Abstract Background Rhizosphere microorganisms can improve soil quality, promote plant growth, and enhance plant health. Despite the isolation of numerous plant growth-promoting rhizobacteria (PGPR) strains, research on how PGPR enhances walnut growth remains limited. Results In this study, the metagenomic sequencing of the rhizosphere soil in 8 major walnut-producing areas in China was conducted to identify 150 shared core amplicon sequence variants. Then, we isolated a strain of Bacillus cereus OTU8977 from the walnut rhizosphere soil and evaluated its potential plant growth-promoting functions. B. cereus OTU8977 can optimize the walnut rhizosphere microecology and promote its growth through its considerable potential in nitrogen fixation, phosphorus solubilization, and potassium dissolution. Transcriptomic analysis of walnut roots revealed that B. cereus OTU8977 promotes the growth of walnuts by enhancing phenylpropanoid biosynthesis and carbohydrate metabolic processes. Conclusions This study identified a strain of Bacillus cereus with multiple plant growth-promoting functions, which significantly enhanced walnut growth. Moreover, the study further elucidated the mechanisms underlying its growth-promoting effects, providing a theoretical foundation for the development of walnut-specific microbial fertilizers. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-025-06812-3. Keywords: Walnut, Metagenomic, PGPR, Bacillus cereus OTU8977, Phenylpropanoid biosynthesis, Carbohydrate metabolic process Background Walnut (Juglans regia L.) is an important woody oil tree species, and its kernel is rich in unsaturated fatty acids, minerals, vitamins, and proteins, providing various health benefits [[46]1, [47]2]. Walnut is widely grown commercially in diverse temperate regions of China [[48]3]. In recent years, due to crude orchard management, the soil environment in walnut orchards has deteriorated, affecting walnut yield, fruit quality, and economic benefits [[49]4]. Recent studies have shown that rhizosphere microorganisms have the potential to improve soil quality and promote plant growth and health [[50]5–[51]7]. This field of research on microbiome factors has already provided new strategies for sustainable cultivation and the management of postharvest pathogens [[52]8–[53]10]. Metagenomic studies have extensively characterized the taxonomic, genomic, and functional profiles of rhizosphere microbial communities [[54]11, [55]12]. The Bai Yang team integrated the cultivable bacterial genomes and metagenomic data from the rhizosphere of various crops to construct the world’s first database of crop rhizosphere bacterial and viral genomes, significantly expanding the number of publicly available crop rhizosphere bacterial genomes [[56]13]. Global metagenomic sequencing of Citrus reticulata rhizosphere revealed that Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia were the core members of the rhizosphere microbiome [[57]14]. Metagenomic shotgun sequencing has unveiled the diversity and metabolic potential of microbial communities on the surface of apple fruits, providing crucial insights into the relationship between the apple fruit microbiota and fruit quality as well as disease resistance [[58]15]. The latest research showed that the core microbiota actively participated in multiple nitrogen metabolic processes and demonstrated a significant correlation with the potential for rhizosphere nitrogen cycling, thereby expanding the traditional plant-centered resource acquisition strategies [[59]16]. Meanwhile, with the development of multi-omics and bioinformatics, the screening and utilization of suitable beneficial PGPR from the core microbiota have become attractive strategies for improving soil environment and promoting plant growth. PGPR colonize plant roots or rhizosphere soil, possessing the ability to dissolve phosphate, nitrogen fixation, potassium dissolution, secretes plant hormones and various metabolites to cope with various abiotic and biotic stresses [[60]17–[61]19]. Numerous studies have reported the roles of Bacillus, Pseudomonas, and other strains as PGPR in alleviating plant biotic and abiotic stress. The plant growth-promoting bacterium Bacillus subtilis expressing a secreted microbial pattern flg22, OKB105 (flg22), enhanced plant growth and plant resistance against two distinct pathogens, Pseudomonas syringae DC3000 ΔhopQ1-1 and Phytophthora parasitica [[62]20]. The PGPR Pseudomonas strain CM11 leads to primary root arrest by activating the PLETHORA 3,5,7-controlled lateral root pathway [[63]21]. The Streptomyces lasalocidi strain JCM 3373^T was identified and secreted indole-3‐carboxaldehyde (ICA1d) to alleviate salt stress and improve soybean root architecture [[64]22]. Nevertheless, there have been limited researches on PGPR improving walnut growth, and the underlying mechanisms remain unclear. Therefore, identifying novel PGPR and elucidating their molecular and physiological effects on walnut growth is essential. Here, we conducted metagenomic sequencing on the rhizosphere soil from eight major walnut-producing areas in China and identified 150 common core Amplicon Sequence Variants (ASVs). Based on correlation analyses between the core microbial community and soil physicochemical properties, as well as functional assessments of isolated strains, we discovered a novel plant growth-promoting rhizobacterium (PGPR), B. cereus OTU8977, which enhances soil physicochemical properties, optimizes microbial community structure, and boosts walnut growth. Additionally, B. cereus OTU8977 regulates gene expression in pathways related to phenylpropanoid biosynthesis and carbohydrate metabolism. Our current findings lay the foundation for developing new microbial inoculants, thereby improving walnut growth and yield. Results Soil physicochemical properties among 8 walnut locations Walnut rhizosphere soil samples were collected from eight representative locations in major walnut-cultivation provinces in northern China to explore differences in soil physicochemical properties and microbial composition characteristics of the rhizosphere microbiome. The soil physicochemical properties vary among these eight locations, with soil pH values ranging from 6.01 (SDTA) to 8.28 (HNJY) and with significant differences in N, P, and K contents (Fig. [65]1). Among these eight locations, the soil available N, P, and K contents in HBXT and LNDL are significantly higher than those in other locations. And there is also a significant difference in soil organic matter content, with the highest being 48.96 g/Kg in HBXT and the lowest being 2.03 g/Kg in XJA. In addition, compared to the lack of significant differences in total P and K contents among the locations, total N content showed significant differences. Fig. 1. [66]Fig. 1 [67]Open in a new tab Physicochemical properties of rhizosphere soil at sampling points. pH, potential of hydrogen; SOM, soil organic matter (g·kg^− 1); TN, TP, and TK indicate total nitrogen, total phosphorus, and total potassium, respectively (g·kg^− 1); AN, AP, and AK indicate available nitrogen, available phosphorus, and available potassium, respectively (mg·kg^− 1). Bars with error bars represent mean ± standard deviation (SD), and different letters indicate significant differences at P < 0.05 The community structure and functional profiles of the walnut rhizosphere Microbiome To examine the microbial community structure and functional profiles of the walnut rhizosphere microbiome, we employed metagenomic sequencing methods. After sequencing, the raw reads obtained totaled 1,892,550,692. Following the removal of low-quality data, the resulting high-quality reads (clean reads) suitable for subsequent accurate analysis amounted to 1,786,866,874. On average, each sample contained 74,452,786 clean reads, yielding an effective data recovery rate of 94.42% (Supplementary Table [68]4). To distinguish the microbial diversity among the eight locations, we first determined the alpha diversity indices, such as the Shannon-Wiener index, Simpson index, and Chao index, and beta diversity metrics including the principal component analysis (PCA) based on the metagenomic data. Our results indicated that the alpha microbial diversity is generally consistent across 8 regions, with no significant differences (Fig. [69]2A). The variation explained by PCA showed that PC1 explained 26.7% of the variation and PC2 explained 16.5% of the variation, indicating low variation in rhizosphere microbial community structure among the eight walnut locations (Fig. [70]2B). These results indicate that the differences in rhizosphere microbial diversity among 8 walnut locations are not significant and may have similar microbial composition and structures. Fig. 2. [71]Fig. 2 [72]Open in a new tab The community structure and functional characteristics of the walnut rhizosphere microbiome. (A) PCA based on metagenomic data. Bars with error bars represent mean ± SD. (B) Comparison of Beta diversity among rhizosphere samples among different sampling sites. (C, D, E) Distribution of rhizosphere samples at the phylum level (C), at the genus level (D), and at the species level (E). (F) Venn diagram of ASV in soil from 8 sampling points. (G) Venn diagram of functional genes among 8 sampling locations. (H) KEGG enrichment analysis of the shared genes among 8 sampling sites Bacterial community composition of the metagenomes was profiled by Kraken2, and a total of 37 phyla, 73 classes, 159 orders, 353 families, 1200 genera, and 4114 species were identified across the 24 samples. The abundance distribution of the bacterial phyla, genera, and species were consistent across locations. Firmicutes, Actinobacteria, Proteobacteria, and Planctomycetes were the most prevalent bacterial phyla, representing 95.25% of the total bacterial community (Fig. [73]2C). The dominant bacterial genera found in the walnut rhizosphere included Nocardioides, Streptomyces, Bradyrhizobium, and Pseudomonas (Fig. [74]2D). Starkeya novella, Streptomyces fradiae, Bradyrhizobium sp. CCBAU 051011, and Pseudomonas resinovorans were the most abundant bacterial species (Fig. [75]2E). Of these species, 150 ASVs shared across the eight locations, with the XJA soil possessing the highest number of unique species (213 ASVs), and the lowest number of unique species in HBXT (168 ASVs) (Fig. [76]2F). Under different kmer lengths, a total of 8,454,237 contigs were assembled from the reads, with a total length of 6,932,831,751 nt. On average, each sample had 352,260 contigs, with an average length of 288,867,990 nt, ranging from 48,664 nt to 456,029 nt (Supplementary Table [77]5). Meanwhile, to understand the microbial functional profiles, the metagenome reads were annotated through comparison with the KEGG database. The functional gene composition differed in the soils from 8 different locations, with a total of 3173 core functional genes shared among these locations (Fig. [78]2G). Of those genes, 79 genes were related to nitrogen metabolism like nrfC and glnA, and 113 genes were related to phosphorus metabolism, such as phoB and phoR (Supplementary Table [79]6). KEGG annotations of these core genes showed that functional genes were mainly involved in various metabolic pathways, ribosome, and oxidative phosphorylation (Fig. [80]2H). B. cereus OTU8977 is a potential plant growth-promoting rhizobacteria To explore the correlation between rhizosphere microbiome and soil physicochemical properties, we linked the abundance of shared species with soil physicochemical properties using the Spearman’s correlation analysis. The bacterial genera were identified as significantly correlated with the soil properties based on the threshold of (|R| > 0.70, P < 0.05) (Supplementary Table [81]7). Among the bacterial genera, Pseudorhodoplanes, Agrobacterium, Bacillus, Terricaulis were identified as key taxa highly correlated with soil properties (Fig. [82]3A). Pseudorhodoplanes was positively correlated with available nitrogen (R = 0.86, P < 0.01), and Agrobacterium was positively correlated with SOM (R = 0.74, P < 0.05), TN (R = 0.76, P < 0.05), AP (R = 0.86, P < 0.01), AK (R = 0.71, P < 0.05). In addition, the AP content was significantly correlated with the enrichment of Bacillus and Terricaulis (Fig. [83]3A). Fig. 3. [84]Fig. 3 [85]Open in a new tab Isolation and identification of rhizosphere-promoting bacteria related to soil properties. (A) Correlation between rhizosphere microbiome and soil physicochemical properties. (B) Phylogenetic tree of 30 isolated strains and comparative analysis in phosphorus solubilization ability, nitrogen fixation ability, potassium release ability, and autotoxin degradation ability. PSA, phosphorus solubilization ability; NFA, nitrogen fixation ability; PRA, potassium release ability; ADA, autotoxin degradation ability. The asterisk indicates a significant difference between the two groups: *p < 0.05; **p < 0.01. (C) Phylogenetic analysis of the strain OTU8977 with other Bacillus species strains Since these bacterial genera were highly enriched in the walnut rhizosphere communities and associated with soil physicochemical properties, we further explored the compositional trends of each individual Operational Taxonomic Unit(OTU)within these genera and identified the PGPR. The rhizobacteria from 8 walnut locations were isolated to identify the nitrogen fixation ability, phosphorus solubilization ability, potassium release ability, and autotoxin degradation ability. A total of 30 rhizobacteria strains were isolated, and 21 strains were assigned as Bacillus (Supplementary Fig. [86]1, Supplementary Table [87]8). Among the isolated strains, OTU8905 was the most abundant strain in the rhizosphere soil, while its growth-promoting function was inferior to that of OTU8977. OTU8977 had the highest plant growth-promoting effects, especially the phosphorus solubilization ability. Based on the molybdenum-antimony colorimetric method, the effective phosphorus content of OTU8977 during liquid culture was measured to be 205.29 mg/L, significantly higher than that of other strains (Fig. [88]3B). Phylogenetic analysis of the strain showed a close relationship with Bacillus cereus [89]KU179332 (Fig. [90]3C). B. cereus OTU8977 improves soil physicochemical properties and promotes walnut growth To experimentally verify whether B. cereus OTU8977 is a key PGPR improving walnut growth, greenhouse and field experiments were conducted. For the greenhouse experiments, we grew walnuts in sterilized soil with B. cereus OTU8977 as the treatment or without treatment as the control. After 90 days of inoculation, the growth and root growth of walnut plants inoculated with B. cereus OTU8977 were significantly better than those of the control group (Fig. [91]4A). Compared with the control group, the plant height and root length of the group inoculated with OTU8977 increased by 17.09% and 14.96%, respectively. Additionally, the fresh root weight, dry root weight, fresh aboveground weight, and dry aboveground weight of the walnut plants in the treatment group increased by 33.48%, 61.68%, 20.87%, and 27.27%, respectively (Fig. [92]4B). Fig. 4. [93]Fig. 4 [94]Open in a new tab B. cereus OTU8977 improved the growth performance of walnut seedlings. (A) Phenotype characteristics of walnut plants under control and B. cereus OTU8977 treatment conditions. (B) B. cereus OTU8977 inoculation significantly improved growth performance of walnut seedlings. (C) B. cereus OTU8977 inoculation significantly improved physiology and biochemical index of walnut trees. A, net photosynthetic rate; E, transpiration rate; Ci, intercellular CO[2] concentration; gsw, stomatal conductance. Bars with error bars represent mean ± SD. The asterisk indicates a significant difference between the two groups: *p < 0.05; **p < 0.01 Next, we explored whether the application of B. cereus OTU8977 is also effective treatments in the field (Supplementary Fig. [95]2). The transpiration rate (E), net photosynthetic rate (A), intercellular CO[2]concentration (Ci), and stomatal conductance (gsw) of walnut plants were measured. Compared with walnuts that were not inoculated with the OTU8977 strain, inoculation with this strain resulted in a 40.70% increase in A, a 36.87% increase in E, a 63.99% increase in Ci, and a significant 162.83% increase in gsw (Fig. [96]4C). These results indicate that B. cereus OTU8977 promotes the growth of walnuts. In order to clarify the mechanisms underlying B. cereus OTU8977 promotes walnut growth, we first tested the physicochemical properties of soil inoculated with B. cereus OTU8977 and the control. The results showed that after inoculation with the OTU8977 strain, the TN content in walnut roots increased by 21.83%, the TP content increased by 14.79%, and the TK content increased by 23.63%. Meanwhile, the AN, AP, and AK contents in the rhizosphere soil increased by 15.52%, 13.65%, and 23.23%, respectively. Inoculating B. cereus OTU8977 optimizes microbial communities To further verify the effects of B. cereus OTU8977 on soil bacterial communities, we undertook 16 S rRNA gene sequencing of treated and control samples. A total of 759,325 sequences clustered into 15,764 ASVs across all six soil samples after quality filtering (Supplementary Table [97]9). The PCA showed that PC1 explained 84.76% of the variation and the bacteria in the soils inoculated with B. cereus OTU8977 were distinctly separated from those in the control (Fig. [98]5A). Then, the observed OTUs, the Chao1 index and the Shannon index at the ASV level were estimated, indicating that inoculation with B. cereus OTU8977 significantly increased bacterial ASV richness compared to the control as shown in Fig. [99]5B. For the bacterial community, 100 phyla, 256 classes, 639 orders, 931 families, and 1696 genera were detected from 6 soil samples (Supplementary Table [100]10). Among them, Proteobacteria, Acidobacteriota, Actinobacteriota, and Gemmatimonadota were the most dominant phyla (Fig. [101]5C), and RB41, Vicinamibacteraceae, Rokubacteriales, and Azovibrio were the most dominant genera (Fig. [102]5D). Although the enriched microbial communities have similar species, the relative abundance of each bacterial group varies. At the level of phyla, the relative abundance of Acidobacteriota in the inoculating B. cereus OTU8977 soils was 20.42%, which was significantly higher than that in control soil (17.94%) (Fig. [103]5E, Supplementary Table [104]11). For genera, the relative abundance of the RB41, Vicinamibacteraceae, and Rokubacteriales in treatment group was higher than that of the control group. Compared to the control group, the biosynthesis of various essential amino acids for plants, such as phenylalanine and tryptophan, was significantly higher in the inoculated group. Additionally, the metabolism of non-essential amino acids like glutamic acid and aspartic acid was also significantly higher. Furthermore, we observed a significant increase in the pentose phosphate pathway (Fig. [105]5F). These results indicate that inoculation with B. cereus OTU8977 exerts potential effects on plant growth and development, as well as metabolic regulation. The above results confirmed that inoculation with B. cereus OTU8977 optimizes the microbial community structure. Fig. 5. [106]Fig. 5 [107]Open in a new tab The impact of B. cereus OTU8977 inoculation on rhizosphere microbiota of walnut trees. (A) PCA of rhizosphere microbiota between the control and B. cereus OTU8977 inoculation groups. (B) The inoculation of B. cereus OTU8977 significantly increased the ASV richness of the bacterial community. Bars with error bars represent mean ± SD. The asterisk indicates a significant difference between the two groups: *p < 0.05. (C, D) The top 15 most abundant bacterial compositions at the phylum level (C) and genus level (D). (E) Analysis of microbial community differences in samples inoculated with or without the strain OTU8977. (F) Functional analysis of microbial communities in the control and treatment groups using PICRUST2 software Transcriptome of walnut root inoculated with B. cereus OTU8977 To investigate the responses of walnut root to B. cereus OTU8977, we conducted a comparative analysis of walnut roots inoculated with B. cereus OTU8977 (T) and the control group (CK) using RNA-seq data. A total of 40.30 Gb clean reads were obtained, with an average of 6.72 Gb per sample (Supplementary Table [108]12). The PCA revealed that PC1 represented 57.9% of the variation and PC2 explained 26.5% of the variation, indicating a significant difference between two treatment groups (Fig. [109]6A). Fig. 6. [110]Fig. 6 [111]Open in a new tab Transcriptome profiling of walnut root inoculated with B. cereus OTU8977. (A) PCA of gene expressions between the control and B. cereus OTU8977 inoculation groups. (B) Volcano plot exhibits gene expression differences between the control and treatment groups. (C) KEGG pathway enrichment analysis of the upregulated genes in the B. cereus OTU8977 inoculation group. (D) GO functional enrichment analysis of the upregulated genes in the B. cereus OTU8977 inoculation group. (E) Heatmap of the differentially expressed genes involved in Phenylpropanoid biosynthesis and Carbohydrate metabolic process between the control and the inoculation groups. Colors from red to blue indicate gene expression from high to low. (F) Relative expression levels of the upregulated genes related to phenylpropanoid biosynthesis and carbohydrate metabolic process pathways. Bars with error bars represent mean ± SD. The asterisk indicates a significant difference between the two groups: *p < 0.05; **p < 0.01; ***p < 0.001. PME12, probable pectinesterase/pectinesterase inhibitor 12; ALDH2C4, aldehyde dehydrogenase family 2 member C4-like; PME68, probable pectinesterase 68; GAUT12, probable galacturonosyltransferase 12; RFS1, probable galactinol-sucrose galactosyltransferase 1; SHT, spermidine hydroxycinnamoyl transferase-like; BGLU, beta-glucosidase BoGH3B-like; CesA7, cellulose synthase A catalytic subunit 7 [UDP-forming]-like; GULO2, L-gulonolactone oxidase 2-like; ADG2, glucose-1-phosphate adenylyltransferase large subunit, chloroplastic/amyloplastic-like; OFUT27, O-fucosyltransferase 27-like; BGLU40, beta-glucosidase 40-like; GPII1, cationic peroxidase 1-like; GPII2, cationic peroxidase 2-like; CTL, endochitinase-like After mapping the clean reads from walnut to the walnut reference genome, we identified 21,798 differentially expressed genes (DEGs) in T versus CK, including 2767 upregulated genes and 1964 downregulated genes (Fig. [112]6B, Supplementary Table [113]13). KEGG enrichment analysis results showed that the upregulated DEGs in the B. cereus OTU8977 treatment group were mainly enriched in metabolic pathways such as Phenylpropanoid biosynthesis, Flavonoid biosynthesis, and Pentose and glucuronate interconversion (Fig. [114]6C, Supplementary Table [115]14). In addition, Gene ontology (GO) functional enrichment analysis showed that the upregulated DEGs were mainly enriched in cell wall organization or biogenesis, cell cycle, polysaccharide metabolic process, flavonoid metabolic process, and carbohydrate metabolic process (Fig. [116]6D, Supplementary Table [117]15). We also found that genes related to phenylpropanoid biosynthesis and carbohydrate metabolic were significantly enriched in walnut roots inoculated with B. cereus OTU8977 (Fig. [118]6E, Supplementary Table [119]16). We conducted qRT-PCR to investigate the expression profiles of genes related to these pathways under inoculated and non-inoculated conditions. The results showed that, compared to the control, the relative expression levels of genes associated with these pathways were significantly upregulated following inoculation with B. cereus OTU8977 (Fig. [120]6F). Discussion The breakthrough progress of high-throughput sequencing technology is profoundly reshaping the field of microbial community research. Through means such as metagenomics and 16 S rRNA amplicon sequencing, it has not only achieved high-throughput analysis of the composition of soil microbial species, the distribution of functional genes, and community dynamics, but also broken through the limitations of traditional culture-dependent methods, significantly expanding the boundaries of detectable microbial groups. Studies have shown that microbial diversity analysis based on deep sequencing has revealed the key functions of soil microorganisms in ecological processes such as carbon and nitrogen cycling, pollutant degradation, and plant health regulation. This provides theoretical support and technical pathways for sustainable agricultural development and the development and utilization of microbial resources [[121]23, [122]24]. In this study, metagenomic sequencing was conducted on the rhizosphere soil from eight major walnut-growing regions in China to investigate the structure and diversity of soil bacterial communities across different sampling sites. The results showed that the main walnut-growing regions have similar bacterial composition and structure, with bacterial communities mainly dominated by the phyla Proteobacteria, Actinobacteria, Firmicutes, and Planctomycetes, and no significant differences in bacterial diversity were observed overall. It was also found that in the northeastern Juglans mandshurica plantations in the Liaodong mountainous area, the dominant bacterial communities in the rhizosphere soil microbiota were Actinobacteria, Proteobacteria, and Acidobacteria [[123]23]. In addition, soybean genotypes can regulate the differences in rhizosphere microbial community structure, and each genotype has a unique rhizosphere microbial community composition [[124]25]. These research findings indicate that, although the rhizosphere microbiome typically fluctuates with environmental changes, certain components of the microbiome can stably assemble in the rhizosphere of specific plant genotypes. We speculate that this phenomenon is likely closely related to the dominant role of plant genetic factors. Changes in the physicochemical properties of soil can significantly affect the composition, abundance, and gene functions of soil bacterial communities, with many bacteria being highly correlated with specific soil factors [[125]26–[126]28]. Studies have found that the assembly of dominant microbial communities in the rhizosphere soil is mainly driven by factors such as soil moisture, inorganic nitrogen, pH, and salinity [[127]29]. There are significant correlations between pH value, soil moisture content, total carbon, total phosphorus, and electrical conductivity and microbial groups, and these indicators are considered key factors in predicting microbial abundance and diversity [[128]30]. Similarly, in this study, Spearman correlation analysis revealed significant positive correlations between Pseudorhodoplanes and AN, Bacillus and AP, and Agromyces and Cystobacter with pH. We speculate that these dominant bacteria may play important roles in enhancing soil fertility and promoting plant nutrient uptake. Investigating the bacterial communities in the rhizosphere of walnuts and their relationships with soil physicochemical properties not only provides a scientific basis for soil improvement but also offers technical support for the selection of beneficial microorganisms with plant growth-promoting functions. PGPR are widely present in the plant rhizosphere soil, which promotes plant growth by producing volatile and secondary metabolites, or by affecting phytohormone homeostasis and signaling transduction [[129]17, [130]19, [131]24, [132]31]. Currently, the rapid development of metagenomic sequencing and bioinformatics provides a convenient approach for the screening of PGPR. The analysis of root-associated microbiota in peanut-maize intercropping systems has suggested that siderophore-secreting Pseudomonas sp. 1502IPR-01 plays a vital role in iron nutrition [[133]32]. Four PGPRs were isolated and characterized to improve the salt tolerance of salt-tolerant rice 86. These strains, identified through metagenomic sequencing and transcriptome analysis, include RL-WG26 (Pseudomonas putida), RL-WG62 (Rossellomorea vietnamensis), RL-WG133 (Bacillus sp.), and RL-WG347 (Bacillus velezensis) [[134]33]. In this study, we isolated thousands of bacterial strains using traditional culture methods and conducted preliminary screening based on colony morphology, size, and color. Subsequently, we determined the taxonomic information of the strains through 16 S rRNA gene sequencing, ultimately identifying 30 strains. These strains cover multiple important bacterial genera, specifically including 22 strains of the family Bacillaceae (16 of which belong to the genus Bacillus), 5 strains of the genus Pseudomonas, 2 strains of the genus Priestia, and 1 strain of the genus Lysinibacillus. This study systematically screened and identified potential beneficial bacteria in the rhizosphere of walnuts and conducted in-depth assessments of their plant growth-promoting functions, providing a rich and high-quality microbial strain resource for the development of walnut-specific microbial fertilizers. The genus Bacillus, a soil-predominant bacterium that is commonly found in soil rhizosphere, has garnered interest in enhancing plant growth through the acquisition of nutrients and production of phytohormones [[135]34, [136]35]. Several species of the genus Bacillus like B. subtilis, B. velezensis, B. megaterium, B. circulans, B. coagulans, etc. are reported as PGPR [[137]36, [138]37]. B. subtilis strain GOT9 as PGPR enhanced tolerance against drought and salt stresses by upregulating the drought-inducible genes in Brassica campestris [[139]38]. The volatile compounds (VCs) emitted by B. velezensis strain SQR9 promoted the absorption of nitrate and ammonium via nitrogen transport and endogenous signaling pathways [[140]39]. Here, strain OTU8977 was identified as highly similar to Bacillus cereus [141]KU179332. This strain exhibited remarkable characteristics in key plant growth-promoting functions such as phosphate solubilization and degradation of autotoxic substances, demonstrating great potential as a plant growth-promoting rhizobacterium. We also conducted abundance analysis on the 30 isolated strains and found that eight strains, including Bacillus cereus, Bacillus pumilus, Bacillus altitudinis, and Bacillus mycoides, had relatively high abundance. Considering both the plant growth-promoting functions and abundance of the strains, we selected Bacillus cereus OTU8977 as the target strain. Recent studies on the rhizosphere of various plants have confirmed that the B. cereus as a PGPR promote plant growth and cope with biotic and abiotic stresses [[142]40–[143]42]. Consistent with these research findings, this study discovered that B. cereus OTU8977 significantly promotes the growth of walnuts by improving the physicochemical properties of the soil, optimizing the photosynthetic efficiency of walnuts, and enhancing the absorption of nitrogen, phosphorus, and potassium by walnut roots. These discoveries lay the foundation for further investigation into the role of PGPR in the growth-promoting mechanisms of walnuts. Although this study has preliminarily evaluated the potential of the strain for agricultural applications and no obvious negative impacts on plants or the environment have been observed, we are also aware of some limitations in the current study. First, we have not yet conducted an in-depth analysis of the strain’s genome to identify potential pathogenicity islands. Second, we have not yet carried out analyses to detect metabolites that may be toxic to mammals. Finally, we have not yet analyzed the antimicrobial resistance of the strain. The lack of these analyses is mainly due to the limitations of our current experimental conditions and technical capabilities. We plan to collaborate with specialized microbiological safety laboratories in future research to fill these gaps. The growth-promoting effects of PGPR on plants are closely related to their interactions with indigenous microorganisms. These interactions can enhance plant growth by improving the structure, diversity, and functionality of the rhizosphere microbial community [[144]43–[145]45]. Our previous studies have shown that inoculation with Bacillus cereus significantly promotes the growth of walnuts. Based on these findings, this study employs high-throughput sequencing technology to further evaluate the impact of Bacillus cereus on the bacterial community in the rhizosphere of walnuts. The study found that after inoculation with Bacillus licheniformis 2R5, the strain altered the soil bacterial community structure by increasing the abundance of plant-beneficial microorganisms (such as Nitrospira) [[146]46]. Inoculation of Bacillus cereus DW019 into potted cherry tomatoes significantly altered the bacterial diversity in the rhizosphere soil and enhanced species richness [[147]42]. Consistent with the aforementioned research findings, our study further revealed that inoculation with Bacillus cereus not only altered the relative abundance of dominant bacteria but also significantly enhanced the diversity of the rhizosphere bacterial community. We speculate that these changes may contribute to improving the availability of soil nutrients, thereby promoting plant growth. These discoveries provide new theoretical evidence for the application of Bacillus in agriculture and enhance our understanding of the mechanisms by which PGPR promote plant growth. During the interaction between plants and microorganisms, transcriptomic profiling has become a dynamic perspective to reveal the potential mechanisms by which PGPR promotes plant growth [[148]47, [149]48]. Transcriptomic profiling results revealed the tobacco-associated PGPR strain Paenibacillus polymyxa YC0136, upregulated hormonal-related and defense-related genes to promote plant growth and yield [[150]49]. Similarly, through transcriptomic profiling, the mechanism by which Bacillus subtilis CNBG-PGPR-1 induces the production of methionine to regulate the ethylene pathway, thereby promoting tomato growth and salt tolerance under both non-saline and saline stress conditions, has been revealed [[151]24]. Currently, due to poor soil conditions and the impact of diseases, both the yield and quality of walnuts in China have declined, and the walnut industry is facing severe challenges [[152]50, [153]51]. Exploring the mechanism by which B. cereus OTU8977 promotes walnut growth is particularly crucial. Studies have shown that phenylpropanoids and flavonoids can participate in plant signaling that drives defense responses, thereby inducing systemic resistance in plants [[154]52, [155]53]. In addition, flavonoids not only have the ability to scavenge reactive oxygen species, but also function as signaling compounds in the interactions between plants and microorganisms. They can promote the establishment of symbiotic relationships between plants and microorganisms and recruit beneficial microorganisms, thereby significantly enhancing the health and nutrient acquisition capabilities of the host plants [[156]54, [157]55]. Consistent with previous studies, the present study conducted a transcriptomic analysis of walnut roots inoculated with Bacillus cereus OTU8977, and the results revealed significant enrichment of genes associated with the biosynthetic pathways of phenylpropanoids and flavonoids, thereby further corroborating the work of predecessors. Additionally, the study identified significant enrichment of genes related to the pentose and glucuronate interconversion pathway. This pathway is closely associated with cell wall metabolism, which plays a crucial role in responding to stress and serves as an important barrier against pathogens [[158]56, [159]57]. Conclusions In this study, rhizosphere soil samples were collected from eight major walnut - growing regions in China. High - throughput sequencing technology was employed to analyze the characteristics of bacterial communities in the rhizosphere soil of walnuts and to explore the relationships between dominant bacteria in the walnut rhizosphere and soil physicochemical properties. A total of 30 potential plant growth - promoting bacteria were isolated from the rhizosphere soil. Considering both the plant growth - promoting functions and the relative abundance of the strains, Bacillus cereus OTU8977 was selected as the target strain. This strain not only significantly enhanced the growth performance of walnuts but also optimized the structure and function of bacterial communities in the walnut rhizosphere. Furthermore, the molecular mechanisms underlying the growth - promoting effects of this strain on walnuts were thoroughly investigated at the level of gene expression. The findings of this paper enrich the research on the microbiology of walnut rhizosphere, identify key beneficial microbial resources, and provide a theoretical basis for the development of walnut - specific microbial fertilizers. Materials and methods Sampling To gain a comprehensive understanding of the rhizosphere conditions of walnuts in various producing areas, we collected samples from representative orchards in the main walnut-producing regions in eight provinces nationwide (Supplementary Table [160]1). In each orchard, 6 vigorous, non-adjacent walnut trees were selected for rhizosphere soil sampling. Specifically, we collected soil from the point 1/3 of the way from the outer edge of the canopy of each tree by selecting 5 points around the canopy. Before sampling, we removed residual plant and animal matter and the loose topsoil (3–5 cm) from the surface. We then dug to a depth of approximately 40–50 cm (adjusted based on the distribution of fine roots) until reaching the area with the densest fine roots. We carefully extracted the roots using a shovel and brushed off the soil tightly adhering to them with a sterile brush. The soil samples from the 5 points of each tree were combined to create a biological replicate, resulting in 6 replicates per orchard. The rhizosphere soil was subsequently divided into two portions: one was transported at 4 °C for immediate soil property measurement, while the other was quickly frozen in liquid nitrogen and stored at -80 °C for future analysis. Measurement of the soil properties The soil properties were determined using the method previously described [[161]58]. The treatment steps for soil samples before determination mainly include air-drying, removal of impurities, grinding, sieving, and digestion (or extraction). Total nitrogen (TN) was measured using the Kjeldahl method, in accordance with NY/T 1121.24–2012 [[162]59, [163]60]. Available nitrogen (AN) was determined by the alkaline hydrolysis diffusion method, in accordance with LY/T 1228–2015 [[164]61, [165]62]. Total phosphorus (TP) and available phosphorus (AP) were assayed by the Mo-Sb colorimetric method, in accordance with GB 9837-88 and NY/T 1121.7–2014, respectively [[166]59, [167]63]. Total potassium (TK) and available potassium (AK) were analyzed using the atomic absorption spectrometry, in accordance with NY/T 87-1988 and NY/T 889–2004, respectively [[168]64–[169]66]. Soil pH was measured using an acidity meter, in accordance with NY/T 1377–2007 [[170]67]. Soil organic matter (SOM) was assessed by the potassium dichromate volumetric-external heating method, in accordance with NY/T 1121.6–2006 [[171]68]. The nitrogen, phosphorus, and potassium in walnut roots were determined using an automatic Kjeldahl nitrogen analyzer, spectrophotometry, and flame atomic absorption spectrophotometry, respectively, in accordance with NY/T 2017 − 2011 [[172]69]. Metagenomic sequencing A library was constructed and sequenced on the Illumina HiSeq platform using Novaseq sequencing. Reads with an average quality score below 20 over every 4 base pairs and reads shorter than 50 bp were removed. The clean reads were then assembled using MEGAHIT v. 1.2.9 with the default k-mer lengths of 21, 41, 61, 81, and 99 [[173]70]. Contigs were generated by assembling the reads using these different k-mer lengths. Subsequently, contigs longer than or equal to 500 bp were counted, and the optimal assembly result was selected from among them. The assembly results were evaluated using QUAST v. 5.1.0rc1 [[174]71]. Species annotation was conducted on microorganisms using Kraken2 v. 2.0.9-beta [[175]72]. KofamScan v. 1.3.0 was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and eggNOG-mapper v. 2.1.10 was used for obtaining orthologous clusters COG and CAZy results [[176]73, [177]74]. Isolation and identification of rhizobacteria Under aseptic conditions, eight soil samples, each weighing 10 g and collected from respective sampling points, were added to flasks containing 90 mL of sterile water along with an appropriate amount of glass beads. The flasks were then shaken at 200 revolutions per minute (rpm) and incubated at 30℃ for 30 min to disperse soil particles and microorganisms. A 1 mL aliquot of the soil suspension was mixed with 9 mL of sterile 0.9% NaCl solution to obtain a 10^− 1 dilution of the soil sample. Subsequently, 1 mL of the 10^− 1 diluted soil suspension was mixed with another 9 mL of sterile 0.9% NaCl solution to prepare a 10^− 2 dilution, and this process was repeated to generate a series of soil dilutions, including 10^− 3, 10^− 4, 10^− 5, and 10^− 6. Using a pipette, 100 µL of the soil dilution was dispensed onto the center of an LB agar plate and spread uniformly. The plates were then incubated at 30℃ for 1 to 3 days. Once colonies formed on the plates, a single colony was scraped off with an inoculation loop and streaked onto another sterile LB solid medium for pure culture. The plates were incubated in a constant-temperature incubator at 30 °C for 24 to 48 h, and the cultures were then frozen and stored in 15% glycerol at − 80 °C for future use. We sequenced the 16 S rRNA genes of rhizosphere bacterial isolates to confirm their taxonomic groups and evolutionary relationships. The full-length 16 S rRNA gene was amplified using the universal bacterial primers 27 F (AGAGTTTGATCCTGGCTCAG) and 1492R (TACGGCTACCTTGTTACGACTT). The sequencing data were analyzed using the method described previously [[178]58]. The sequencing results were submitted to the GenBank database for homology comparison. Strains with high homology to type strains were selected, and the alignment explorer program in MEGA 7.0 was used for multiple sequence homology analysis [[179]75]. A phylogenetic tree of the tested strains was constructed using the neighbor-joining method, which helped determine the phylogenetic positions of the strains. Determination of the growth-promoting ability of strains Using a microorganism nitrogenase ELISA research kit (TW-reagent, Shanghai, China), the double-antibody sandwich method was employed to determine the level of nitrogenase in the samples. The molybdenum-antimony colorimetry method is utilized to assess the phosphorus solubilization ability of each bacterial strain, in accordance with NY/T 1847–2010 [[180]76]. The soluble potassium content is measured using a flame spectrophotometer manufactured by Shanghai INESA Scientific Instrument Co., Ltd. (INESA, Shanghai, China), in accordance with NY 882–2004 [[181]77]. With juglone (5-Hydroxy-1,4-naphthalenedione) serving as the sole nutrient source, each bacterial strain was cultivated under shaking conditions, and the degradation ability of autotoxins by each strain is determined using a UV-Vis spectrophotometer [[182]78, [183]79]. Pot experiment The experiment was conducted using uniformly grown ‘Xiangling’ plants that were planted in 2000 g of sterilized soil. The experiment was set up with three replicates, and three walnut seedlings were selected for each replicate. The upper inner diameter, lower inner diameter, and height of the pots were 21 cm, 15 cm, and 16 cm, respectively. The soil used in the experiment was sourced from the forestry comprehensive experimental base of Shandong Agricultural University. Two loops of the bacterial culture were inoculated into LB liquid medium and incubated at 28 °C with shaking at 200 r/min overnight. The bacterial suspension was then diluted with sterile water to a final concentration of 1 × 10⁸ CFU/mL [[184]42]. When the plants reached a height of approximately 10 cm, 200 mL of the B. cereus OTU8977 bacterial suspension was inoculated into the rhizosphere of each walnut seedling, with sterile water inoculation used as the control treatment. This inoculation process was repeated three times, with a 7-day interval between each inoculation. After 90 days, plant height, root length, and biomass were measured for both the treated and control groups. Measurement of photosynthetic parameters After inoculating the B. cereus OTU8977 bacterial suspension into the roots of walnut plants (with ‘Jin RS-1’ as the rootstock), the net photosynthetic rate (A), stomatal conductance (gsw), intercellular CO[2]concentration (Ci), and transpiration rate (E) of fully expanded walnut leaves were measured using the LI-6800XT Portable Photosynthesis System (Li-Cor, USA). The measurements were conducted between 9:00 and 11:00 in the morning [[185]80]. DNA extraction, 16 S rRNA gene amplicon sequencing The previous study has already described in detail the operational procedures for DNA extraction, PCR amplification, library preparation, and sequencing [[186]58]. Transcriptome analysis To collect bacterial suspensions, treated and untreated walnut roots were subjected to transcriptome sequencing. Total RNA extraction, library construction, and sequencing were conducted by KeGene Science & Technology Co., Ltd. (KeGene, Taian, China). The transcriptome analysis software HISAT2 v. 2.2.1 was used for aligning the sequences to the walnut Chandler v. 2.0 genome [[187]81, [188]82]. The software featureCounts v. 2.0.3 [[189]83] was employed to quantitatively analyze the expression of known genes. Differential expression analysis of genes was performed using the DESeq2 v. 1.34.0, with the criteria of an adjusted P Value (FDR) ≤ 0.05 and a fold change greater than 2 (i.e.,|log2foldchange| ≥ 1) for screening differentially expressed genes and transcripts [[190]84]. These genes were then used for gene function enrichment analysis according to the previous description [[191]85]. qRT-PCR analysis The cDNA was synthesized using the first-strand cDNA Synthesis kit (Vazyme, [192]https://bio.vazyme.com). We used BEACON DESIGNER 7 (Premier Biosoft, [193]http://www.premierbiosoft.com) to design the primers used for Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). The primers were synthesized by Sangon Biotech Co., Ltd. (Sangon, Shanghai, China). The qRT-PCR was performed using the 2× SYBR Blue Mix. The 18 S rRNA gene of walnut was used as the internal reference gene, and the relative expression amount of the target gene was calculated according to the 2^−ΔΔCt (Software IQ5 2.0). The primers are listed in Supplementary Table [194]2. Statistical analyses Data were expressed as mean value ± standard deviation (SD). For comparisons between two groups, a Student’s t-test was conducted; for comparisons involving more than two groups, a one-way analysis of variance (ANOVA) was employed, followed by Tukey’s post-hoc test. GraphPad Prism 8.00 software was utilized for data processing. Significant values are denoted as * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). Electronic supplementary material Below is the link to the electronic supplementary material. [195]12870_2025_6812_MOESM1_ESM.tif^ (3MB, tif) Supplementary Material 1: Electrophoresis pattern of PCR products of 16S rDNA sequences from 30 bacterial isolates obtained from the rhizosphere of walnut trees. M stands for Trans2K^TM PlusDNA Marker. The lanes 1-30 are the amplification products of the 16S rDNA sequences of strains OTU8837, OTU9019, OTU8869, OTU8947, OTU8905, OTU8951, OTU8863, OTU8895, OTU8849, OTU8983, OTU8827, OTU8903, OTU8985, OTU8845, OTU8909, OTU9017, OTU8969, OTU8975, OTU8977, OTU8997, OTU8917, OTU8989, OTU8943, OTU8949, OTU8893, OTU9021, OTU8881, OTU8859, OTU8913, and OTU8897, respectively. [196]12870_2025_6812_MOESM2_ESM.tif^ (5.5MB, tif) Supplementary Material 2: Comparative experiment under field conditions between inoculated and non-inoculated plants with B. cereus OTU8977. [197]Supplementary Material 3^ (626.1KB, xlsx) Acknowledgements