectively enhanced soil nutrient availability, as evidenced by notable increases in NH 4 + -N, NO 3 − -N, and AK concentrations, along with elevated S-Ure activity in the rhizosphere microenvironment. These synergistic improvements in soil biochemical properties resulted in measurable physiological outcomes, such as increased stem and root biomass, thus demonstrating substantial growth promotion effects. Overall, our findings indicate that PGPR inoculation under PASP supplementation represents an effective agroecological strategy to enhance plant growth promotion through rhizosphere microbiome engineering. However, the experiment was limited to pot conditions, and the lack of field validation may restrict the practical extrapolation of conclusions. Although correlation analysis revealed the association between microbial communities and nutrients, specific action pathways were not clarified through functional assays (such as strain colonization verification or metabolomics), requiring further studies to deepen mechanism elucidation. Declarations Funding This work was supported by the Sichuan Provincial Science and Technology Program Breeding Research Project (Grants No. 2021YFYZ0005 and 2021YFYZ0019), the National Modern Agricultural Industry Technology System Sichuan Tuber Crops Innovation Team Project (Grant No. sccxtd-2025-09), and the Sichuan Natural Science Foundation (Grant No. 2022NSFSC0014). Competing interests The authors declare no competing financial or non-financial interests. Author Contribution All authors contributed to the study’s conception and design. X.Z. performed research design, methodology selection, sample collection, data analysis, and manuscript drafting. H.Y.M. and K.Q.Z. contributed to research design and methodology. C.L., R.L.L., and J.L. conducted sample collection and data analysis. H.N.C. reviewed and revised the manuscript. S.L.Z. provided overall project planning, experimental facilities, funding acquisition, and supervision. All authors read and approved the final manuscript. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. References Arjun A, Lee KE, Aaqil KM, Kang SM, Bishnu A, Muhammad I, Rahmatullah J, Kim KM, Lee IJ (2020) Effect of silicate and phosphate solubilizing rhizobacterium Enterobacter ludwigii GAK2 on Oryza sativa L. under cadmium stress. 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Proceedings of the National Academy of Sciences of the United States of America 115 (22): E5213-E5222. https://doi.org/10.1073/pnas.1722335115 Sughra H, Tahir N, Shoib NM, Iqra L, Jawad SM, Rabisa Z, Sajjad MM, Asma I (2021) Rhizosphere engineering with plant growth-promoting microorganisms for agriculture and ecological sustainability. Frontiers in Sustainable Food Systems 5: 617157. https://doi.org/10.3389/fsufs.2021.617157 Sun XL, Xu ZH, Xie JY, Hesselberg TV, Tan TM, Zheng DY, Strube ML, Dragoš A, Shen QR, Zhang RF, Kovács ÁT (2021) Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions. The ISME Journal 16(3): 774-787. http://dx.doi.org/10.1038/S41396-021-01125-3 Sun B, Sun CY, Fu WJ, Fu HJ, ShuHL, Wu MF, Guo Q, Lai HX (2024) Bacillus siamensis orchestrates plant gene reprogramming and rhizosphere microbiome reshaping to bolster maize crop performance. Soil Science Society of America Journal 88(6): 2031-2045. https://doi.org/10.1002/saj2.20768 Sun WL, Shahrajabian MH (2025) Biostimulant and beyond: Bacillus spp., the important plant growth-promoting rhizobacteria (PGPR)-based biostimulant for sustainable agriculture. Earth Systems and Environment (prepublish): 1-34. http://dx.doi.org/10.1007/S41748-024-00552-4 Wang QF, Chu CB, Zhao Z, Wu SH, Zhou DP (2024) Pseudomonas fluorescens enriched by Bacillus velezensis containing agricultural waste promotes strawberry growth by microbial interaction in plant rhizosphere. Land Degradation & Development 35(7): 2476-2488. http://dx.doi.org/10.1002/LDR.5074 Woo HJ, Kim IY, Chhetri G, Park SH, Lee HJ, Yook SB, Seo TG (2024) Two novel bacterial species, Rhodanobacter lycopersici sp. nov.Si-c and Rhodanobacter geophilus sp. nov.S2-g, isolated from the rhizosphere of Solanum lycopersicum with plant growth-promoting traits. Microorganisms 12(11): 2227. http://dx.doi.org/10.3390/MICROORGANISMS12112227 Xie CS, Wu YT, Wu ZH, Cao H, Huang XH, Cui F, Meng S, Chen J (2025) Bacillus velezensis TCS001 enhances the resistance of hickory to Phytophthora cinnamomi and reshapes the rhizosphere microbial community. Agriculture 15(2): 193-193. https://doi.org/10.3390/AGRICULTURE15020193 Yang W, Zhao YN, Yang Y, Zhang MS, Mao XX, Guo YJ, Li XY, Tao B, Qi YZ, Ma L, Liu WJ, Li BW, Di HJ (2022) Co-application of biochar and microbial inoculants increases soil phosphorus and potassium fertility and improves soil health and tomato growth. Journal of Soils and Sediments 23 (2): 947-957. https://doi.org/10.1007/s11368-022-03347-0 Zhong NQ, Liu N, Zhao P, Cai DQ, Song SW, Chao YP (2018) Current status and challenges for potato chemical fertilizer & pesticide reductions in China. Chinese Science Bulletin 63 (17): 1693-1702. https://doi.org/10.1360/N972017-01325 Zhao Y, Yan CB, Hu FC, Luo ZW, Zhang SQ, Xiao M, Chen Z, Fan HY (2022) Intercropping pinto peanut in litchi orchard effectively improved soil available potassium content, optimized soil bacterial community structure, and advanced bacterial community diversity. Frontiers in Microbiology 13: 868312. https://doi.org/10.3389/FMICB.2022.868312 Zhang SJ, Gu WH, Bai JF, Dong B, Zhao J, Zhuang XN, Shih K (2023) Influence of sludge-based biochar on the soil physicochemical properties and the growth of Brassica chinensis L. Journal of Soil Science and Plant Nutrition 23(4): 4886-4898. https://doi.org/10.1007/S42729-023-01384-3 Zhao Q, Wang RY, Song Y, Lu J, Zhou BJ, Song F, Zhang LJ, Huang QQ, Gong J, Lei JJ, Dong SM, Gu Q, Borriss R, Gao XW, Wu HJ (2024) Pyoluteorin-deficient Pseudomonas protegens improves cooperation with Bacillus velezensis, biofilm formation, co-colonizing, and reshapes rhizosphere microbiome. NPJ Biofilms and Microbiomes 10 (1): 145. https://doi.org/10.1038/S41522-024-00627-0 Zheng SH, Ni K, Chai HL, Ning QY, Cheng C, Kang HJ, Ruan JY (2024) Comparative research on monitoring methods for nitrate nitrogen leaching in tea plantation soils. Scientific Reports 14 (1): 20747. https://doi.org/10.1038/S41598-024-71081-3 Zulfiqar B, Raza MAS, Akhtar M, Zhang N, Hussain M, Ahmad J, Maksoud MA.A, Ebaid H, Iqba Rl, Aslam MU, Tayeb MA.E, Su SM (2024) Combined application of biochar and silicon nanoparticles enhance soil and wheat productivity under drought: Insights into physiological and antioxidant defense mechanisms. Current Plant Biology 40: 100424. https://doi.org/10.1016/j.cpb.2024.100424 Additional Declarations No competing interests reported. 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. 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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-6917360","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475588160,"identity":"c6a886e5-3c46-4449-9777-c6e47ec30c05","order_by":0,"name":"Xin Zhou","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhou","suffix":""},{"id":475588161,"identity":"a09ccf65-3541-4836-9fe4-ce1e9cc4804a","order_by":1,"name":"Haiyan Ma","email":"","orcid":"","institution":"Yibin Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Ma","suffix":""},{"id":475588162,"identity":"7cd1d475-87b4-474e-9c7a-76f70d07e5af","order_by":2,"name":"Chao Luo","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Luo","suffix":""},{"id":475588164,"identity":"6cf8963e-2fdf-4a41-9e8a-67bc65b4dfe3","order_by":3,"name":"Hafsa Nazir Cheema","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hafsa","middleName":"Nazir","lastName":"Cheema","suffix":""},{"id":475588165,"identity":"90172cfb-5bd9-401e-b09e-2431dc309f1f","order_by":4,"name":"Ruilin Liu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ruilin","middleName":"","lastName":"Liu","suffix":""},{"id":475588167,"identity":"ebc27e47-ec18-45da-a557-7f929fdb0e93","order_by":5,"name":"Jing Li","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":475588168,"identity":"21bb2a7c-2a6f-43c1-821f-7806c01ce204","order_by":6,"name":"Kaiqin Zhang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kaiqin","middleName":"","lastName":"Zhang","suffix":""},{"id":475588169,"identity":"e58f9739-52b9-45f3-b095-606675beac0d","order_by":7,"name":"Shunlin Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIie3RsQrCMBCA4YRCXA5dr7ToKwSF0qHQB3GJFDrp5NohTi6+TN8gEripD9C9q4MvoJhNt8RNMP/+kbscY7HYD7Zg9jo9nh2ImQ4k6YkaCYLyOZhAIu1QIIikWqIKnYyoRWdApFM/sq7aegW/WFtKzEFk7bFk1B60jyRIu1FJ90q2L5Br6ydidZNoVOIGGwIJsGGdauMIQiBBRs2Ga3If4HZRIbvUxp2S665enW0/3rvKTz6T4ad5k29FLBaL/Ucv6y86ce+GZX4AAAAASUVORK5CYII=","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shunlin","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-06-17 20:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6917360/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6917360/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85393964,"identity":"426ac3f8-a285-44f4-a58a-6f6283e91347","added_by":"auto","created_at":"2025-06-25 10:54:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193702,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of PGPR inoculation on potato seedling growth under PASP application: \u003cstrong\u003e(a) \u003c/strong\u003ePlant height (cm), \u003cstrong\u003e(b)\u003c/strong\u003e stem diameter (mm), \u003cstrong\u003e(c)\u003c/strong\u003e root length (cm), \u003cstrong\u003e(d)\u003c/strong\u003edry weight partitioning (roots, stems, leaves) (g plant⁻¹) and \u003cstrong\u003e(f)\u003c/strong\u003ePhenotype of potato seedlings . Data are presented as means ± SD (n = 6). Lowercase letters indicate significant differences among treatments (ANOVA, Duncan’s test, p \u0026lt; 0.05). CK: Control (no treatment); S0P1: PASP only; S1P0: PGPR only; S1P1: PASP + PGPR.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/c46cfe3a367d4b6f64fdf673.png"},{"id":85393959,"identity":"6478dedd-62c1-47e4-a947-e816a0398983","added_by":"auto","created_at":"2025-06-25 10:54:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112688,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PGPR inoculation on soil biochemical properties under PASP application: \u003cstrong\u003e(a)\u003c/strong\u003e Ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) (mg kg⁻¹), \u003cstrong\u003e(b)\u003c/strong\u003e nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N) (mg kg⁻¹), \u003cstrong\u003e(c)\u003c/strong\u003e available potassium (AK) (mg kg⁻¹), and \u003cstrong\u003e(d)\u003c/strong\u003e soil urease (S-Ure) activity (U g⁻¹). Data are presented as means ± SD (n = 6). Different letters indicate significant differences (ANOVA, Duncan’s test,\u0026nbsp;p\u0026nbsp;\u0026lt; 0.05). CK: Control (no treatment); S0P1: PASP only; S1P0: PGPR only; S1P1: PASP + PGPR.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/24e0109d87ecfbe035c8d467.png"},{"id":85393942,"identity":"7175550d-836f-4b30-9e61-b38e0d8bfaeb","added_by":"auto","created_at":"2025-06-25 10:54:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127243,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PGPR inoculation on the rhizosphere microbial community structure and function under PASP application:\u003cstrong\u003e (a)\u003c/strong\u003e α-diversity (Chao1 index, *p \u0026lt; 0.05), \u003cstrong\u003e(b)\u003c/strong\u003e α-diversity (Shannon index, *p \u0026lt; 0.05), \u003cstrong\u003e(c) \u003c/strong\u003eprincipal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity, \u003cstrong\u003e(d) \u003c/strong\u003eVenn diagram of OTU distribution, and \u003cstrong\u003e(e)\u003c/strong\u003e KEGG pathway composition of the top 10 functional categories (Level 2). S0P1: PASP only; S1P1: PASP + PGPR.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/2fb3961333107c64334c5b84.png"},{"id":85394875,"identity":"3c9cfd34-0a08-4a32-bc9a-76ee7c382b4f","added_by":"auto","created_at":"2025-06-25 11:02:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":250064,"visible":true,"origin":"","legend":"\u003cp\u003eTaxonomic shifts in rhizosphere microbial composition induced by PGPR inoculation under PASP application:\u003cstrong\u003e (a)\u003c/strong\u003e Phylum-level relative abundance (top 10 taxa), \u003cstrong\u003e(b)\u003c/strong\u003egenus-level composition (top 20 taxa; bubble size reflects abundance), \u003cstrong\u003e(c)\u003c/strong\u003edifferentially abundant genera (top 20 taxa; asterisks indicate significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01), and\u003cstrong\u003e (d)\u003c/strong\u003erandom forest analysis of the top 30 genera. S0P1: PASP only; S1P1: PASP + PGPR.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/af3ab3bed6ae48df3fd2f730.png"},{"id":85393966,"identity":"83e06c01-1e7e-40e4-8627-5604bb725cf8","added_by":"auto","created_at":"2025-06-25 10:54:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":199997,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between potato seedling biomass and rhizosphere soil biochemical properties. Red and gray gradients denote positive (r \u0026gt; 0) and negative (r \u0026lt; 0) correlations, respectively. Asterisks indicate significance (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001; Pearson’s correlation). DR: Root dry weight, DS: Stem dry weight, DL: Leaf dry weight, NH4: Ammonium nitrogen, NO3: Nitrate nitrogen, AK: Available potassium, Ure: Soil urease activity.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/310e4c065b5399167e724dfd.png"},{"id":85393955,"identity":"fd5db215-deb2-4c98-a8cf-097ee4a6684b","added_by":"auto","created_at":"2025-06-25 10:54:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":120882,"visible":true,"origin":"","legend":"\u003cp\u003eInteractions between environmental factors and microbial diversity: \u003cstrong\u003e(a) \u003c/strong\u003eRedundancy analysis (RDA) of microbial communities (OTU abundance) and soil properties, \u003cstrong\u003e(b) \u003c/strong\u003evariance partitioning analysis (VPA) quantifying environmental contributions to microbial variation (Residuals: unexplained variance), and\u003cstrong\u003e (c)\u003c/strong\u003e heatmap of genus-environment correlations (top 20 genera; asterisks: *p \u0026lt; 0.05). S0P1: PASP only; S1P1: PASP + PGPR.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/74e9722ea4a59a97e5daad89.png"},{"id":85393954,"identity":"e414edde-6779-427a-830f-23802f1208c7","added_by":"auto","created_at":"2025-06-25 10:54:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":338427,"visible":true,"origin":"","legend":"\u003cp\u003eInteractive pattern diagram of microbe - soil biochemical properties - plant growth triad\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/fd1fe067e7b3114150894331.png"},{"id":92532106,"identity":"6126c678-897f-46bf-864e-7428823f3b75","added_by":"auto","created_at":"2025-09-30 16:46:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2375851,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6917360/v1/ea9578c1-9af9-4143-8a23-9232822ffb6a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enterobacter asburiae S13 enhances the promoting effect of polyaspartic acid on potato growth by improving rhizosphere nutrient availability and reshaping microbial community","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePotato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.), the fourth-largest staple crop globally, with a cultivation area of approximately 18.9\u0026nbsp;million hectares (He et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), plays a crucial role in ensuring food security and sustainable agricultural development. In China, efforts to enhance potato yield have historically relied on intensive fertilizer and pesticide applications, which exacerbate environmental issues such as soil degradation, declining fertility, and non-point source pollution (Zhong et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Addressing these challenges requires eco-friendly strategies to optimize nutrient use and improve soil health.\u003c/p\u003e \u003cp\u003ePolyaspartic acid (PASP), a biodegradable amino acid-based polymer naturally found in snails and mollusks, can also be synthesized through the thermal polycondensation of aspartic acid (Obst \u0026amp; Steinb\u0026uuml;chel, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). As a fertilizer synergist, PASP has shown considerable potential in enhancing crop growth and yield by chelating rhizosphere nutrients (Machingura et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), stimulating microbial activity (Liu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and regulating plant metabolism via its degradation product, aspartic acid (Machingura et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, PASP application has been reported to improve nitrogen assimilation pathways in potatoes, promoting growth under low-nitrogen conditions (Cheema et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, the synergistic effects of PASP with microbial inoculants, particularly plant growth-promoting rhizobacteria (PGPR), remain unexplored.\u003c/p\u003e \u003cp\u003ePGPR are root-colonizing microorganisms that promote plant growth through various mechanisms (Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), including nutrient solubilization and modulation of soil-plant-microbe interactions ( Sughra et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For example, phosphate- and silicate-solubilizing strains enhance the availability of essential elements in the rhizosphere (Arjun et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), while nitrogen-fixing bacteria improve plant nitrogen assimilation efficiency (Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). PGPR that colonize mineral surfaces proliferate by forming biofilms composed of extracellular polymers, such as cellulose, proteins, and galactose, while releasing metabolites, including organic acids, proteins, and enzymes (Pankaj et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Maurya et al., 2024). The dynamic chemical reactions within these biofilms create favorable conditions for PGPR to assimilate inorganic nutrients, facilitating mineral weathering and enhancing plant nutrient absorption (Pankaj et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The restructuring of the rhizosphere microbial community is primarily driven by changes in the composition and concentration of soil exudates. Exogenously applied PGPR influence interspecies interactions through their secreted products (Zhao et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and can also induce host plants to alter the composition and quantity of root exudates (Sun et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This modulation recruits complementary microbial taxa, collectively promoting plant growth.\u003c/p\u003e \u003cp\u003eThe combined use of growth-promoting agents has emerged as an effective strategy to enhance crop resilience and productivity (A.Rahou et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, the combination of biochar and nano-silicon improves drought resistance in wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) (Zulfiqar et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), while microbial fertilizers integrated with biochar enhance soil phosphorus and potassium fertility, promoting tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) growth (Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite these advancements, the effects of PGPR inoculation on rhizosphere nutrient dynamics, microbial community structure, and plant performance\u0026mdash;particularly under PASP-supplemented cultivation systems\u0026mdash;remain underexplored.\u003c/p\u003e \u003cp\u003eA pot experiment was conducted to assess the effects of PGPR inoculation combined with PASP application on potato growth, rhizosphere nutrient content, and microbial community composition. We hypothesized that PGPR inoculation would synergize with PASP to (1) increase potato biomass, (2) enhance rhizosphere nutrient availability, and (3) shift the microbial community toward beneficial taxa. This study elucidates the mechanisms underlying plant-soil-microbe interactions in the context of combined PASP-PGPR application, providing both theoretical and practical insights for developing sustainable \"fertilizer synergist-microbial inoculant\" strategies in agriculture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Site and Materials\u003c/h2\u003e \u003cp\u003eThe pot experiment was conducted at the Chengdu Academy of Agricultural and Forestry Sciences, Sichuan Province, China (30\u0026deg;42\u0026prime;32\u0026Prime;N, 103\u0026deg;51\u0026prime;49\u0026Prime;E; altitude 540 m). Potato cultivation began in late December 2023 and concluded in May 2024, with pots maintained under open-air conditions. The potato variety used was \"Chuanyu 50,\" known for its strong field growth potential, resistance to late blight, and high resistance to viral diseases. Seed tubers were provided by the Key Laboratory of Crop Cultivation and Farming Systems, College of Agronomy, Sichuan Agricultural University. The PGPR strain S13, identified as \u003cem\u003eEnterobacter asburiae\u003c/em\u003e (NCBI accession number SUB14859840) through 16S rRNA sequencing, was supplied by the same laboratory. PASP (purity: analytical grade [AR], active ingredient\u0026thinsp;\u0026gt;\u0026thinsp;99.7%) was obtained from Zhengzhou Guanda Chemical Products Co., Ltd.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePot Experiment Design\u003c/h3\u003e\n\u003cp\u003eThe experiment was designed using a completely randomized design with four treatments: (1) untreated control (CK); (2) control with PGPR inoculation only (S1P0); (3) PASP application without PGPR inoculation (S0P1); (4) combined application of PASP and PGPR inoculation (S1P1). Each treatment consisted of 15 pots, totaling 60 pots. Vermiculite and perlite (3:1 v/v) were used as the growth substrate, filled to 4/5 of the pots (370 mm upper diameter \u0026times; 240 mm height).\u003c/p\u003e\n\u003ch3\u003eSeed Preparation and Inoculation\u003c/h3\u003e\n\u003cp\u003eSeed tubers (~\u0026thinsp;50 g each) were surface sterilized with 0.1% NaClO for 20 min, rinsed with distilled water, and air-dried. For PGPR inoculation (S1P0 and S1P1), tubers were immersed in a bacterial suspension for 30 min, while the control groups (S0P1 and CK) were received with sterile water. Through preliminary germination tests, a working concentration of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e CFU\u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e was selected for the bacterial suspension \u003cb\u003e(Supplemental Fig.\u0026nbsp;1)\u003c/b\u003e. The bacterial suspension was prepared by culturing a single colony of \u003cem\u003eE. asburiae\u003c/em\u003e in LB broth (37\u0026deg;C, 170 rpm, 24 h), adjusting the concentration to 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU\u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.8\u0026thinsp;~\u0026thinsp;1.0), and diluting 1:10,000 in sterile water (The final concentration is 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e CFU\u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eFertilization and Cultivation\u003c/h3\u003e\n\u003cp\u003eA potassium sulfate compound fertilizer (N: P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e: K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16:6:8; total nutrients\u0026thinsp;\u0026ge;\u0026thinsp;40%) was applied at a rate of 10 g per pot. PASP (0.5 g per pot, 5% of the fertilizer mass) was dissolved in sterile water and administered through root irrigation. Routine watering and manual weeding were conducted throughout the growth period.\u003c/p\u003e\n\u003ch3\u003eSample Collection and Processing\u003c/h3\u003e\n\u003cp\u003ePlant Samples\u003c/p\u003e \u003cp\u003eTen days post-emergence, nine uniformly growing plants per treatment were harvested. Three plants were used for the collection of phenotypic images. Six plants were used for growth parameter analysis, including stem diameter, root length, and dry weight determination. Stem diameter was measured at the base using vernier calipers, while root length was recorded as the distance from the root origin to the longest tip. For dry weight measurement, roots, stems, and leaves were separated, oven-dried at 105\u0026deg;C for 30 min, followed by drying at 80\u0026deg;C until a constant weight was achieved, and then weighed.\u003c/p\u003e \u003cp\u003eRhizosphere Soil Samples\u003c/p\u003e \u003cp\u003eRhizosphere soil was collected from six plants per treatment by gently removing loosely adhered soil and brushing off tightly bound soil from the roots. The samples were then divided into two subsets: one was flash-frozen in liquid nitrogen (-80\u0026deg;C) for microbial analysis, while the other was air-dried, ground, and sieved through a 60-mesh for biochemical assays.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSoil Biochemical Analysis\u003c/h2\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAmmonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N): Extracted using 2 \u003cem\u003eM\u003c/em\u003e KCl and quantified via indophenol blue colorimetry (UV-756 spectrophotometer, 625 nm) (Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eNitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N): Extracted with 2 \u003cem\u003eM\u003c/em\u003e KCl, acidified with 10% H\u003csub\u003e2\u003c/sub\u003eSO4, and quantified using dual-wavelength spectrophotometry (UV-756 spectrophotometer, 220 nm and 275 nm) (Zheng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAvailable potassium (AK): Extracted with 1 \u003cem\u003eM\u003c/em\u003e CH\u003csub\u003e3\u003c/sub\u003eCOONH\u003csub\u003e4\u003c/sub\u003e (pH 7.0) and analyzed using flame photometry (FP6400) (Zhao et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSoli Urease activity (S-Ure): Measured using a commercial kit (Jiangsu Adison Biotechnology Co., Cat#ADS-F-TR001) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRhizosphere Microbiome Sequencing of Potato\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA was extracted from soil samples using the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified with primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were analyzed by agarose gel electrophoresis and purified using the D6492-00 OMEGA Cycle Pure Kit (Noblebio Technology Co., Ltd., Beijing). Sequencing was performed on the Illumina Novaseq 6000 platform with paired-end (2\u0026times;250 bp) reads. To obtain high-quality sequencing data, raw data were first filtered using Trimmomatic v0.33 software, followed by primer sequence identification and removal using Cutadapt 1.9.1 software. Denoising, assembly, and chimera removal were then performed using the DADA2 plugin in QIIME2 software, resulting in high-quality sequences for subsequent analysis (Bolyen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Callahan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003ePlant growth parameters and soil biochemical properties were analyzed using one-way ANOVA followed by Duncan's multiple range test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in SPSS Statistics 26. Data visualization was performed using GraphPad Prism 9.5.\u003c/p\u003e \u003cp\u003eFor microbial community analysis, α-diversity indices (Chao1 and Shannon) were calculated using QIIME2 (v2023.2). Operational taxonomic units (OTUs) were clustered at a 97% similarity threshold using USEARCH (v10.0) in R, with low-abundance OTUs (\u0026lt;\u0026thinsp;0.005% of total sequences) filtered out. β-Diversity was visualized via principal coordinate analysis (PCoA), PERMANOVA and ANOSIM analyses based on Bray-Curtis dissimilarity. Functional profiling of microbial communities was inferred using PICRUSt2 against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Taxonomic annotation was performed in QIIME2 with reference to the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Differential abundance of taxa between groups was assessed using Metastats (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Nizigiyimana et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Feature taxa contributing to community differences were identified via random forest analysis using the RandomForest package (v4.6-10) in R (ntree\u0026thinsp;=\u0026thinsp;500). Relationships between microbial diversity and environmental factors were evaluated through redundancy analysis (RDA) and variance partitioning analysis (VPA) implemented in the vegan package (v2.3) in R (Rui et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of PGPR Inoculation on Potato Seedling Growth\u003c/h2\u003e \u003cp\u003eTo assess the effects of PGPR inoculation on the growth of potato seedlings and potato yield under PASP application, we measured growth indices and the dry weights of various plant parts. Specifically, the S1P1 treatment showed a 70.83% increase in plant height \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, a 38.43% increase in stem diameter \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, and a 41.12% increase in root length (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e compared with the S0P1 treatment. S1P1 treatment also notably enhanced the biomass of both roots and stems (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the dry weights of roots and stems increasing by 50.00% and 45.83%, respectively, relative to the S0P1 treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of PGPR Inoculation on Rhizosphere Soil Biochemical Properties\u003c/h2\u003e \u003cp\u003eTo assess the effects of PGPR inoculation under PASP application on rhizosphere soil chemistry and enzyme activity, we measured soil nutrient content and urease activity. Compared to the S0P1 treatment, the S0P1 treatment significantly enhanced NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, AK \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, and S-Ure activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e in the rhizosphere, increased NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and AK by 40.00%, 57.70%, and 47.56%, respectively, with all differences being statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of PGPR Inoculation on Rhizosphere Microbial Community Structure and Function\u003c/h2\u003e \u003cp\u003eTo further assess the effect of PGPR on the rhizosphere soil microenvironment under PASP application, we conducted 16S rRNA sequencing for the S0P1 and S1P1 treatments. Results showed that PGPR inoculation under PASP application altered the bacterial community diversity. Specifically, PGPR inoculation under PASP application decreased α-diversity indices (Chao1 and Shannon) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. PCoA based on Bray - Curtis dissimilarity indicated a significant change in β - diversity (p\u0026thinsp;=\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, suggesting a reorganization of the microbial community structure. Venn analysis of OTU abundance showed that the PGPR-inoculated group under PASP application had fewer unique OTUs (3,713) compared to the PASP-alone group, with only 122 shared OTUs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. However, KEGG pathway analysis did not show significant changes in microbial metabolic functions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of PGPR Inoculation on Rhizosphere Microbial Composition\u003c/h2\u003e \u003cp\u003eAt the phylum level, PGPR inoculation increased the relative abundances of \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eAcidobacteriota\u003c/em\u003e while decreasing those of \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eActinobacteria\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. At the genus level, PGPR inoculation enriched beneficial taxa such as \u003cem\u003ePaucibacter\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eMassilia\u003c/em\u003e, while reducing the abundance of \u003cem\u003eDuganella\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, and \u003cem\u003ePedobacter\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Genus-level enrichment analysis revealed distinct microbial signatures, with several taxa showing statistically significant variations in relative abundance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Specifically, the inoculated group exhibited marked increases in \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e, along with significant reductions \u003cem\u003eShinella\u003c/em\u003e, \u003cem\u003ePseudarcicella\u003c/em\u003e, and \u003cem\u003eMarmoricola\u003c/em\u003e. Furthermore, taxa such as \u003cem\u003eRhodanobacter\u003c/em\u003e, \u003cem\u003eHirschia\u003c/em\u003e, and \u003cem\u003eJatrophihabitans\u003c/em\u003e displayed significant abundance differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Notably, genera such as \u003cem\u003eTuricibacter\u003c/em\u003e (a genus associated with host metabolic regulation) and \u003cem\u003eRhodanobacter\u003c/em\u003e (a denitrification-related taxon), whose abundances increased, along with genera like \u003cem\u003eShinella\u003c/em\u003e (a potential nitrogen-cycling participant) and \u003cem\u003eMarmoricola\u003c/em\u003e (a stress-responsive genus), whose abundances decreased, were identified by Random Forest analysis as major drivers of microbial community restructuring between treatments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCorrelations Between Seedling Biomass and Rhizosphere Soil Properties\u003c/h2\u003e \u003cp\u003eCorrelation analysis showed that root dry weight was significantly positively correlated with AK (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas S-Ure activity exhibited strong positive correlations with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInteractions Between Soil Biochemical Properties and Microbial Diversity\u003c/h2\u003e \u003cp\u003eRDA analysis of rhizosphere microbial communities and environmental factors indicated that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content had the greatest influence on microbial community structure, followed by AK content, while NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content and S-Ure activity made marginal contributions. Notably, bacterial communities in PGPR-inoculated treatments under PASP application exhibited positive correlations with all environmental factors, whereas non-inoculated groups showed negative correlations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. VPA further quantified the contributions of individual environmental factors to microbial community variation. AK and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N explained 1.22% and 0.61% of the total variation, respectively. Additionally, the combined effects of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and S-Ure activity accounted for 0.62% of the microbial community variation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. At the genus level, correlation analysis between dominant taxa and environmental factors revealed that beneficial genera such as \u003cem\u003ePaucibacter\u003c/em\u003e, \u003cem\u003eMassilia\u003c/em\u003e, and \u003cem\u003eMethylophilus\u003c/em\u003e were positively correlated with nutrient availability (e.g., NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, AK), whereas genera including \u003cem\u003eDuganella\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, and \u003cem\u003ePedobacter\u003c/em\u003e showed negative correlations. Notably, \u003cem\u003ePedobacter\u003c/em\u003e and \u003cem\u003eJanthinobacterium\u003c/em\u003e exhibited significant negative correlations with AK (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting their sensitivity to potassium dynamics in PASP-supplemented systems \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePGPR Inoculation Enhances Potato Growth Through Rhizosphere Nutrient Modulation\u003c/h2\u003e \u003cp\u003eMost PGPR strains exhibit growth-promoting properties. For instance, \u003cem\u003eBacillus sp.\u003c/em\u003e can stimulate plant growth by producing plant hormones such as IAA and solubilizing inorganic phosphorus (Sun and Shahrajabian, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), while \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e HL14-3 induces ABA production in cucumbers, enhancing their growth under drought stress (Qin et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, PGPR inoculation under PASP application increased the plant height, stem diameter, root length, and dry matter accumulation in the stems and roots of potato seedlings \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, confirming the growth-promoting efficacy of PGPR as an inoculant. To explore the mechanism by which PGPR promotes potato growth, we measured the nutrient content in the rhizosphere soil and analyzed its correlation with dry matter accumulation in different plant parts. The results indicated that PGPR inoculation increased the levels of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and AK in the rhizosphere soil of potato seedlings \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, with a significant positive correlation observed between AK content and root dry matter accumulation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Mechanistically, plant growth and development are strongly correlated with rhizosphere nutrient availability. For example, soil nitrogen content influences the absorption and utilization of potassium in rapeseed (\u003cem\u003eBrassica napus\u003c/em\u003e L.), with high nitrogen conditions alleviating the effects of low potassium stress on photosynthesis, while low nitrogen conditions require more potassium to maintain normal photosynthetic function (Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Soil potassium content can alter root architecture, under low potassium conditions, tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e L. Kuntze) roots secrete organic acids to solubilize potassium salts, whereas under high potassium conditions, they upregulate genes related to cellulose degradation, thereby increasing total root length and fine root proportion in response to soil potassium gradients (Ruan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings suggest that the increased biomass of potato seedlings in this study may be linked to changes in soil nitrogen and potassium content, with the increase in soil potassium content potentially triggering root response mechanisms that enhance root length and dry weight.\u003c/p\u003e \u003cp\u003e \u003cem\u003eThe changes in soil biochemical properties are the result of the combined action of rhizosphere microbial communities mediated by PGPR inoculation\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePGPR can enhance nutrient availability in the plant rhizosphere, increasing the content of available nitrogen, potassium, and phosphorus (Razack et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For example, \u003cem\u003eAzoarcus sp.\u003c/em\u003e BH72 has been shown to increase nitrogen content in the rhizosphere of kallar grass (\u003cem\u003eLeptochloa fusca\u003c/em\u003e L. Kunth) under low nitrogen conditions (Igiehon and Babalola, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while \u003cem\u003ePseudomonas sp.\u003c/em\u003e and \u003cem\u003eBacillus sp.\u003c/em\u003e can secrete oxalic acid, citric acid, and phytase to solubilize organic or inorganic phosphorus, thereby enhancing its availability to plants (Rawat et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, PGPR inoculation significantly increased the levels of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, AK, and S-Ure activity in the rhizosphere soil of potatoes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, which is consistent with previous findings. Furthermore, RDA combined with VPA revealed that changes in soil biochemical characteristics within the potato rhizosphere were predominantly driven by the synergistic effects of PGPR-inoculated rhizosphere microbial communities. The analysis indicated that AK content was the most significantly affected parameter, followed by NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Subsequent correlation analysis between microbial diversity indices and edaphic factors showed that these changes were associated with specific functional taxa, such as \u003cem\u003ePaucibacter\u003c/em\u003e (a genus recognized for its metabolic versatility in carbon cycling and xenobiotic degradation), \u003cem\u003eMassilia\u003c/em\u003e (a rhizosphere-competent genus involved in root exudate utilization and phytohormone modulation), and \u003cem\u003eMethylophilus\u003c/em\u003e (a methylotrophic bacterium contributing to single-carbon metabolism and nitrogen mineralization) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Studies have shown that the addition of exogenous PGPR can increase the abundance of beneficial microorganisms in the microbial community, thereby promoting plant growth (Xie et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For example, the use of \u003cem\u003eBacillus velezensis\u003c/em\u003e SQR9 increased the relative abundance of \u003cem\u003ePseudomonas sp.\u003c/em\u003e and \u003cem\u003eBacillus sp.\u003c/em\u003e in the rhizosphere of cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.) and promoted cucumber growth in association with the native strain \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (Sun et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Inoculation with \u003cem\u003eBacillus velezensis\u003c/em\u003e containing agricultural waste enhanced the biofilm formation and colonization ability of native \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e, promoting strawberry (\u003cem\u003eFragaria \u0026times; ananassa\u003c/em\u003e) growth (Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This suggests that changes in rhizosphere soil nutrient content are not solely attributed to a single microbial species but are likely the result of the combined action of rhizosphere microbial communities mediated by exogenous PGPR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe introduction of exogenous microorganisms can reshape the structure of the rhizosphere soil microbial community\u003c/h2\u003e \u003cp\u003ePrevious studies have demonstrated that the use of microbial inoculants can not only directly affect host plants but also regulate the structure and composition of soil microbial communities, thereby synergistically promoting plant growth, disease resistance, and stress tolerance (Xie et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, PGPR inoculation reduced the richness and diversity of soil microbial communities under PASP application, but increased the compositional differences between samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, indicating that PGPR inoculation significantly impacted the soil microbial community structure, consistent with previous findings. Although there were no significant changes in α - diversity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, the changes in β - diversity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e and the dynamics of key species \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e jointly validated the rhizosphere reshaping hypothesis. This aligns with the ecological tenet: Function transcends taxonomy in soil-microbe dialogues (Pankaj et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The reduction in microbial community richness and diversity, alongside the increase in compositional differences between treatments, may be related to PGPR colonization activities in the potato rhizosphere. The introduction of exogenous microorganisms may stimulate the host plant to produce specific secretions that favor their colonization, enhancing their competitive advantage in nutrient and ecological niche competition (Nicolle et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Segura and Ramos, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For instance, inoculation with \u003cem\u003ePseudomonas simiae\u003c/em\u003e WCS417 can induce the secretion of scopoletin by Arabidopsis (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e L. Heynh) roots, facilitating the colonization of this strain while inhibiting the growth of pathogens and other native microbial communities (Stringlis et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, PGPR inoculation increased the abundance of phyla such as \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eAcidobacteriota\u003c/em\u003e in the potato rhizosphere soil, as well as the abundance of genera like \u003cem\u003ePaucibacter\u003c/em\u003e and \u003cem\u003eMassilia\u003c/em\u003e. Random Forest analysis revealed that the increased abundance of genera such as \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eRhodanobacter\u003c/em\u003e significantly influenced the differences in microbial community composition between groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These increased microbial populations often exhibit strong growth-promoting and soil nutrient-activating abilities. For example, \u003cem\u003eMassilia\u003c/em\u003e strains JJY03 and JJY04 can solubilize phosphate and produce IAA (Chhetri et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), while \u003cem\u003eRhodanobacter\u003c/em\u003e strains Si-c and S2-g demonstrate robust phosphate solubilization and nitrogen fixation capabilities (Woo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This suggests that PGPR inoculation promotes the recruitment of beneficial microorganisms associated with growth promotion, thereby reshaping the structure of the potato rhizosphere microbial community.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the inoculation of PGPR under PASP application led to a significant restructuring of the rhizosphere microbial community composition and architecture in potato, along with the selective enrichment of beneficial microbial taxa. The PGPR-mediated microbial consortia collectively enhanced soil nutrient availability, as evidenced by notable increases in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and AK concentrations, along with elevated S-Ure activity in the rhizosphere microenvironment. These synergistic improvements in soil biochemical properties resulted in measurable physiological outcomes, such as increased stem and root biomass, thus demonstrating substantial growth promotion effects. Overall, our findings indicate that PGPR inoculation under PASP supplementation represents an effective agroecological strategy to enhance plant growth promotion through rhizosphere microbiome engineering. However, the experiment was limited to pot conditions, and the lack of field validation may restrict the practical extrapolation of conclusions. Although correlation analysis revealed the association between microbial communities and nutrients, specific action pathways were not clarified through functional assays (such as strain colonization verification or metabolomics), requiring further studies to deepen mechanism elucidation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Sichuan Provincial Science and Technology Program Breeding Research Project (Grants No. 2021YFYZ0005 and 2021YFYZ0019), the National Modern Agricultural Industry Technology System Sichuan Tuber Crops Innovation Team Project (Grant No. sccxtd-2025-09), and the Sichuan Natural Science Foundation (Grant No. 2022NSFSC0014).\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eThe authors declare no competing financial or non-financial interests.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study\u0026rsquo;s conception and design. X.Z. performed research design, methodology selection, sample collection, data analysis, and manuscript drafting. H.Y.M. and K.Q.Z. contributed to research design and methodology. C.L., R.L.L., and J.L. conducted sample collection and data analysis. H.N.C. reviewed and revised the manuscript. S.L.Z. provided overall project planning, experimental facilities, funding acquisition, and supervision. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArjun A, Lee KE, Aaqil KM, Kang SM, Bishnu A, Muhammad I, Rahmatullah J, Kim KM, Lee IJ (2020) Effect of silicate and phosphate solubilizing rhizobacterium Enterobacter ludwigii GAK2 on Oryza sativa L. under cadmium stress. \u003cem\u003eJournal of Microbiology and Biotechnology\u003c/em\u003e 30 (1): 118-126. https://doi.org/10.4014/jmb.1906.06010\u003c/li\u003e\n\u003cli\u003eA.Rahou Y, Douira A, Tahiri AI, Cherkaoui EM, Benkirane R, Meddich A (2022) Application of plant growth-promoting rhizobacteria combined with compost as a management strategy against Verticillium dahliae in tomato. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e 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https://doi.org/10.1016/j.cpb.2024.100424\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Potato, Plant Growth-Promoting Rhizobacteria, Polyaspartic Acid, Soil Nutrients, Microbial Community","lastPublishedDoi":"10.21203/rs.3.rs-6917360/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6917360/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolyaspartic acid (PASP), as an environmentally friendly fertilizer synergist, has been widely applied in agricultural production. However, the effects of combined application of PASP and microbial inoculants have not been fully investigated. Through integrating pot experiments, soil biochemical property analysis, and microbiome sequencing, this study revealed that inoculation with \u003cem\u003eEnterobacter asburiae\u003c/em\u003e S13 improved the plant height (70.83%), stem diameter (38.43%), root length (41.12%), and root-shoot biomass (50.00\u0026ndash;45.83%) of potato seedlings under PASP application. Meanwhile, it simultaneously enhanced the contents of ammonium nitrogen (40.00%), nitrate nitrogen (57.70%), available potassium (47.56%), and urease activity in rhizosphere soil. 16S rRNA sequencing showed that Enterobacter asburiae S13 addition enriched beneficial microbial communities (e.g., \u003cem\u003ePaucibacter\u003c/em\u003e, \u003cem\u003eMassilia\u003c/em\u003e) and suppressed potential competitive taxa (e.g., \u003cem\u003eDuganella\u003c/em\u003e, \u003cem\u003ePedobacter\u003c/em\u003e). Redundancy analysis (RDA) indicated that available potassium and ammonium nitrogen were the core factors driving microbial community structure changes. These results elucidated the causal relationship between rhizosphere nutrient dynamics and microbial community reshaping under the combined application of PGPR and PASP, providing theoretical and technical support for sustainable fertilization strategies in agriculture.\u003c/p\u003e","manuscriptTitle":"Enterobacter asburiae S13 enhances the promoting effect of polyaspartic acid on potato growth by improving rhizosphere nutrient availability and reshaping microbial community","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 10:53:39","doi":"10.21203/rs.3.rs-6917360/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":"b60c1514-eb9c-4ded-9517-4d2c161001ca","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-30T16:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-25 10:53:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6917360","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6917360","identity":"rs-6917360","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}