Integrated analysis of the rhizosphere microbiome and transcriptome reveals the growth-promoting mechanism of plant growth-promoting rhizobacterium XYY in peach plants

Integrated analysis of the rhizosphere microbiome and transcriptome reveals the growth-promoting mechanism of plant growth-promoting rhizobacterium XYY in peach plants | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 11 December 2025 V1 Latest version Share on Integrated analysis of the rhizosphere microbiome and transcriptome reveals the growth-promoting mechanism of plant growth-promoting rhizobacterium XYY in peach plants Authors : Guangyuan Liu , Yanyan Li , Tianyu Dong , Jian Guo , Yuansong Xiao , Huaifeng Gao , Yangyang Gao , Qiuju Chen , Jingjing Luo , Zixuan Li , Huitian Wei , and Futian Peng [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176542759.92935639/v1 246 views 125 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Plant growth-promoting rhizobacteria (PGPR) represent promising eco-friendly alternatives for sustainable agriculture; however, the underlying mechanisms by which they enhance peach tree growth remain poorly understood. In this study, an Agrobacterium strain, designated XYY, exhibiting multiple plant growth-promoting traits, was isolated and selected from soil that had been long-term treated with bag-controlled slow-release fertilizer. By constructing a green fluorescent protein (GFP)-labeled derivative strain and integrating pot experiments with transcriptomic and microbiome analyses, we demonstrated that XYY effectively colonizes the rhizosphere and significantly promotes the growth of peach seedlings, increasing plant height and root area by 25.4% and 32.3%, respectively. The strain also improved rhizosphere soil fertility and enhanced key soil enzyme activities. Transcriptomic analysis revealed that Agrobacterium XYY activates pathways associated with auxin, cytokinin, and gibberellin biosynthesis, as well as photosynthesis and growth-related metabolic processes, while suppressing energetically costly defense pathways, including those involved in ethylene, jasmonic acid, and monoterpene biosynthesis. At the soil ecological level, XYY reshaped the structure of the rhizosphere microbial community, enriched beneficial bacterial taxa such as Arthrobacter and Actinomycetota, and enhanced the metabolic potential of the microbial community. This study elucidates the synergistic growth-promoting mechanism of Agrobacterium XYY within the ”microbe–soil–plant” system, providing a theoretical foundation for the development and application of biofertilizers in fruit tree cultivation. Integrated analysis of the rhizosphere microbiome and transcriptome reveals the growth-promoting mechanism of plant growth-promoting rhizobacterium XYY in peach plants Guangyuan Liu 1 , Yanyan Li 1 , Tianyu Dong 1 , Jian Guo 1 , Yuansong Xiao 1 , Huaifeng Gao 1 , Yangyang Gao 1 , Qiuju Chen 1 , Jingjing Luo 1 , Zixuan Li 1 , Huitian Wei 1 , Futian Peng 1* 1 College of Horticulture Science and Engineering, Shandong Agricultural University, Taian 271018, China *. Corresponding author. Futian Peng College of Horticulture Science and Engineering, Shandong Agricultural University, Taian 271018, China E-mail address: [email protected] (FT.Peng) Abstract Plant growth-promoting rhizobacteria (PGPR) represent promising eco-friendly alternatives for sustainable agriculture; however, the underlying mechanisms by which they enhance peach tree growth remain poorly understood. In this study, an Agrobacterium strain, designated XYY, exhibiting multiple plant growth-promoting traits, was isolated and selected from soil that had been long-term treated with bag-controlled slow-release fertilizer. By constructing a green fluorescent protein (GFP)-labeled derivative strain and integrating pot experiments with transcriptomic and microbiome analyses, we demonstrated that XYY effectively colonizes the rhizosphere and significantly promotes the growth of peach seedlings, increasing plant height and root area by 25.4% and 32.3%, respectively. The strain also improved rhizosphere soil fertility and enhanced key soil enzyme activities. Transcriptomic analysis revealed that Agrobacterium XYY activates pathways associated with auxin, cytokinin, and gibberellin biosynthesis, as well as photosynthesis and growth-related metabolic processes, while suppressing energetically costly defense pathways, including those involved in ethylene, jasmonic acid, and monoterpene biosynthesis. At the soil ecological level, XYY reshaped the structure of the rhizosphere microbial community, enriched beneficial bacterial taxa such as Arthrobacter and Actinomycetota, and enhanced the metabolic potential of the microbial community. This study elucidates the synergistic growth-promoting mechanism of Agrobacterium XYY within the ”microbe–soil–plant” system, providing a theoretical foundation for the development and application of biofertilizers in fruit tree cultivation. Key words: PGPR, Rhizosphere microbiome, Transcriptomics, Peach, Growth-promoting mechanism 1.Introduction The invention and wide application of chemical fertilizers have made significant contributions to ensuring global food security. However, in intensive agricultural production, especially in fruit tree cultivation systems such as peach trees, long-term excessive application of chemical fertilizers has led to serious environmental problems such as soil acidification, water eutrophication, and biodiversity decline, seriously threatening the sustainable development of agriculture (Hossain et al., 2022, Schulte-Uebbing et al., 2022). Currently, exploring environmentally friendly strategies to achieve reduced fertilizer application with increased efficiency has become a key issue that urgently needs to be addressed in the agricultural field (Mariotte et al., 2018).The application of a laboratory-developed bag-controlled slow-release fertilizer—with an N:P₂O₅:K₂O ratio of 22:7.3:21.5, a total nutrient content exceeding 50%, and each bag weighing 95 g with perforations to regulate nutrient release—in peach orchards has been shown to maintain a high nitrogen use efficiency under low-nitrogen conditions, increasing it by 37.73% compared to conventional fertilization methods. Furthermore, the fertilizer exhibits a prolonged release duration, with nutrient release kinetics well synchronized with the growth demands of fruit trees, thereby achieving the principle of ”single application, sustained supply” (Zhang et al., 2021). Within this fertilization framework, this study identified beneficial microbial strains and investigated novel strategies to enhance efficiency and reduce input requirements. Using plant growth-promoting rhizobacteria (PGPR) to replace chemical fertilizer input is a highly promising approach. PGPR are beneficial bacterial groups that promote plant growth through direct or indirect effects (Mitra et al., 2025). Their direct growth-promoting mechanisms mainly include biological nitrogen fixation, activation of insoluble phosphorus and potassium nutrients in the soil, synthesis of plant hormones, and activation of plant growth metabolic pathways, etc. (Oribhabor et al., 2025); indirect mechanisms include inducing plants to produce iron carriers or antibiotics to inhibit soil-borne pathogens, improving soil physical and chemical properties, and reshaping the structure and function of soil microbial communities, etc. (Gao et al., 2022). Therefore, PGPR are regarded as the key link connecting the ”microbial-soil-plant” system, and their metabolic activities can promote the coordinated improvement of plant health and soil fertility, thus receiving extensive attention from researchers in recent years. Common PGPR groups include Pseudomonas, Rhizobium, Bacillus, Azospirillum, etc. (de Andrade et al., 2023). A fluorescent Pseudomonas (VSMKU3054) can promote the elongation of tomato roots and stems by producing IAA (Suresh et al., 2025). Similarly, PGPR strains isolated from the tomato rhizosphere have also been found to have the ability to secrete IAA, promoting the development of lateral roots and root hairs and increasing the root absorption area (Dashti et al., 2021). Inoculation with specific PGPR can significantly increase the germination rate, survival rate, and nitrogen, phosphorus, and potassium levels in rice plants (Israr et al., 2016). In addition to the growth-promoting functions of the strains themselves, PGPR can also directly regulate plant gene expression. Inoculation with PGPR usually induces the up-regulation of genes related to plant growth pathways and the down-regulation of genes in defense-related pathways. For example, after inoculation with B. subtilis MBI600, more than 1000 genes in the cucumber root system were differentially expressed, significantly up-regulating genes related to the synthesis of hormones such as auxin and cytokinin, down-regulating key genes in the SA signaling pathway (such as NPR1 , PR1 ), and simultaneously inhibiting the over-activation of JA and ethylene pathways, thereby balancing the allocation of growth and defense resources (Samaras et al., 2022). Another study also demonstrated that Bacillus BT22 upregulated the growth hormone pathway-related genes in Arabidopsis thaliana and downregulated the expression of genes related to ethylene and jasmonic acid synthesis (Liu et al., 2023b). These transcriptome-level studies indicate that PGPR strains promote plant growth and development, reduce the investment in high-energy-consuming defense, and reallocate more energy and resources to growth and basic metabolic pathways, ultimately resulting in increased biomass. This strategy is often referred to as ”metabolic thrift” (Huot et al., 2014). In addition to the above direct growth-promoting mechanisms, PGPR can also indirectly promote plant health by influencing soil physical and chemical properties and reshaping the structure of the rhizosphere microbial community. PGPR can increase the content of soil elements and soil nutrient levels that are beneficial to plant growth, demonstrating its potential in reducing the input of chemical fertilizers (Timofeeva et al., 2023). For instance, a study on cucumbers found that the application of PGPR significantly increased the activities of urease and phosphatase in the soil, accelerating the transformation rate of key elements such as nitrogen and phosphorus from unavailable to available forms (Feng et al., 2025). Maheshwari et al.’s research also confirmed that the combined application of efficient potassium-solubilizing bacteria (Agrobacterium pusense and Bacillus paralicheniformis) isolated from the rhizosphere of bananas with nitrogen-fixing and phosphorus-solubilizing bacteria increased the content of nitrogen, phosphorus, and potassium in the soil under the condition of reducing NPK chemical fertilizers by 25% (Bright et al., 2025). This highlights the great application prospects of PGPR as microbial inoculants. In terms of microbial community regulation, PGPR strains, as key ecological nodes, can regulate the composition and function of indigenous microorganisms by competing for nutrient sites, producing specific signal molecules, or altering the rhizosphere physical and chemical environment, thereby driving the rhizosphere microbial community structure and function towards a direction more conducive to plant growth (Song et al., 2025). For example, in a study on the remediation of cadmium-contaminated soil, the colonization of Bacillus megaterium NCT-2 activated the nitrogen cycle in the rhizosphere, promoting the transformation of the rhizosphere microbial community structure and function, and specifically enriching beneficial rare microbial groups such as Lysobacter and Microbacterium to help remediate cadmium pollution (Chi et al., 2025). This change in community structure has improved the overall soil health. Although significant progress has been made in research on PGPR, the majority of studies remain focused on the preliminary characterization of plant growth-promoting traits of bacterial strains. Reports on Agrobacterium species as PGPR in peach trees are scarce, and there remains a lack of comprehensive analysis regarding their functional roles within the ”microbial-soil-plant” system. This study aims to elucidate the growth-promoting mechanisms of an Agrobacterium strain, designated XYY, isolated from the soil of a peach orchard with long-term application of bag-controlled slow-release fertilizer. By integrating physiological assessments, plant transcriptomic profiling, and soil microbiomic analyses, this investigation addresses three key scientific questions: (1) How does colonization by Agrobacterium strain XYY influence the growth phenotypes of peach seedlings and the physicochemical properties of the soil? (2) Through which key signaling pathways does strain XYY mediate interactions to facilitate the reallocation of ”growth-defense” resources in peach trees? (3) How does strain XYY alter the structure and function of the rhizosphere microbial community in peach seedlings, thereby enhancing soil nutrient utilization efficiency? 2. Materials and methods 2. 1 Isolation, identification and in vitro plant growth-promoting characteristics of strain XYY The experimental strain was isolated from rhizosphere soil of peach trees at the Horticultural Experiment Station of Shandong Agricultural University in Tai’an, Shandong Province, China (36.16°N, 117.156°E). The soil had been subjected to long-term application of bag-controlled slow-release fertilizer, a practice previously demonstrated in laboratory studies to enhance peach tree growth and improve fruit quality (Shou-shi et al., 2008). After analyzing and comparing the rhizosphere microbiome of this orchard with that of the conventionally fertilized orchard, a dominant strain that was significantly enriched and had excellent nitrogen-fixing ability was screened out and named XYY. Whole-genome sequencing was subsequently conducted by Sangon Biotech (Shanghai) Co., Ltd. to determine the taxonomic identity of the strain, and screen the Ri plasmid for the presence of pathogenic genes to exclude potential bacterial pathogenicity. Physiological and biochemical characteristics—including urease activity, starch hydrolysis, hydrogen sulfide production, catalase activity, lipase hydrolysis, Gram staining reaction, and gelatin liquefaction were determined according to standard protocols outlined in *Bergey’s Manual of Systematic Bacteriology*(Krieg and Holt, 1984). Functional gene annotation was performed based on the KEGG database following whole-genome analysis to identify genes associated with plant growth-promoting traits. In vitro assays were conducted to evaluate the strain’s plant growth-promoting properties, with all experiments performed in triplicate. Nitrogen fixation potential was initially screened on Ashby’s nitrogen-free agar medium and quantitatively assessed using the acetylene reduction assay (Montes-Luz et al., 2023). Phosphate solubilization was qualitatively evaluated on Pikovskaya’s (PKO) agar medium; quantitative analysis was carried out in NBRIP broth with a 1% (v/v) inoculum, incubated at 28 °C and 180 rpm for 7 days. Samples were collected periodically, centrifuged, and the pH and available phosphorus concentration in the supernatant were measured (Amri et al., 2023). Potassium solubilization capacity was quantified using the hydrogen peroxide ashing method. Indole-3-acetic acid (IAA) production was determined spectrophotometrically using the Salkowski reagent, with absorbance measured at 530 nm. Biofilm formation was quantified using the 96-well microtiter plate assay with crystal violet staining (Haney et al., 2021). Siderophore production was detected using the chrome azurol S (CAS) agar plate method (Rathod et al., 2024). Detailed methodological descriptions are provided in the supplementary materials. 2. 2 Construction of GFP-labeled strains To investigate the stable colonization of strain XYY in the rhizosphere of peach seedlings, a fluorescently labeled derivative was constructed using the pCAMBIA1300 plasmid, which carries kanamycin resistance and the green fluorescent protein (GFP) gene. The primer sequences used for verification were F: 5′-TCGCAAGACCCTTCCTCTA-3′ and R: 5′-GATGGGCACCACCCCGGT-3′. Competent cells of strain XYY were prepared and preserved at −80 °C in an ultra-low temperature freezer(Kong et al., 2009, Zhang et al., 2022). Plasmid transformation was performed via electroporation according to established protocols (Yuan et al., 2003). The transformed bacterial suspension was plated onto LB agar supplemented with 50 µg/mL kanamycin and incubated at 28 °C for 48 hours. The PCR reaction procedure is as follows: pre-denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute. Finally, there is a final extension at 72°C for 8 minutes, and the reaction is stored at 4°C. The PCR products are detected by agarose gel electrophoresis (150 V, 15 minutes). Individual colonies were subsequently selected for PCR confirmation. To verify that the GFP tag did not alter the intrinsic properties of the strain, the growth curves and in vitro plant growth-promoting traits of the tagged strain and the wild-type strain were compared following five consecutive passages. The successfully constructed GFP-labeled strain was inoculated onto peach seedlings, and small root samples were collected on days 8, 15, 22, and 29 post-inoculation. Following staining and sample preparation, bacterial colonization in the root zone was observed and documented using a Nikon Ti2-U fluorescence microscope (Hölscher et al., 2016). 2. 3 Pot Experiment Design This study utilized Prunus davidiana (Carrière) Franch seeds as experimental materials. The seeds were surface-sterilized using 70%–75% (v/v) ethanol for 1–3 minutes, followed by three rinses with sterile distilled water. Subsequently, the seeds were germinated and transplanted into potting soil. The experimental soil was collected from the Horticultural Experiment Station of Shandong Agricultural University in Tai’an City, Shandong Province, China (36.16°N, 117.156°E), where it had been subjected to long-term application of bag-controlled slow-release fertilizer. The soil’s physicochemical properties were as follows: pH 7.06, organic matter content 22.5 g/kg, alkali-hydrolyzable nitrogen 128 mg/kg, available phosphorus 69.8 mg/kg, and available potassium 106 mg/kg. The soil was naturally air-dried, sieved through a 3 mm mesh, and then packed into pots at a rate of 1.5 kg per pot (diameter 15 cm × height 12 cm). Strain XYY was cultivated in Luria–Bertani (LB) medium (containing the following components (g·L⁻¹): NaCl 10.0; yeast extract 5.0; tryptone 10.0) at 28°C and 180 rpm until reaching the logarithmic growth phase. Bacterial cells were harvested by centrifugation, resuspended in sterile water, and adjusted to an OD₆₀₀ of 0.8 to prepare a 250 mL bacterial suspension. Uniformly growing peach seedlings were selected and randomly assigned to two groups: CK (control group, treated with sterile water without Agrobacterium inoculation) and XYY (experimental group, inoculated with Agrobacterium strain XYY via root irrigation). Each group consisted of 10 biological replicates. Inoculation was performed through root irrigation once every seven days for a total of four applications, with routine watering maintained between treatments. All plants were grown in a controlled greenhouse environment (25°C, 12-h light/12-h dark photoperiod) for 30 days. 2. 4 Assessment of Plant Growth and Physiological Characteristics After 30 days of cultivation, growth parameters of the peach seedling groups, including plant height, root length, root dry weight and root area, were recorded. Leaf SPAD values were measured using a portable chlorophyll meter (KONICA MINOLTA SPAD-502PLUS, Japan). Root systems were gently rinsed with sterile distilled water and scanned using a flatbed scanner (Epson Perfection V700/750, Japan), and total root length as well as average root area was analyzed with the WinRHIZO root analysis system (Regent Instruments Inc., Canada). Leaf nitrogen content was determined using an automatic Kjeldahl nitrogen analyzer (K0860, Hanon Instruments, China); phosphorus content was quantified via the vanadomolybdate blue colorimetric method (Murphy and Riley, 1962); and potassium content was assessed using a flame spectrophotometer (Model 721S, Lengguang, China). Photosynthetic pigment contents chlorophyll a, chlorophyll b, and carotenoids were determined spectrophotometrically. Root activity was evaluated using the triphenyltetrazolium chloride (TTC) reduction method (Xu et al., 2024). Absorbance was read at 485 nm, and TTC reduction intensity was subsequently calculated. 2. 5 Assessment of Soil Physicochemical Properties and Enzyme Activities Rhizosphere soil samples collected after 30 days of cultivation were homogenized and stored at -80 °C. Soil pH was measured using a PHSJ-4F pH meter. Alkali-hydrolyzable nitrogen (AN) content was determined via the alkaline hydrolysis diffusion method, and soil organic matter (SOM) content was quantified using the potassium dichromate volumetric method. Available phosphorus (AP) content was analyzed by the molybdenum blue colorimetric method with a Shimadzu UV-2550 ultraviolet-visible spectrophotometer (Kyoto, Japan) (Sarker et al., 2014). Available potassium (AK) content was determined by flame photometry. The activities of soil catalase, urease, acid phosphatase, and sucrase were assayed using the same spectrophotometer (Shimadzu UV-2550, Kyoto, Japan) (Cordero et al., 2019). Statistical analyses were performed using SPSS 26.0, with significance determined by Student’s t-test (for two groups) . 2. 6 RNA Sequencing Analysis of Peach Seedling Root Systems After 30 days of cultivation, root tissue samples were collected from peach seedlings, with three biological replicates established for each treatment. All samples were immediately stored at -80°C and subsequently shipped to Wuhan Metware Biotechnology Co., Ltd. for transcriptome sequencing. Total RNA was extracted using the RNAiso Plus kit (TaKaRa, Japan). RNA integrity was assessed using the Agilent 2100 Bioanalyzer system, with a minimum RNA Integrity Number (RIN) of 8.0 required. RNA purity and concentration were determined using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), with an OD₂₆₀/OD₂₈₀ ratio between 1.8 and 2.0 and an OD₂₆₀/OD₂₃₀ ratio of at least 2.0 to ensure suitability for downstream sequencing. For library construction, mRNA was enriched and fragmented to yield insert sizes of approximately 300 bp, followed by reverse transcription into cDNA. Sequencing adapters were then ligated, and the resulting libraries were evaluated for concentration and fragment size distribution using the Qubit Fluorometer (Thermo Fisher Scientific, USA) and the Qsep400 Bioanalyzer (Optic Protein Solutions, Taiwan) to confirm compliance with sequencing quality standards. Final single-stranded circular DNA libraries were sequenced on the MGI platform using 150 bp paired-end (PE150) sequencing, with a minimum of 6 Gb high-quality data generated per sample. Raw sequencing reads underwent stringent quality control to remove adapter sequences, low-quality reads (Q20 < 90%), and reads with a high proportion of undetermined bases (N bases), yielding clean reads for downstream analysis. Clean reads were aligned to the peach reference genome (Prunus persica, Ensembl Plants release-52) using HISAT2 (v2.0.4), and transcript assembly was performed with StringTie (v1.3.4). Gene expression levels were quantified using featureCounts and normalized as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Differentially expressed genes (DEGs) were identified using DESeq2, with a threshold of adjusted p-value < 0.01 and |log₂FoldChange| ≥ 1 (Benjamini-Hochberg correction). Finally, GO functional annotation and KEGG pathway enrichment analysis were performed on the differentially expressed genes using databases (GOATOOLS and KEGG Release 107). Significantly enriched GO terms and KEGG pathways were determined based on the corrected Q value (Q value < 0.05). The specific KEGG pathway diagrams were drawn using WPS Office and Adobe Photoshop 2025 software. 2. 7 Analysis of Rhizosphere Soil Metagenomic Sequencing Data After 30 days of cultivation, rhizosphere soil samples were collected from peach seedlings in each treatment group using the root-shaking method. Soil particles adhering to the root surface within a 1-mm distance were carefully retrieved. Three independent biological replicates were randomly sampled and immediately stored at –80 °C. Subsequently, the samples were transported to Wuhan Meiji Biotechnology Co., Ltd. for microbial community analysis. Total soil DNA was extracted using the Meiji Biological Soil DNA Extraction Kit, and DNA concentration was quantified using a Qubit Fluorometer (Thermo Fisher Scientific, USA). Library construction involved the following steps: DNA fragment end repair, 3’ adenylation (”A-tailing”), adapter ligation, PCR amplification, and purification. The resulting libraries were quantified using the Qubit system, and qualified libraries were sequenced on the MGI DNBSEQ-T7 platform to obtain comprehensive microbial genetic information. Raw sequencing data underwent stringent quality control to remove low-quality reads, adapter sequences, and contamination from the peach host genome, yielding high-quality clean data. Subsequent bioinformatics analyses were conducted based on the QIIME2 (v2023.2) software for the following analyses： Alpha diversity was assessed using the Shannon and Chao1 indices to evaluate microbial community evenness and richness, respectively. Beta diversity was analyzed using Bray-Curtis dissimilarity coupled with PERMANOVA to test for significant differences in community structure among groups. The top 10 most abundant taxa at the phylum and genus levels were selected for visualization, while the remaining taxa were aggregated into an ”Others” category, and relative abundance bar plots were generated. Biomarker species differentially represented between groups were identified using the LEfSe method, with a significance threshold of LDA score ≥ 4 and P < 0.05. Subsequently, correlation analysis between differentially abundant phylum-level taxa and environmental factors was conducted based on Bray-Curtis distance, with a variance inflation factor (VIF) set at 10.0. Finally, functional profiling of the microbial communities was performed by comparing enriched genes across treatments in the KEGG database(eggNOG-mapper), enabling annotation of metabolic pathways and inference of microbial community functional characteristics. 2. 8 Data analysis The dataset preparation and data visualization were performed using Excel 2019 (Microsoft Corporation, USA) and GraphPad Prism 10 (GraphPad Software, San Diego, USA), respectively. Prior to conducting Student’s t-test to evaluate the effects of XYY strain colonization on peach seedling traits and soil properties, the normality of the data was assessed using the Shapiro-Wilk test, and homogeneity of variance between groups was evaluated using Levene’s test. Upon confirmation that the data met the assumptions of normality and homogeneity of variance, Student’s t-test was applied to determine significant differences between the control and treatment groups, with statistical significance defined at P < 0.05. 3. Results 3. 1 Physiological and Biochemical Characterization of Strain XYY and Its Plant Growth-Promoting Traits The bacterial phylogenetic tree indicates that strain XYY belongs to the genus Agrobacterium. The closest matching strain retrieved from the NT database is Agrobacterium rhizogenes K599 (GCA_002005205.3), with an average nucleotide identity (ANI) value of 99.96%, which exceeds the 95% threshold for species-level classification, thereby supporting its identification at the species level (Fig. 1A). Whole-genome analysis revealed that the T-DNA region of the Ri plasmid in this strain lacks key pathogenic genes (iaaM, iaaH, ags) and rol genes, while retaining a complete vir gene region. These genomic findings confirm the absence of pathogenic potential in this strain. Physiological and biochemical characteristics of the strain are summarized in (Table S1): XYY is a Gram-negative bacterium capable of urea hydrolysis, starch degradation, hydrogen sulfide production, catalase activity, lipase activity, and gelatin liquefaction (Fig. S1). These capabilities demonstrate that bacteria can utilize diverse organic substrates, including urea, starch, lipids, and proteins in the plant rhizosphere environment, and by mediating the biogeochemical cycling of key elements such as carbon, nitrogen, and sulfur, contribute to soil nutrient dynamics. Plate-based assays demonstrated that XYY exhibits nitrogen fixation, phosphate solubilization, potassium solubilization, and siderophore production capabilities (Fig. 1B), preliminarily confirming its plant growth-promoting potential. Quantitative analyses revealed that XYY produces biofilm with an absorbance value of 2.37 ± 0.17 (OD₅₈₀), displays nitrogenase activity of 108.01 ± 6.69 nmol C₂H₄·h⁻¹·mL⁻¹, and synthesizes indole-3-acetic acid (IAA) at a concentration of 16.20 ± 1.82 μg/mL (Table S2). In potassium solubilization assays, the water-soluble potassium content after H₂O₂ treatment reached 49.00 ± 2.22 μg/mL, representing a 29.3% increase compared to the control. Temporal assessment of phosphorus solubilization showed that over a 7-day incubation period, the culture pH was negatively correlated with available phosphorus levels (Fig. 1C), with phosphorus availability increasing by 64% relative to day one (p < 0.01). KEGG pathway annotation indicated that bacterial genes were predominantly enriched in cellular processes, environmental information processing, and metabolic pathways. Notably, a high number of genes were mapped to biofilm formation (quorum sensing), membrane transport, amino acid metabolism, and acetate metabolism (Fig. 1D). Additionally, a total of 41 functional genes associated with siderophore biosynthesis, IAA production, phosphorus metabolism, and stress tolerance were identified (Table S3). 3. 2 Agrobacterium XYY colonizes the root system, thereby promoting the growth of peach seedlings. The XYY transformation strain harboring the pCAMBIA1300 plasmid was confirmed by PCR amplification, which yielded a distinct band consistent with the positive control (Fig. S2), thereby verifying the successful construction of the GFP-labeled strain. No significant differences were observed in growth curve profiles, nitrogenase activity, IAA production capacity, or biofilm formation levels between the GFP-labeled and wild-type strains, indicating that the fluorescent tag did not impair the plant growth-promoting properties of Agrobacterium XYY. Following a 30-day treatment period, peach seedling growth parameters were measured and samples collected. The results demonstrated that both shoot and root growth in seedlings inoculated with Agrobacterium XYY were significantly enhanced compared to the CK control group (Fig. 2A–C). Scanning fluorescence electron microscopy was employed to examine the root systems of peach seedlings at various time points, revealing detectable fluorescence in all samples (Fig. 2D). Furthermore, fluorescence intensity increased with prolonged treatment duration. These findings confirm the successful generation of the GFP-labeled strain and substantiate the effective colonization of Agrobacterium XYY within the peach seedling root system. Relative to the CK group, plant height, root length, root dry weight, and root area in the experimental group increased by 25.4% (P < 0.05), 9.4% (P < 0.05), 16.9% (P < 0.05), and 32.3% (P < 0.05), respectively (Table S4). Concurrently, XYY treatment improved photosynthesis-related traits in peach seedlings (Fig. 2E–H). The SPAD value increased by 16.3% (P < 0.05) relative to the control, while chlorophyll a and chlorophyll b contents rose by 25.5% and 11.2% (P < 0.05), respectively. Carotenoid content did not exhibit significant changes. These alterations in photosynthetic pigments may be attributed to bacterial-induced reallocation of energy resources for photosynthesis(Mao et al., 2025, Croce and van Amerongen, 2013). The observed increase in pigment levels is consistent with the significant upregulation of photosynthetic pathways identified in subsequent transcriptome analysis. Additionally, leaf nitrogen, phosphorus, and potassium contents, as well as root activity, were significantly higher in the experimental group (Fig. 2I–L), increasing by 27.6% (P < 0.05), 27.5% (P < 0.05), 15.8% (P < 0.05), and 31.3% (P < 0.05), respectively, compared to the CK group. These findings indicate that strain XYY enhances nutrient uptake and promotes the growth and development of peach seedlings. 3. 3 Effects of Agrobacterium XYY Inoculation on Soil Nutrient Content and Enzyme Activity Analysis of rhizosphere soil from the pot experiment revealed that, compared with the control group, soil inoculated with strain XYY exhibited a 21.9% (P < 0.05) increase in alkali-hydrolyzable nitrogen content and a 21.0% (P < 0.05) increase in available phosphorus content. The pH was lower than that of the control, while changes in soil organic matter and available potassium content were not statistically significant (Fig. 3A–E). The observed enhancement in soil nutrient levels represents a significant finding, indicating that the beneficial effects of the bacterium extend beyond plant growth promotion to include improvement of the rhizosphere environment. Application of strain XYY enhanced soil nutrient availability and improved energy supply to the plant by increasing root absorption surface area, thereby promoting aboveground growth of peach seedlings. Soil enzyme activity assays (Fig. 3F–I) demonstrated that XYY inoculation significantly increased the activities of key enzymes: urease, acid phosphatase, and sucrase increased by 18.1% (P < 0.05), 22.9% (P < 0.05), and 16.8% (P < 0.05), respectively. In contrast, no significant difference was observed in soil catalase activity. 3. 4 Agrobacterium XYY promotes plant growth through the regulation of gene expression. Under Agrobacterium XYY colonization, a total of 3,474 differentially expressed genes (DEGs) were identified in the root system of peach seedlings, including 1,383 up-regulated and 2,091 down-regulated genes. Principal component analysis (PCA) confirmed high reproducibility across biological replicates (Fig. 4A–B). Gene Ontology (GO) enrichment analysis revealed that DEGs were predominantly enriched in biological processes (1,609 genes) and molecular functions (806 genes). Among biological processes , the most significantly represented functional modules included regulation of cell communication (87 genes), regulation of signal transduction (85 genes), signal regulation (85 genes), and response to organic cyclic compounds (85 genes). In terms of molecular functions, DEGs were primarily associated with ADP binding (103 genes) and hexosyltransferase activity (94 genes) (Fig. 4C). KEGG pathway enrichment analysis indicated significant differential expression in several key pathways, including the MAPK signaling pathway—plant , plant hormone signal transduction , monoterpene biosynthesis , and photosynthesis (Fig. 4D). Within the plant hormone signal transduction pathway, genes involved in auxin, cytokinin, and gibberellin biosynthesis and signaling were significantly up-regulated. Specifically, 12 and 23 genes were significantly up-regulated in the photosynthesis and MAPK signaling pathways, respectively. Additionally, multiple metabolic pathways related to growth—including starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, pentose phosphate pathway, and zeatin biosynthesis showed a general trend of up-regulation. Conversely, genes in ethylene and jasmonic acid biosynthesis pathways, as well as those involved in monoterpene biosynthesis, alpha-linolenic acid metabolism, and glutathione metabolism, exhibited down-regulation. These transcriptional changes suggest that Agrobacterium XYY promotes auxin and gibberellin synthesis while modulating resource allocation between growth and defense, thereby redirecting metabolic energy toward developmental processes. To elucidate the molecular mechanisms underlying XYY-mediated growth promotion, hormone signaling and photosynthesis pathways directly linked to plant growth were further analyzed (Fernández-Cancelo et al., 2023). Reported that multiple genes in the auxin biosynthesis pathway were significantly up-regulated (Fig. 5A), primarily enriched in the AUX1 , ARF , SAUR , and TMK gene families. Notably, the LOC18784874 gene, encoding an auxin influx transporter in the AUX1 family, was significantly up-regulated, indicating enhanced cellular uptake of auxin(Yang et al., 2025b). Four SAUR family genes also showed significant up-regulation, with fold changes of 1.44, 1.98, 2.44, and 2.93, respectively. As key transcriptional regulators of auxin responses, ARF genes act as molecular switches to initiate downstream gene expression. In this study, three ARF genes were up-regulated, suggesting activation of auxin-responsive transcription and enhanced auxin signaling. In the cytokinin signaling pathway, two CRE1 family genes (involved in signal perception), five B-ARR family genes (core transcription factors), and one A-ARR family gene (a negative feedback regulator) were significantly up-regulated (Fig. 5B), indicating enhanced cytokinin signal transduction(Müller and Sheen, 2007). The up-regulation of A-ARR may serve as a feedback mechanism to prevent overactivation due to excessive cytokinin accumulation(Liu et al., 2020, Zhou et al., 2024). Similarly, the gibberellin (GA) signaling pathway was activated in the experimental group. Seven genes encoding DELLA proteins negative regulators of GA signaling, including LOC18779041 , LOC18775089 , and LOC18768743 were down-regulated (Fig. 5C), suggesting that XYY inoculation may alleviate GA signaling repression. Concurrently(Hirano et al., 2008), three GID1 receptor genes were up-regulated by 1.63-, 3.35-, and 1.57-fold, respectively, and five genes of the TF family that promote the downstream synthesis of gibberellin were upregulated by 2.64-, 1.61-, 2.12-, 1.12-, and 2.76-fold(Yoshida et al., 2014, Murase et al., 2008), respectively, further supporting enhanced GA responsiveness. Interestingly, genes in the ethylene and jasmonic acid signaling pathways exhibited a general down-regulation. Following XYY treatment, five ERF1/2 genes in the ethylene pathway were down-regulated, while two JAZ and nine MYC2 genes—key components of jasmonic acid signaling were significantly down-regulated (Fig. 5D–E), indicating suppression of defense-related pathways. Furthermore, in photosynthetic pathways (ko00195, ko00196), a total of 17 genes were significantly up-regulated (Fig. 5F, Fig. S3), primarily localized to Photosystem II (PSII), Photosystem I (PSI), and the light-harvesting chlorophyll protein complex (LHC) (Rong et al., 2025). The heatmap illustrating the correlations between the eight differentially expressed genes and plant phenotypes revealed that the significantly up-regulated genes were positively correlated with most phenotypic traits, particularly root activity, chlorophyll a content, and SPAD value, showing highly significant associations (Fig. S4). In contrast, the two significantly down-regulated genes, LOC1877883 and LOC18771053 , exhibited significant negative correlations with multiple core phenotypic traits, suggesting a direct relationship between transcriptomic changes and phenotypic variation in plants. 3. 5 The colonization by Agrobacterium XYY alters the structure and function of soil microbial communities. Metagenomic analysis revealed that, compared with the control group (CK), the XYY strain treatment showed no significant difference in α-diversity based on the Chao1 index, but exhibited a significant difference in the Shannon index (p < 0.05) (Fig. 6A–B). With respect to β-diversity, principal component analysis (PCA) indicated significant separation between microbial communities under the two treatments (Fig. 6C). Bar plots illustrated compositional differences in microbial communities at the phylum and genus levels across treatments (Fig. 6D–E). At the phylum level, the relative abundance of Actinomycetota increased by 10.3% (p < 0.01) in the XYY treatment relative to the CK. At the genus level, the abundances of f__Rhizobiaceae;g__Agrobacterium and f__Micrococcaceae;g__Arthrobacter significantly increased by 32.3% (p < 0.01) and 22.3% (p < 0.01), respectively. The elevated abundance of Agrobacterium indicates successful colonization of the rhizosphere soil by Agrobacterium XYY, which is consistent with prior construction and expression results of the GFP-labeled strain. LEfSe analysis identified Micrococcales, Micrococcaceae, Arthrobacter, Actinomycetes, Rhizobiaceae, Hyphomicrobiales, and Agrobacterium as taxa with higher relative abundances in the experimental group and significant contributions to intergroup dissimilarity (Fig. 6F). The correlation analysis between microbial species and environmental factors at the genus level in the experimental group (Fig. 7G) reveals significant associations among soil physicochemical properties, enzyme activities, and microbial community structure. Agrobacterium rhizogenes was significantly associated with multiple soil parameters. It exhibited a strong correlation with soil pH (Mantel test, P < 0.01, correlation coefficient ≥ 0.6), indicating that strain XYY induces soil acidification through metabolic activities such as organic acid secretion. Furthermore, Agrobacterium rhizogenes showed significant correlations with alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), urease activity (UA), acid phosphatase activity (ACP), and sucrase activity (SA) (Mantel test, 0.01 < P < 0.05, correlation coefficient ≥ 0.4), demonstrating its direct involvement in nutrient mobilization and enhancement of enzymatic activities. Similarly, Arthrobacter was significantly correlated with the same set of indicators (Mantel test, 0.01 < P < 0.05, correlation coefficient ≥ 0.4), suggesting active participation in nutrient cycling and soil enzyme regulation rather than passive adaptation. These findings indicate that Arthrobacter and Agrobacterium rhizogenes play complementary roles in modulating the rhizosphere soil microenvironment. KEGG functional annotation of the soil microbial community revealed enrichment of metabolic pathways such as purine metabolism (map00230), pyrimidine metabolism (map00240), amino acid biosynthesis (map01230), and nucleotide metabolism (map01232) in the experimental group (Fig. 6H), indicating an active metabolic state characterized by enhanced nucleic acid and protein synthesis. This suggests that the microbial community was in a growth-promoting phase, likely supported by favorable environmental conditions (Wang et al., 2025a, Mishra et al., 2025). Enrichment of ABC transporters (map02010) implies active nutrient uptake and environmental adaptation by the microbial community. These enhanced metabolic functions may be associated with the increased abundances of Arthrobacter, Actinomycetota, and Agrobacterium, indicating that inoculation with Agrobacterium XYY not only altered the structure of the rhizosphere microbiome but also stimulated microbial activity, thereby enhancing nutrient acquisition and accumulation in the aboveground plant tissues. 4. Discussion 4. 1 The plant growth-promoting potential of Agrobacterium XYY and its colonization capacity within the root system Plant growth-promoting rhizobacteria (PGPR) have emerged as a focal point in agricultural research due to their capacity to enhance plant growth and reduce reliance on chemical fertilizers. In this study, an safe and non-pathogenic Agrobacterium strain was isolated from soil that had been subjected to long-term laboratory treatment with controlled-release fertilizer and designated XYY. Effective PGPR strains typically exhibit multiple plant growth-promoting traits, and successful colonization of the rhizosphere is essential for the expression of these beneficial functions. In vitro assays revealed that strain XYY possesses the ability to fix nitrogen, solubilize phosphorus and potassium, and produce indole-3-acetic acid (IAA), along with notable capabilities in siderophore production and biofilm formation. These findings provide a theoretical foundation for understanding its growth-promoting effects and rhizospheric adaptability: siderophores enable the strain to compete for iron under iron-limited rhizosphere conditions, while biofilm formation facilitates adhesion and stable colonization on root surfaces. Together, these traits support the survival and proliferation of the strain in the rhizosphere, thereby establishing the basis for its subsequent plant growth-promoting activities. The experimental progression from in vitro characterization to pot trials aligns with established methodologies in prior studies (Zhang et al., 2025b). Whole-genome analysis reveals that strain XYY harbors multiple key functional genes associated with phosphorus solubilization (e.g., pstS , pstA , and pstB ), auxin biosynthesis (e.g., aro and ald ), siderophore production, and stress response. For instance, the pstS gene encodes a high-affinity phosphate-binding protein that is upregulated under low-phosphorus conditions, enhancing the strain’s capacity to acquire inorganic phosphorus and thereby ensuring adequate phosphorus supply for energy metabolism and cellular biosynthesis (Allenby et al., 2004). Additionally, the aro gene participates in the shikimate pathway-mediated synthesis of tryptophan, a precursor of IAA, while the ald gene may catalyze the oxidation of indole-3-acetaldehyde to IAA (Rawle et al., 2020). The presence of these functional genes provides genetic evidence supporting the multifaceted growth-promoting properties observed in strain XYY. For PGPR strains to exert sustained beneficial effects, efficient rhizosphere colonization is critical. To assess the colonization capability of XYY, a green fluorescent protein (GFP)-labeled derivative was constructed. Fluorescence imaging revealed consistent signals on the root systems of peach seedlings at 8, 15, 22, and 29 days post-inoculation, indicating successful and persistent rhizospheric colonization. Metagenomic data further confirmed the stable presence of XYY in rhizosphere soil, corroborating the GFP labeling results and collectively demonstrating its robust rhizosphere adaptability. Similar GFP-based tracking approaches have been employed to monitor the colonization of Bacillus subtilis N-1-gfp in rice tissues (Liu et al., 2023a) and the movement of pathogenic bacteria in tomato plants (Chalupowicz et al., 2011). In conclusion, strain XYY exhibits multiple in vitro growth-promoting traits, carries corresponding functional genes in its genome, and demonstrates efficient and durable rhizosphere colonization. These attributes collectively establish a solid scientific foundation for its significant promotion of peach seedling growth observed in subsequent pot experiments, positioning it as a promising candidate for the development of microbial inoculants tailored for fruit tree cultivation. 4. 2 Inoculation with Agrobacterium XYY promoted the growth of peach seedlings and enhanced soil nutrient availability. The growth-promoting mechanisms of plant growth-promoting rhizobacteria (PGPR) commonly encompass nitrogen fixation, phosphorus and potassium solubilization, phytohormone production, and enhancement of root system development (Arif et al., 2020). High-performing PGPR strains not only directly stimulate plant growth but also reduce dependence on chemical fertilizers, thereby contributing to improved soil health (Jabborova et al., 2021). This study confirmed that Agrobacterium XYY has significant plant growth-promoting potential, expanding the root system’s contact area with the soil and thereby enhancing the absorption efficiency of nutrients and water. This observation aligns with findings from other cropping systems; for example, Bacillus aryabhattai LAD improved nutrient absorption in maize by modifying root system architecture (Deng et al., 2022). Moreover, the enhancement of photosynthetic performance provides physiological evidence of the strain’s beneficial effects and indicates that chlorophyll a—acting as the primary pigment in the photosynthetic reaction centers—directly converts light energy into chemical energy (ATP and NADPH), driving the photosynthetic process, whereas chlorophyll b primarily assists chlorophyll a in light harvesting and broadens the spectral range of light absorption. Together, these pigments constitute photosystem I (PSI), photosystem II (PSII), and the light-harvesting complex II (LHCII) (Mao et al., 2025, Croce and van Amerongen, 2013). Similarly, studies have shown that Bacillus halodurans enhances the maximum photochemical efficiency of both photosystems II and I in barley and upregulates sugar metabolism pathways to supply additional photosynthetic substrates (Slatni et al., 2025). This study revealed a significant increase in leaf nitrogen, phosphorus, and potassium concentrations in peach seedlings inoculated with strain XYY. As essential macronutrients for plant physiological processes, elevated leaf levels of these elements reflect an improved overall nutritional status. This outcome suggests that XYY enhances the acquisition of nutrients from the soil via the root system and facilitates their translocation and assimilation into aboveground tissues. Supporting evidence comes from El Ifa et al., who reported that PGPR application significantly increased phosphorus content in barley leaves (El Ifa et al., 2024). Leaf elemental composition serves as a critical link connecting aboveground plant growth with rhizosphere microbial activity. Strain XYY enhances the availability of key nutrients to the host plant, ultimately reflected in elevated leaf nitrogen, phosphorus, and potassium concentrations. Concurrently, the observed increase in root activity is closely associated with enhanced root growth and surface area in peach seedlings (Yue et al., 2019), underscoring the pivotal role of Agrobacterium XYY in mediating plant-microbe interactions. Furthermore, analysis of rhizosphere soil physicochemical properties demonstrated that XYY inoculation increased nutrient content and key enzyme activities in the rhizosphere of peach seedlings, fostering a more fertile rhizosphere environment conducive to vigorous plant growth. This finding is consistent with previous reports: for instance, a composite inoculant containing Pseudomonas putida and Bacillus flexus significantly enhanced soil organic carbon, available nitrogen, phosphorus, potassium, and multiple enzyme activities (Zhang et al., 2025a); additionally, under a 25% reduction in chemical fertilizer input, various Bacillus species effectively mobilized soil phosphorus and potassium. In this study, we hypothesize that metabolic products of strain XYY may stimulate soil sucrase activity, and the secretion of acidic metabolites could enhance acid phosphatase activity—consistent with the slight decrease in soil pH observed in our experiment (Rawat et al., 2021, Nehls and Plassard, 2018). In conclusion, Agrobacterium XYY not only directly promotes the growth of peach seedlings and enhances their physiological status, but also indirectly improves plant growth conditions by stimulating soil enzyme activity, accelerating nutrient cycling and transformation, and increasing the fertility potential of the rhizosphere soil. This finding provides a scientific foundation for its development as a potential microbial fertilizer, supporting the goal of ”reducing inputs while enhancing efficiency” in orchard management systems. 4. 3 Agrobacterium XYY modulates root metabolic pathway-related genes to promote the growth of peach seedlings. This study through transcriptomic analysis, revealed that inoculation with Agrobacterium XYY significantly altered the gene expression profile of peach seedlings, resulting in the identification of 3,474 differentially expressed genes. Pathway enrichment analysis of these genes systematically demonstrated that Agrobacterium XYY promotes plant growth via a ”metabolic thrift” molecular mechanism, which involves the coordination of hormone signaling networks, enhancement of photosynthetic capacity, and optimization of carbon resource allocation. In the up-regulated pathways, the hormone signal transduction pathways related to plant growth, such as auxin, cytokinin and gibberellin signaling pathways, were significantly enriched. Auxin, a key regulator of root development, exhibited increased expression of critical gene families such as ARF and SAUR . The SAUR family acts as a direct downstream effector in the auxin signaling cascade and plays a pivotal role in promoting cell elongation. Previous studies have demonstrated that PpSAUR5 in peach promotes plant growth (Li et al., 2024), while activated ARF transcription factors (e.g., ARF7 and ARF19 ) can directly bind to the promoter regions of SAUR genes to initiate their transcription, thereby regulating directional growth responses (Wang et al., 2020). This finding is highly consistent with the observed increase in root surface area in XYY-treated seedlings. Additionally, upregulation of the cytokinin receptor gene CRE1 and its downstream response regulator B-ARR indicates enhanced cell division signaling (Heyl and Schmülling, 2003). In the gibberellin pathway, elevated expression of GID1 receptors facilitates the degradation of DELLA repressor proteins, thereby releasing their inhibitory effect on growth and activating downstream growth-related gene programs (Fukazawa et al., 2021, Zhang et al.). The concurrent enrichment of these three major growth-promoting hormonal pathways suggests that Agrobacterium XYY may establish a favorable hormonal environment by either directly producing phytohormones or stimulating endogenous biosynthesis pathways in the host plant. Concurrently, photosynthesis-related pathways were significantly upregulated. Enhanced expression of gene families such as Psa (photosystem I) and Psb (photosystem II) reflects improved light harvesting efficiency and electron transport capacity. This molecular evidence supports the observed increases in photosynthetic pigment content and SPAD values in leaves of XYY-inoculated seedlings, providing a robust foundation of energy and biomass accumulation for vigorous growth. Moreover, the upregulation of core metabolic pathways indicates a strategic reallocation of resources toward growth and development. Enhanced starch and sucrose metabolism suggests accelerated conversion and translocation of photosynthates, supplying essential carbon skeletons and energy to rapidly growing tissues (Lastdrager et al., 2014). Upregulation of the pentose phosphate pathway implies increased production of NADPH, a crucial cofactor for reductive biosynthesis (Zhou et al., 2025b). Simultaneously, active amino sugar and nucleotide sugar metabolism provides key precursors for cell wall biosynthesis and remodeling. Increased zeatin biosynthesis further corroborates heightened cell division activity. The coordinated activation of these pathways indicates that Agrobacterium XYY not only triggers growth-promoting hormonal signals but also enhances the mobilization and efficient utilization of carbon resources, ultimately manifesting in superior growth performance, including increased plant height and root length. It is worth noting that this study also found that the expression of multiple defense-related pathways’ genes showed a downward trend. Key regulatory genes such as ERF1/2 in the ethylene pathway and JAZ and MYC2 in the jasmonate pathway were significantly downregulated, suggesting reduced levels of these defense hormones (Zhou et al., 2025a). In addition, the monoterpene and glutathione metabolic pathways were inhibited, which are related to biotic stress resistance and antioxidant defense (Xi-Hua et al., 2010) . This collectively indicates that the levels of both biotic and abiotic stress in the rhizosphere of seedlings inoculated with XYY are relatively low. The significant correlation between differentially expressed genes and the growth phenotypes of peach seedlings proved the direct response of plants to the changes at the transcriptome level. The inoculation of Agrobacterium rhizogenes XYY directly promoted the growth and development of plants by activating and inhibiting the expression of related genes. In conclusion, the inoculation of Agrobacterium XYY induced significant transcriptome reprogramming in peach seedlings. It efficiently reallocated resources, on the one hand, by activating growth hormone signaling and enhancing photosynthesis and other growth metabolic pathways; on the other hand, by downregulating high-energy-consuming defense pathways to save resource utilization. This ”metabolic thrift” type of expression regulation reallocated carbon, nitrogen, and energy resources originally used for defense to growth metabolism, ultimately promoting the growth and development of peach seedlings in a coordinated manner. 4. 4 Inoculation with Agrobacterium XYY significantly reshaped the structure of the rhizosphere soil microbial community. The rhizosphere soil microbial community is a central component of the plant-soil system, and its structural and functional dynamics are closely linked to plant growth, development, and soil ecological stability (Liu et al., 2025). PGPR strains isolated from local environments often exhibit enhanced colonization efficiency and greater soil modification potential in native soils, offering more pronounced benefits for crop growth (Jiang et al., 2023). This study demonstrates that inoculation of locally derived Agrobacterium XYY in peach seedlings, combined with soil metagenomic sequencing, significantly reshaped the structure and altered the function of the rhizosphere microbial community, providing critical insights into the mechanism of Agrobacterium-mediated rhizosphere microecological regulation. At the community structure level, Agrobacterium XYY inoculation exerted a significant influence across multiple taxonomic levels. Previous studies have demonstrated that Agrobacterium inoculation can promote the proliferation of closely related rhizosphere microorganisms through mechanisms such as niche competition and signal molecule secretion, thereby facilitating the assembly of beneficial microbial communities (Thamvithayakorn et al., 2024). At the phylum level, the relative abundance of Actinobacteria in the inoculated treatment group increased significantly by 10.3% compared to the control group (CK) (p < 0.01). Actinobacteria represent a key functional group in soil ecosystems, playing essential roles in organic matter decomposition and carbon and nitrogen cycling. Moreover, they produce diverse secondary metabolites, including antibiotics and siderophores, which contribute to pathogen suppression and enhanced nutrient uptake in plants (Palaniyandi et al., 2013). Notably, Actinobacteria isolated from sugarcane have been shown to synthesize bioactive compounds with antimicrobial properties (Wang et al., 2021). At the genus level, Agrobacterium XYY application led to significant enrichment of specific functional genera. The abundance of g__Agrobacterium increased by 32.3% (p < 0.01), indicating successful colonization of the inoculated strain in the rhizosphere. Concurrently, the abundance of g__Arthrobacter rose by 22.3% relative to the control group (p < 0.01). Arthrobacter species are recognized for their robust stress tolerance and environmental adaptability, with capabilities in degrading organic pollutants and promoting plant growth under adverse soil conditions (Barnawal et al., 2014). For example, Arthrobacter DUT_AHX, isolated from nitrobenzene-contaminated sludge, can utilize nitrobenzene as its sole carbon source under high-salt conditions (Ai et al., 2009). LEfSe analysis identified the primary taxa driving community differentiation, all of which were enriched in the treatment group and contributed most significantly to structural divergence. The increase in Actinomycetes aligns with the observed rise in Actinomycetota at the phylum level. Micrococcales have been previously associated with positive effects on plant growth; for example, Xu et al. reported a significant positive correlation between Micrococcales abundance and soil nitrate nitrogen content under green manure application, highlighting their role in nitrogen cycling (Xu et al., 2023). And other studies have documented their involvement in bioremediation of contaminated soils (Giovanella et al., 2021). Furthermore, certain Hyphomicrobiales species exhibit strong capabilities in degrading chloromethane and participating in nitrogen metabolism, contributing to microbial adaptation and remediation in polluted environments (Wang et al., 2025b, Bartoli et al., 2025). Rhizobiaceae, as a key player in organic matter decomposition and transformation, has also been linked to enhanced soil carbon sequestration—for instance, its abundance increases following the return of peach branch residues to soil (Liu et al., 2024). The selective enrichment of these beneficial taxa underscores the targeted regulatory effect of Agrobacterium XYY on dominant functional groups(Segata et al., 2011), reshaping the core composition of the rhizosphere microbiome and fostering a more favorable micro-ecological environment for peach seedling development. Correlation analysis between microbial community structure and environmental factors revealed a significant relationship between soil physicochemical properties and microbial composition. The inoculation of Agrobacterium XYY enhanced the activities of soil urease, sucrase, and acid phosphatase. Urease and sucrase activities showed significant positive correlations with soil organic carbon, total nitrogen, and labile humus content, indicating their potential as sensitive indicators of soil fertility(Kravkaz Kuşcu et al., 2018). Meanwhile, acid phosphatase (ACP) activity was closely associated with soil phosphorus transformation efficiency and the expression of microbial functional genes involved in phosphate metabolism(Yang et al., 2025a), thereby promoting the cycling of carbon, nitrogen, and phosphorus in the soil and increasing the levels of alkali-hydrolyzable nitrogen and available phosphorus. The enrichment of Arthrobacter exhibited a significant positive correlation (P < 0.05) with these improvements. Arthrobacter further promoted nutrient mobilization and enzyme secretion in the modified rhizosphere environment, and together with Agrobacterium XYY, synergistically enhanced soil nutrient availability and enzymatic activities. These findings underscore the close linkage between soil physicochemical characteristics and microbial community dynamics, clarify the influence of environmental factors in shaping microbial assemblages, and provide a theoretical foundation for the rational design of synthetic microbial consortia centered on Agrobacterium XYY in future applications. At the functional level, Agrobacterium XYY inoculation significantly enriched KEGG pathways related to purine metabolism (map00230), pyrimidine metabolism (map00240), amino acid biosynthesis (map01230), and nucleotide metabolism (map01232) in the rhizosphere microbiome. Purine and pyrimidine metabolism are fundamental to nucleic acid synthesis and energy transfer in microorganisms; their enrichment indicates heightened microbial proliferation and metabolic activity in the treated rhizosphere (Zheng et al., 2024, Zhang et al., 2024). Enhanced amino acid biosynthesis not only supports microbial growth by supplying essential nitrogen but also facilitates plant nitrogen nutrition through microbe-plant exchange mechanisms (Zheng et al., 2024). The coordinated upregulation of these core metabolic pathways indicates that Agrobacterium XYY does not merely alter community composition but actively enhances the rhizosphere microbiome’s capacity for nutrient cycling and resource provisioning. In summary, Agrobacterium XYY inoculation effectively modulates the rhizosphere microbial community by enriching beneficial taxa and key differential groups, while redirecting microbial metabolic functions toward pathways involved in material cycling and nutrient synthesis. This dual structural and functional optimization contributes to the establishment of a more resilient and productive rhizosphere microecosystem. These findings provide novel insights into the interactions between Agrobacterium and the rhizosphere microbiome and offer theoretical support and practical guidance for developing microbial-based alternatives to chemical fertilizers, improving soil health, and promoting sustainable fruit tree cultivation. Future studies should extend these observations to field trials, examining the relationship between microbial community shifts following XYY inoculation and peach tree growth performance as well as soil physicochemical properties. 5. Conclusions This study for the first time elucidates the synergistic plant growth-promoting mechanism of Agrobacterium within the ”microbe–soil–plant” system in peach, and pioneers the integration of transcriptomic and metagenomic approaches to dissect its underlying molecular pathways. The strain is found to possess multiple functional traits, including nitrogen fixation, phosphorus and potassium solubilization, indole-3-acetic acid (IAA) production, and siderophore synthesis. Its robust colonization capacity is further confirmed through the construction of GFP-labeled strains. Pot experiments demonstrate that inoculation with Agrobacterium XYY significantly enhances plant height, root length, root surface area, leaf nitrogen, phosphorus, and potassium concentrations, as well as photosynthetic performance in peach seedlings. Additionally, positive effects are observed on soil nutrient levels and related enzymatic activities. Transcriptomic analysis reveals up regulation of pathways associated with growth and metabolism, while high energy-consuming defense-related pathways such as those involved in jasmonic acid signaling and monoterpenoid biosynthesis are down regulated, indicating a ”metabolic thrift” effect conferred by the growth-promoting strain. Furthermore, Agrobacterium XYY reshapes the structure and function of the soil microbial community by significantly enriching beneficial taxa such as Actinomycetota and Arthrobacter, and by activating metabolic pathways linked to rhizosphere microorganisms, thereby enhancing community activity and functional potential (Fig. 7 presents the graphical workflow of the study). Although this study clarifies the growth-promoting efficacy of Agrobacterium XYY under controlled pot conditions, the complexity of field environments poses greater challenges for practical application. Therefore, future research focuses on leveraging this strain as a core component, integrating isolated Arthrobacter strains to construct synthetic microbial communities. By combining metabolomics with field-scale orchard trials, we aim to systematically evaluate the strain’s performance in reducing fertilizer inputs while improving agricultural efficiency, thus enabling a substantive transition of its growth-promoting effects from laboratory (”pot”) to real-world (”field”) settings. Acknowledgements This experiment was supported by the China Agriculture Research System (No. CARS-30-2-02). Conflicts of Interest The authors declare no conflicts of interes. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. reference AI, H.-X., ZHOU, J.-T. & Lü, H. 2009. A novel salt-tolerant Micrococcus sp. DUT_AHX capable of degrading nitrobenzene. Journal of Central South University of Technology, 16 , 230–235.ALLENBY, N. E. E., APOS, CONNOR, N., PRáGAI, Z., CARTER, N. M., MIETHKE, M., ENGELMANN, S., HECKER, M., WIPAT, A., WARD, A. C. & HARWOOD, C. R. 2004. 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Glucose-1-phosphate promotes compartmentalization of glycogen with the pentose phosphate pathway in CD8+ memory T cells. Molecular Cell, 85 , 2535–2549.e10. Supplementary Material File (figure.docx) Download 3.38 MB Information & Authors Information Version history V1 Version 1 11 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords development growth-promoting mechanism pgpr rhizosphere microbiome transcriptome transcriptomics Authors Affiliations Guangyuan Liu Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Yanyan Li Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Tianyu Dong Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Jian Guo Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Yuansong Xiao Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Huaifeng Gao Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Yangyang Gao Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Qiuju Chen Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Jingjing Luo Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Zixuan Li Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Huitian Wei Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Futian Peng [email protected] Shandong Agricultural University College of Horticulture Science and Engineering View all articles by this author Metrics & Citations Metrics Article Usage 246 views 125 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Guangyuan Liu, Yanyan Li, Tianyu Dong, et al. Integrated analysis of the rhizosphere microbiome and transcriptome reveals the growth-promoting mechanism of plant growth-promoting rhizobacterium XYY in peach plants. Authorea . 11 December 2025. DOI: https://doi.org/10.22541/au.176542759.92935639/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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