A driver bacterial strain and trait-complementary partners enhance photosynthesis and growth in ryegrass

A driver bacterial strain and trait-complementary partners enhance photosynthesis and growth in ryegrass | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A driver bacterial strain and trait-complementary partners enhance photosynthesis and growth in ryegrass Jianjun Deng, Yu Kang, Libo Fu, Yingxue Wang, Tong Su, Weichen Hou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8828630/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background and Aims Forage grasses whose yield is almost entirely leaf-based rely on high canopy photosynthesis and rapid leaf regrowth, yet how they recruit root-associated bacteria to support this aboveground performance remains unclear. Methods Soil samples from ryegrass rhizospheres and bulk soils were analyzed through 16S rRNA sequencing and bacterial isolation, followed by pot and plate experiments to assess the plant growth-promoting effects and interactions of selected strains, along with their transcriptomic responses. Results We identify a small set of root-recruited bacterial strains in wild ryegrass that consistently enhance plant growth and leaf greenness. Each strain individually increased biomass and chlorophyll content, but they differed in plant-beneficial functions such as hormone production and nutrient mobilization. When assembled into single- and multi-strain communities, their effects on plant performance were strong and non-additive: Arthrobacter pascens , the field-dominant rhizosphere strain of wild ryegrass, generated disproportionate gains in growth and chlorophyll, whereas other members acted as complementary helpers that further amplified plant responses in mixtures. Conclusion Our work illustrates how leaf‐dominated crops can harness naturally recruited microbial allies to enhance photosynthetic capacity and leaf production, providing insights for designing microbial inoculants for forage grasses. Forage grasses Rhizosphere bacteria Bacterial interactions Plant-microbe interaction Plant performance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plant species with contrasting life histories, dominant organs, and resource-use strategies tend to recruit microbial partners whose functions match their physiological requirements. Legumes serve as the canonical example: they form highly specific symbioses with nitrogen-fixing rhizobia, which is particularly advantageous in nitrogen-limited soils (Westhoek et al. 2021 ; Ashrafi et al. 2022 ; Cui et al. 2023 ). In contrast, many mycorrhizal trees and cereals rely on arbuscular or ectomycorrhizal fungi to expand root absorptive surfaces and mobilize limiting phosphorus, thereby helping to meet their phosphorus demand (Martin et al. 2016 ; Yang et al. 2022 ; Zhao et al. 2024 ). Non-mycorrhizal plants (e.g. Brassicaceae), which have lost key genes required to form mycorrhizal symbioses, instead enrich distinctive bacterial consortia that fulfil some of the functions typically provided by mycorrhizae—contributing to nitrogen and phosphorus supply (Zhang et al. 2022 ; Liu et al. 2024 ), producing phytohormones that regulate root growth (Zhang et al. 2023 ; Shaffique et al. 2023 ), and providing protection against soil-borne pathogens (Liu et al. 2021 ). Species whose biomass and harvestable yield are concentrated in leaves form a distinct life-history type. In these leaf-dominated plants, including many forage grasses, performance is tightly linked to canopy photosynthesis and rapid replacement of defoliated tissue (Pavlů et al. 2021 ; Costa et al. 2022 ; Fan et al. 2024 ). Perennial ryegrass ( Lolium spp.) exemplifies this strategy: in temperate pasture systems it is repeatedly grazed or mown and must quickly rebuild green leaf area to maintain yield and nutritive value (Tubritt et al. 2021 ). These characteristics make ryegrass particularly sensitive to processes that govern nutrient supply and photosynthetic capacity (Amri et al. 2022 ; Ihtisham et al. 2023 ; Brito et al. 2023 ), and thus an attractive system for studying how root-associated microbes support leaf growth. Previous work has characterized rhizosphere and endophytic microbiomes of ryegrass and identified plant growth-promoting microbes that can enhance biomass production or stress tolerance when inoculated (Ke et al., 2021 ; Liu et al., 2025a ; Maimaitiyiming et al., 2025 ). However, we still lack field-based assessments of which beneficial bacteria ryegrass preferentially recruits to its rhizosphere, how these partners interact within consortia, and which functional traits enable them to support leaf growth and photosynthetic capacity. Here, we used wild ryegrass growing in agricultural fields as a model to examine how root-recruited bacteria support leaf growth and photosynthetic performance. We first compared rhizosphere and bulk soils to identify bacterial taxa that are preferentially associated with ryegrass roots under field conditions. We then isolated these taxa and quantified a suite of seven plant-beneficial traits—ACC deaminase activity, siderophore production, indole-3-acetic acid production, potassium solubilization, inorganic phosphate solubilization, organic phosphate mineralization and nitrogen fixation—as well as their pairwise interactions. Finally, we constructed all single-strain and multi-strain communities to identify key driver strains within consortia and compared their effects on ryegrass growth, leaf greenness and transcriptional regulation. Building on the life-history framework outlined above, we hypothesized that leaf-dominated ryegrass preferentially recruits rhizosphere bacteria whose functional traits enhance leaf photosynthetic capacity, and that these root-recruited partners provide the greatest benefits when combined into trait-complementary consortia. Materials and methods Ryegrass rhizosphere and bulk soil sampling Field sampling was conducted in Dongxing Village, Gongzhuling, Changchun, Jilin Province, China (124°48′11″E, 43°37′5″N). The site has a temperate continental monsoon climate, with a mean annual temperature of 5.6℃ and mean annual precipitation of 594.8 mm. The soil is representative of thin-layer black soil in the region, where rotary tillage is the predominant management practice. Before the experiment, soil organic carbon was 11.79 g·kg⁻¹, and soil texture comprised 22.53% sand, 48.92% silt, and 28.55% clay. Ryegrass was established by manual sowing on May 8, 2022, in six plots (2 m × 2 m) separated by 1m buffer zones. No crops were planted at the site prior to ryegrass cultivation. Soil sampling was performed on October 1, 2023. Whole ryegrass plants were carefully uprooted, and loosely attached soil was gently shaken off. Rhizosphere soil was then collected by brushing soil tightly adhering to the soils (within 2 mm of the root surface) into sterile containers using sterile brushes. In parallel, bulk soil was collected from the 0–20 cm layer at locations away from the ryegrass root system. Rhizosphere and bulk soils were homogenized separately, transported to the laboratory on ice, and sieved through a 2 mm mesh to remove plant debris and stones. Each soil sample was split into two subsamples: one was stored at − 80°C for DNA extraction, and the other (fresh soil) was used immediately for bacterial isolation and cultivation. Soil DNA extraction, amplicon sequencing, and bioinformatics Total DNA was extracted from 0.5 g of soil using the FastDNA® Spin Kit for Soil (MP Biomedicals, US) according to the manufacturer’s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using barcoded primers 341F (CCTACGGGNGGCWGCAG) and 785R (GACTACHVGGGTATCTAATCC (Lee et al. 1993 ; Muyzer et al. 1993 ). PCR reactions contained 15 µL Phusion® High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.2 µM of each primer, and about 10 ng template DNA. The cycling program was 98℃ for 1 min, followed by 30 cycles of 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation at 72℃ for 30 s; followed by a final extension at 72℃ for 5 min. Amplicons were checked on a 2% agarose gel (mixed with 1× loading buffer containing SYBR Green), pooled at equimolar concentrations, and purified using a Universal DNA Purification Kit (Tiangen, China; DP214). Sequencing libraries were generated using NEB Next® Ultra™ II FS DNA PCR- Free Library Prep Kit (New England Biolabs, USA; E7430L) following the manufacturer’s recommendations, with index sequences added during library construction. Libraries were quantified using Qubit and real-time PCR and assessed for size distribution using a Bioanalyzer. Pooled libraries were sequenced on an Illumina platform using paired-end chemistry. Raw paired-end reads were demultiplexed by unique barcodes, and barcode/primer sequences were removed. Paired reads were merged with FLASH (V1.2. 1 1, http://ccb.jhu.edu/software/FLASH/ ) (Magoč and Salzberg 2011 ). Quality filtering was performed using fastp v0.23.1 to obtain high-quality clean reads, and chimeric sequences were identified against the SILVA 16S reference database using the UCHIME algorithm and removed. Amplicon sequence variants (ASVs) were inferred in QIIME2 (v2020.06) using the DADA2 (default) or deblur denoising workflow. Taxonomic assignment of ASVs was performed in QIIME2 using the SILVA database. Isolation, cultivation, and identification of bacterial strains To isolate cultivable bacteria, 1 g of fresh ryegrass rhizosphere soil was suspended in 9 mL sterile water, shaken at 200 rpm and 30℃ for 1 h, and allowed to settle for 10 min. The supernatant was serially diluted to 10 − 7 . Aliquots (100 µL) from the 10 − 5 , 10 − 6 , and 10 − 7 dilutions were spread onto TSB agar plates in triplicate and incubated at 30℃ for 2–7 days. Morphologically distinct colonies were picked and purified by repeated streaking. Pure isolates were then grown in 20 mL TSB broth at 30°C and 200 rpm for 1–3 days, with cultures split for identification and long-term preservation. For strain identification, genomic DNA was extracted from 1 mL bacterial culture using the TIANamp Bacteria DNA Kit (Tiangen, China) following the manufacturer’s protocol. Nearly full-length 16S rRNA genes (V1 − V9) were amplified using primers 27F (5′-AGAGTTTGATCCTGGCTC-3′) and 1492R (5′-CGGCTACCTTGTTACGACTT-3′) under the following conditions: 94°C for 5 min; 35 cycles of 94°C for 1 min, 56°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were sequenced by Beijing Tsingke Biotech Co., Ltd. (Beijing, China), and taxonomic identities were determined by BLAST searches against the NCBI database. For preservation, isolates were stored at − 80°C in 30% (v/v) glycerol. Based on amplicon sequencing and isolate identification, five indicator strains ( A. pascens , C. indoltheticum , A. tamlense , N. lianchengensis , and M. makkahensis ) were selected for subsequent experiments. Pot experiment with single-strain inoculation A pot experiment was conducted using non-sterilized field soil collected from the sampling site. Six treatments were established: inoculation with one of five individual bacterial strains ( A. pascens , C. indoltheticum , A. tamlense , N. lianchengensis , and M. makkahensis ) or a non-inoculated control receiving sterile water only. For inoculated treatments, each strain was applied to achieve a final density of 10⁷ CFU g⁻¹ dry soil. Each treatment included three independent replicate pots (11 × 11 × 10 cm), and each pot was sown with 1.5 g perennial ryegrass seeds. Ryegrass height was measured at 10, 20, and 30 days after sowing. Leaf chlorophyll content was determined on day 10 using the acetone–ethanol extraction method as described by Ritchie (Ritchie 2006 ). Assays for plant growth-promoting traits of the five indicator strains We evaluated seven plant growth-promoting traits of the seven indicator strains: ACC deaminase activity, siderophore production, indole-3-acetic acid (IAA) production, potassium solubilization, inorganic phosphate solubilization, organic phosphate mineralization, and nitrogen-fixing potential. Bacterial activation and preparation Strains preserved at -80℃ in glycerol stocks were revived by inoculating 1% (v/v) into 20 mL TSB broth and incubating at 30°C and 200 rpm until cultures reached OD₆₀₀ ≈ 0.8. These activated cultures were used for all assays described below. ACC deaminase activity ACC deaminase activity was assessed based on growth on ACC as the sole nitrogen source (Ali et al. 2014 ). Briefly, 50 µL of activated culture was inoculated into 5 mL DF minimal medium and incubated at 30°C and 200 rpm for 24 h. Subsequently, 40 µL of culture was transferred into 1 mL ADF medium containing ACC; DF medium without ACC served as the negative control. Cultures were incubated in 24-well plates for 48–72 h, and growth was quantified by measuring OD₆₀₀. Strains showing growth in ACC-containing ADF medium were considered ACC deaminase-positive. All treatments were run in triplicate. Siderophore production Siderophore production was assessed on CAS agar plates (Murakami et al. 2021 ). Activated cultures (10 µL) were spotted onto CAS plates (three replicates per strain) and incubated at 30°C for 3–7 days. The diameter of the clear halo (D) and the colony diameter (d) were measured; strains with D/d > 1 were scored as positive. IAA production IAA production was quantified using King’s medium supplemented with tryptophan (0.2 g L⁻¹) and the Salkowski colorimetric assay (Feng et al. 2024 ). Cultures were inoculated at 1% (v/v) and incubated at 30°C and 200 rpm for 3 days (three replicates per strain). Cultures were centrifuged at 12,000 rpm for 10 min, and 1 mL supernatant was mixed with an equal volume of Salkowski reagent in a 24-well plate. After incubation in the dark at room temperature for 30 min, absorbance was measured at 530 nm. IAA concentrations were calculated from an IAA standard curve; detectable IAA indicated a positive result. Potassium solubilization and phosphorus transformation Potassium solubilization, inorganic phosphate solubilization, and organic phosphate mineralization were evaluated using plate assays on potassium-solubilizing medium, PKO (inorganic P) medium, and Mongina (organic P) medium, respectively (Zhou et al. 2012 ; Setiawati and Mutmainnah 2016 ; Wang et al. 2022 ). Activated cultures (10 µL) were spotted onto each medium (three replicates per strain) and incubated at 30°C for 3–7 days. Halo (D) and colony (d) diameters were measured, and strains with D/d > 1 were considered positive for the corresponding trait. Nitrogen-fixing potential (acetylene reduction assay) Nitrogen fixation potential was assessed using the acetylene reduction assay (ARA) (Montes-Luz et al. 2023 ). Briefly, 1 mL activated culture was transferred into a 100 mL serum bottle, amended with 2 mL of 0.1 mol L⁻¹ glucose, and sealed with a butyl rubber stopper and aluminum cap. A 5 mL headspace sample was withdrawn and replaced with 5 mL acetylene gas, and bottles were incubated at 28°C for 2 days. After incubation, 500 µL headspace gas was sampled and ethylene production was quantified by gas chromatography. ARA was calculated as: where K is the ratio of ethylene to acetylene peak height, T is absolute temperature, X is ambient temperature during measurement, Y is atmospheric pressure during measurement, Z is the injected acetylene volume (mL), W is sample mass (g) (or volume for liquid samples), and t is the incubation time after acetylene injection (h). Strains with ARA > 0 were considered to have nitrogen-fixing potential. Pairwise interactions among indicator strains Indicator strains stored at − 80°C in glycerol were revived by inoculating 1% (v/v) into 20 mL TSB broth and incubating at 30°C with shaking (200 rpm) until cultures reached OD₆₀₀ ≈ 0.5. Each culture was then split into two fractions to prepare (i) washed cell suspensions and (ii) cell-free supernatants. For washed cell suspensions, one fraction was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in an equal volume of sterile water. This washing step was repeated twice to remove residual TSB, yielding a medium-free bacterial suspension. For cell-free supernatants, the second fraction was centrifuged at 8,000 rpm for 10 min; the supernatant was collected and passed through a 0.22 µm membrane filter to obtain sterile, cell-free filtrates. To quantify pairwise effects mediated by extracellular products, equal volumes of each strain’s washed cell suspension were mixed with (i) the sterile supernatant from each of the other four strains, and (ii) sterile TSB broth as a control. All mixtures were incubated at 30°C and 200 rpm for 4 h, after which cultures were serially diluted and plated to determine viable cell density (CFU). For each focal strain, interaction strength was calculated as the difference in CFU between growth in TSB supplemented with a heterologous sterile supernatant and growth in TSB alone; this “difference in bacterial concentration” was used to characterize pairwise interactions. Plate assays to test the effects of bacterial consortia on ryegrass performance Perennial ryegrass seeds were surface-sterilized by immersion in 70% ethanol for 1 min, followed by 5 min in a disinfectant solution containing 10% (v/v) sodium hypochlorite and 0.1% (w/v) SDS. Seeds were then rinsed ten times with sterile water, suspended in 0.15% agarose, and pre-incubated at 4°C in the dark for 3 days. Bacterial inoculants were prepared by scraping single colonies from TSB agar plates and resuspending them in sterile water. For single-strain treatments, suspensions were adjusted to a common OD₆₀₀ of 0.01. For multi-strain consortia, equal volumes of the component strains were combined and diluted to a final OD₆₀₀ of 0.01 (approximately 0.5 × 10⁷ CFU mL⁻¹). Consortia were inoculated directly onto individual seeds by applying 2 µL of bacterial suspension per seed on 20 × 20 cm TSB agar plates. Each plate contained 20 seeds, and each treatment was replicated three times. Plates were incubated for 14 days under controlled conditions (22°C, 60% relative humidity, 100 µE m⁻² s⁻¹ light intensity, and a 12 h light : 12 h dark photoperiod). Pot experiment with inoculation of optimal bacterial consortia and transcriptome analysis Pot experiments were conducted using non-sterilized field soil collected from the sampling site. Five treatments were established: four optimal bacterial consortia (1245, 124, 12345, and 1) and a sterile-water control. For each inoculated treatment, the consortium was applied to achieve a final density of 10⁷ CFU g⁻¹ dry soil. Each treatment included three independent replicate pots (11 × 11 × 10 cm), and each pot was sown with 1.5 g of perennial ryegrass seeds. Leaf chlorophyll content was measured 10 days after sowing, and plant tissues were collected for transcriptome sequencing. RNA extraction and quality control Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) and treated with RNase-free DNase I (Takara, Kusatsu, Japan). RNA degradation and contamination were checked on 1% agarose gels. RNA quantity and integrity were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and purity was assessed with a NanoDrop spectrophotometer (Thermo Scientific, DE, USA). Library construction and Illumina sequencing For each sample, 1.5 µg of total RNA was used for library preparation with the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, USA), following the manufacturer’s protocol, with index codes added to label individual samples. Briefly, mRNA was enriched from total RNA using poly(T) oligo-attached magnetic beads and fragmented in NEBNext First Strand Synthesis Reaction Buffer (5×) with divalent cations at elevated temperature. First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H−), followed by second-strand synthesis using DNA Polymerase I and RNase H. Double-stranded cDNA ends were repaired and blunted, 3′ ends were adenylated, and NEBNext adaptors (hairpin-loop structure) were ligated. Libraries were size-selected (target insert size ~ 200–250 bp) using AMPure XP beads (Beckman Coulter, USA). USER enzyme (NEB, USA) treatment (37°C for 15 min, then 95°C for 5 min) was performed prior to PCR enrichment using Phusion High-Fidelity DNA polymerase with universal and index primers. Final libraries were purified with AMPure XP and evaluated on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NovaSeq 6000 platform (paired-end 150 bp) by Beijing Allwegene Technology Co., Ltd. (Beijing, China). Read processing, mapping, and gene expression quantification Raw FASTQ reads were processed using in-house Perl scripts to remove adaptor contamination, poly-N reads, and low-quality reads, generating clean reads. Quality metrics including Q20, Q30, GC content, and sequence duplication levels were calculated, and all downstream analyses were based on high-quality clean reads. Clean reads were aligned to the reference genome using STAR. Only reads with perfect matches or a single mismatch were retained for subsequent analyses and annotation. Aligned BAM files were processed using Picard-tools v1.41 and SAMtools v0.1.18 to sort reads, remove PCR duplicates, and merge alignments for each sample. Gene-level read counts were generated with HTSeq v0.5.4p3, and gene expression was normalized as fragments per kilobase of transcript per million mapped reads (FPKM). Differential expression and pathway enrichment analysis Differential expression was assessed by comparing each consortium treatment to the control (1245 vs control, 124 vs control, 12345 vs control, and 1 vs control) using DESeq (R package v1.10.1), which models read counts with a negative binomial distribution. P-values were adjusted for multiple testing using the Benjamini–Hochberg method, and genes with an adjusted P -value < 0.05 were considered differentially expressed. KEGG pathway enrichment of differentially expressed genes was performed using KOBAS (Mao et al. 2005 ). Statistical analyses For the field survey, bacterial α-diversity was quantified using the Shannon index. Community β-diversity was calculated based on Bray-Curtis dissimilarity and visualized by principle coordinates analysis (PCoA) to evaluate differences in bacterial community composition between ryegrass rhizosphere and bulk soils. PCoA was implemented in R using the vegan package (Oksanen et al. 2013 ). To identify bacterial genera that best discriminated between rhizosphere and bulk soil, we performed random forest analysis using the randomForest package in R (RColorBrewer and Liaw 2018 ). Genera were ranked by mean decrease in accuracy, with higher values indicating greater importance for classification. Model performance and feature selection were assessed using 10,000 trees and the rfcv function, and cross-validation curves were visualized with matplot function. We employed the Shapiro-Wilk test to assess whether the data conformed to a normal distribution. For datasets that adhered to a normal distribution, we applied one-way ANOVA to analyze variance and employed LSD test for post hoc multiple comparisons. Conversely, for datasets that did not follow a normal distribution, we utilized the Kruskal-Wallis test, a non-parametric method, and conducted multiple comparisons using Dunn’s Test. In the context of pot and plate experiments, the treatment effects on variables such as ryegrass height, chlorophyll content, OD600 (ADF-DF), siderophore production (CAS halo ratio, D/d), IAA concentration, acetylene reduction activity (ARA), and plant performance were evaluated using these statistical approaches, ensuring a significance level of P < 0.05. A composite plant performance index was calculated by min–max standardizing seedling height, fresh weight, and chlorophyll content to a 0–1 scale and then averaging the standardized values across the three traits. For plate assays, hierarchical partitioning was employed to quantify the independent contribution of each bacterial strain to variation in plant performance. Analysis were conducted in R using rdacca.hp with supporting functions from vegan package (Lai et al. 2022 ). Results Perennial ryegrass recruits beneficial bacteria to facilitate photosynthesis We compared bacterial communities in the ryegrass rhizosphere and adjacent bulk soil (Fig. 1 a). Bacterial α-diversity (Shannon index) was significantly lower in the rhizosphere than in bulk soil, indicating selective filtering of soil bacteria near roots (Fig. S1 a). Community composition also differed clearly between two compartments, with rhizosphere and bulk soil formed forming two well-separated clusters (Bray-Curtis, R 2 = 0.29, P < 0.01, Fig. S1 b). At the phylum level, Actinobacteria were a dominant group in both the rhizosphere and bulk soil. Notably, their relative abundance was 10 percentage points higher in the rhizosphere than in bulk soil (Fig. S1 c). We identified 20 indicator genera that distinguished rhizosphere from bulk soil, including 9 enriched in the rhizosphere and 11 enriched in bulk soil (Fig. 1 b; Fig. S2). Ten-fold cross-validation with five repeats showed that model error stabilized with the 10 most relevant genera (Fig. S3). We therefore focused on the top 10 ranked genera, of which six were significantly enriched in the ryegrass rhizosphere. From these, we isolated representative strains from five genera: Arthrobacter ( A. pascens ), Nocardioides ( N. lianchengensis ), Microvirga ( M. makkahensis ), Aeromicrobium ( A. tamlense ), and Chryseobacterium ( C. indoltheticum ). To test whether rhizosphere-enriched bacteria benefit ryegrass, we inoculated indicator isolates into natural soil planted with ryegrass (Fig. 1 d). Compared with the control, inoculation with A. pascens , C. indoltheticum , A. tamlense , N. lianchengensis , and M. makkahensis significantly increased ryegrass height and the total chlorophyll content of ryegrass (Fig. 1 e; Fig. 1 f; Fig. S4). The five indicator strains exhibit plant growth-promoting traits To explore how the indicator bacteria may promote ryegrass growth, we screened seven plant growth-promoting functions in the five isolates (Fig. 2 a). Three strains— A. tamlense , N. lianchengensis and M. makkahensis —showed ACC deaminase activity, with the strongest response in A. tamlense (Fig. 2 b). Siderophore production was detected in C. indoltheticum and N. lianchengensis , with C. indoltheticum exhibiting the larger halo (Fig. 2 b). IAA production was observed in A. pascens , C. indoltheticum and A. tamlense , and was highest in A. pascens (Fig. 2 b). In addition, all five strains reduced acetylene to ethylene, indicating nitrogen-fixing potential, with activity ranking C. indoltheticum > N. lianchengensis > A. pascens > A. tamlense > M. makkahensis (Fig. 2 b). Interactions among indicator bacteria shape combinatorial effects on ryegrass performance Pairwise interaction assays revealed a largely facilitative network among four strains— A. pascens , C. indoltheticum , N. lianchengensis and M. makkahensis —which generally promoted each other’s growth (Fig. 3 a, b). In contrast, A. tamlense showed consistent antagonistic effects on the other four strains, although its own growth could be supported by them (Fig. 3 b). Notably, C. indoltheticum was most strongly stimulated by A. pascens and M. makkahensis (Fig. 3 a). We next evaluated all possible strain combinations for their effects on ryegrass performance (A composite index integrating seedling height, fresh weight, and chlorophyll content.). Plant performance varied substantially across consortia, with consortia 1245 ( A. pascens , C. indoltheticum , N. lianchengensis , M. makkahensis ), 124 ( A. pascens , C. indoltheticum , N. lianchengensis ), 12345 ( A. pascens , C. indoltheticum , A. tamlense , N. lianchengensis , M. makkahensis ), and strain 1 alone ( A. pascens ) showing relatively higher performance (Fig. 3 c; Fig. S5). Across combinations, consortia containing A. pascens tended to outperform those built around the other strains (Fig. 3 d). Diversity effects were strain-dependent: performance increased with consortium richness when C. indoltheticum or M. makkahensis was included, whereas consortia containing A. pascens , A. tamlense , or N. lianchengensis showed a non-linear pattern, reaching a minimum at three-member consortia (Fig. S6). Variance decomposition and hierarchical partitioning identified A. pascens , A. tamlense and C. indoltheticum as the strongest contributors to enhanced ryegrass performance, with A. pascens as the primary driver (Fig. 3 e). Bacterial consortia enhance photosynthesis-related traits and transcriptional signals We next examined the mechanisms by which the optimal consortia (1245, 124, 12345, and strain 1) promote ryegrass growth (Fig. 4 a). Compared to the control, all four treatments significantly increased total chlorophyll, Chlorophyll a and chlorophyll b contents. (Fig. 4 b). Transcriptomic profiling showed extensive transcriptional reprogramming in ryegrass leaves following inoculation, with thousands of genes up- or downregulated across treatments (Fig. S8). KEGG enrichment of upregulated genes consistently highlighted photosynthesis-related pathways, particularly photosynthesis-antenna proteins and photosynthesis, across all four consortia (Fig. 4 c–f). In contrast, downregulated genes were enriched in lipid-related pathways, with α-linolenic acid metabolism repeatedly suppressed across treatments (Fig. S9a–d). Collectively, these results indicate that the optimal consortia promote ryegrass growth primarily by enhancing photosynthesis-associated processes while concomitantly repressing linolenic acid metabolism. Discussion Our study demonstrates that the bacterial α-diversity in the rhizosphere of ryegrass is significantly lower than in the adjacent bulk soil, with the two communities forming distinctly clusters. This is consistent with the well-established rhizosphere effect, where plant roots recruit and shape microbial communities primarily through the release of soluble carbon sources and signaling molecules (Singh et al. 2022 ; Panchal et al. 2022 ). These environmental changes create selective pressures, leading to the enrichment of specific microbial groups while reducing overall diversity (Ling et al. 2022 ). Notably, Actinobacteria dominate both the rhizosphere and bulk soil, with a higher relative abundance observed in the rhizosphere. This finding aligns with previous studies that have shown Actinobacteria to be a dominant group in the rhizosphere of several plant species, including ryegrass (Lagos et al. 2014 ; Borowik et al. 2020 ). This enrichment of Actinobacteria can be attributed to several factors, including their ability to use complex substrates, their tolerance to drought and low-nutrient conditions, and their rich secondary metabolism and antagonistic potential (Boukhatem et al. 2022 ; Fu et al. 2022 ). These characteristics allow them to thrive in the challenging rhizosphere environment, where competition for resources is high. Furthermore, our study identified Arthrobacter pascens , a strain within the Actinobacteria phylum, as a key contributor to plant performance in ryegrass rhizosphere. We found that A. pascens significantly promoted plant growth, likely through its production of IAA, which has been shown in previous studies to stimulate root elongation and enhance overall plant growth (Tian et al. 2008 ; Li et al. 2018 ; Etesami and Glick 2024 ). This suggests that A. pascens may serve as keystone bacteria in the ryegrass rhizosphere, directly influencing plant growth and enhancing community-level functional outputs by promoting other beneficial strains. The five representative strains from rhizosphere-enriched groups significantly enhanced ryegrass growth, particularly increasing plant height and chlorophyll content. These strains exhibited a range of plant growth-promoting traits, such as ACC deaminase activity, which modulates ethylene levels to promote root and shoot growth (Gamalero et al. 2023 ); IAA production, which promotes the elongation of plant cells and stimulates root growth (Mohite 2013 ); and siderophore production, which improves iron bioavailability (Wang et al. 2024 ), essential for chlorophyll formation (Therby-Vale et al. 2022 ). Additionally, all five strains exhibited acetylene reduction activity, indicating nitrogen-fixing potential, which further supports increased chlorophyll content and overall plant growth (Muhammad et al. 2022 ). We propose that the synergistic effects of bacterial-derived IAA and siderophores contribute to the increase in chlorophyll content and photosynthetic efficiency (Salazar-Iribe and De-la-Peña 2020 ; Brick et al. 2025 ). Furthermore, the potential contributions from nitrogen fixation and ACC deaminase regulation may further support leaf development and photosynthesis (Mu and Chen 2021 ; Chandwani and Amaresan 2022 ), leading to improved plant performance. These findings highlight the importance of these bacteria’s functional traits in regulating plant metabolic processes, which are essential for optimizing growth and productivity. Our analysis of bacterial interactions revealed both cooperative and antagonistic behaviors that collectively influence plant performance. Except for A. tamlense , the other four rhizosphere-enriched strains promoted each other’s growth, forming a mutually beneficial network. In contrast, A. tamlense exhibited antagonistic effects on the other strains, though its growth was still supported by them. This asymmetric mutualism-antagonism network implies the importance of balancing positive and negative interactions to prevent the overgrowth of any single strain, ensuring community stability and ecological resilience (Lopes et al. 2024 ). Further analysis of multi-strain consortia showed that combinations centered around A. pascens resulted in the best plant performance, even with single-strain inoculation. However, multi-strain consortia exhibited a nonlinear response, with a performance dip at three-strain combinations (Fig. S6). This can be explained by a balance between functional complementarity and resource competition: when bacterial richness was low (fewer than four strains), competition limited plant benefits, but as richness increased, complementary functions outweighed the competitive costs, enhancing plant growth. This supports the concept of functional complementarity, where diverse microbial communities provide synergistic effects, particularly in complex environments (Sarsan et al. 2021 ; Puente-Sánchez et al. 2024 ). Additionally, increasing community richness, including two other strains, led to improved plant performance, consistent with previous reports linking bacterial diversity to plant growth (Weidner et al. 2015 ; Wei et al. 2015 ; Laforest-Lapointe et al. 2017 ). The optimal bacterial consortia significantly enhanced chlorophyll content, particularly chlorophyll b , reflecting an increase in the plant’s photosynthetic capacity. Transcriptomic analysis further confirmed that the upregulated genes were predominantly involved in the photosynthesis-antenna proteins and photosynthesis pathways, suggesting that bacterial inoculation enhances plant growth by improving light capture and electron transport. The increase in chlorophyll b correlates with the expansion of light-harvesting complexes, optimizing light energy absorption (Lokstein et al. 2021 ). Furthermore, a consistent downregulation of α-linolenic acid metabolism was observed across all treatments. This pathway is a precursor for jasmonic acid (JA) and other oxylipins (Mosblech et al. 2009 ). The inhibition of this pathway suggests a shift in the growth-defense trade-off: when beneficial microbes colonize, plants may downregulate JA-related pathways to reduce investment in defense, thereby reallocating resources towards photosynthesis and growth, ultimately achieving higher plant performance (He et al., 2022 ; Liu et al., 2025b ). Conclusion This study highlights the critical role of A. pascens in promoting ryegrass growth, particularly through IAA production. While A. pascens has been identified as a keystone bacterium in the ryegrass rhizosphere, the exact mechanisms by which ryegrass recruits this beneficial microbe remain unclear and warrant further investigation. Furthermore, it is still unknown whether A. pascens is widely enriched in the rhizosphere of other leaf-dominated plants, such as forage grasses. Alongside the role of individual bacteria, our findings suggest that the composition and number of strains in synthetic microbial consortia play a crucial role in optimizing plant performance. Identifying the optimal number of strains for maximum plant benefit is an intriguing and underexplored area that requires further investigation. Understanding how microbial richness and functional complementarity affect plant growth will be essential for improving microbial management strategies. This deeper understanding could lead to more targeted, effective applications of microbial consortia in sustainable agricultural practices. Declarations Acknowledgments Ting Liu and Jiaguo Jiao were supported by the National Key R&D Program (2024YFD1501803 and 2023YFD1901401). Weichen Hou was supported by Postgraduate Research & Practice Innovation Program of Jiangsu Province (Project No. KYCX25_0998). Libo Fu and Yingxue Wang were supported by the Earmarked fund for Modern Agroindustry Technology Research System-Green manure (CARS-22). Conflicts of interest The authors declare that they have no conflict of interest. References Ali SZ, Sandhya V, Venkateswar Rao L (2014) Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8828630","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590111085,"identity":"a4af68f0-6a41-452f-8a34-bad98c6f2215","order_by":0,"name":"Jianjun Deng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Deng","suffix":""},{"id":590111086,"identity":"c638cae8-4603-4f3b-a310-45cd64f856cd","order_by":1,"name":"Yu Kang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Kang","suffix":""},{"id":590111087,"identity":"a7a4c71a-ab4b-479e-9f4c-391dacdf0b5d","order_by":2,"name":"Libo Fu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Libo","middleName":"","lastName":"Fu","suffix":""},{"id":590111088,"identity":"86ab7d8a-63c4-402b-83ce-b7e20e56bacb","order_by":3,"name":"Yingxue Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yingxue","middleName":"","lastName":"Wang","suffix":""},{"id":590111089,"identity":"9476a166-d56e-4537-bd99-9d44d900faf2","order_by":4,"name":"Tong Su","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Su","suffix":""},{"id":590111090,"identity":"b6c723e4-8fe0-473b-a76f-7ced4e59c930","order_by":5,"name":"Weichen Hou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Weichen","middleName":"","lastName":"Hou","suffix":""},{"id":590111091,"identity":"58c7e05a-a221-481a-9c39-d4344aad43c6","order_by":6,"name":"Huixin Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huixin","middleName":"","lastName":"Li","suffix":""},{"id":590111092,"identity":"2b55662c-b777-441e-bdd3-a24e1e7b8846","order_by":7,"name":"Jiaguo Jiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACxmYwxcbDwMB8gEECzEkgWgtbAnFakACPAZRBQAtzO/Pj1zwVfDL87T3fHljUHGbgZ88xYPi5A5/D2Mysec6w8UicObvdQOLYYQbJnjcGjL1n8PrFzJi3DeiXG7nbJCTYDjMY3MgxYGZsw6eF/RtYi/yNnGcSEv8OM9gT1sJj/BikBWg4m4RkG9AWCcJayhjnAP1ieOaYmYRkXzrQU88KDvbi0WLYf3zzhzcVx+zljjc/k5b4Zi3H35688cFPfFoaGNiAEXgMzGEGsnhAjAO4NTAwyAMVfmBgqIG48gM+paNgFIyCUTBiAQDjm0lI9G2uyAAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Jiaguo","middleName":"","lastName":"Jiao","suffix":""},{"id":590111093,"identity":"72453f52-05fe-4848-b1a0-854d2997f835","order_by":8,"name":"Ting Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-02-09 09:41:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8828630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8828630/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102833373,"identity":"14e58b75-6302-46b6-a229-d950598239eb","added_by":"auto","created_at":"2026-02-17 10:28:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2980899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRoot-recruited rhizosphere bacteria from field-grown ryegrass promote plant growth and chlorophyll accumulation.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Schematic of field sampling of wild ryegrass rhizosphere and bulk soil for differential bacterial abundance analysis. Twelve composite samples were collected as six paired rhizosphere-bulk soil sets (n = 6 per group); each sample comprised soil pooled from 20 ryegrass plants spaced at least 5 m apart in the field. (\u003cstrong\u003eb\u003c/strong\u003e) Indicator bacterial genera distinguishing rhizosphere and bulk soil identified by random forest. The top 20 genera are shown and ranked by mean decrease in model accuracy. (\u003cstrong\u003ec\u003c/strong\u003e) Relative abundance of rhizosphere-enriched indicator genera. Bars show mean ± standard deviation. A checkmark (√) to the left of a genus indicates that strains from this genus were isolated from the ryegrass, whereas a cross (×) indicates genera for which no isolates were obtained. (\u003cstrong\u003ed\u003c/strong\u003e) Schematic of pot inoculation with five rhizosphere-enriched, plant-beneficial strains. Specifically, the five bacterial isolates are \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e, and \u003cem\u003eM. makkahensis\u003c/em\u003e, which are taxonomically affiliated with Actinobacteria, Bacteroidetes, Actinobacteria, Actinobacteria, and Proteobacteria, respectively. (\u003cstrong\u003ee, f\u003c/strong\u003e) Effects of single-strain inoculation on plant height (\u003cstrong\u003ee\u003c/strong\u003e) and leaf chlorophyll content (\u003cstrong\u003ef\u003c/strong\u003e). Plant height is the average height of four ryegrass plants per pot, calculated across three sampling times (days 10, 20 and 30); leaf chlorophyll content was measured on day 10. Different lowercase letters indicate significant differences between treatments (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8828630/v1/83088e97cdc92c6478a28cfd.png"},{"id":102833372,"identity":"6fc0e70a-6a61-444d-b6a8-e7822e97e76c","added_by":"auto","created_at":"2026-02-17 10:28:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1850307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTraits profiling shows complementary plant-beneficial functions among rhizosphere-enriched bacterial strains.\u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic of assays used to validate seven common plant-beneficial functions in the five strains. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of plant-beneficial functions for each strain (n = 6 per strain). Bars show means ± standard deviation. Different lowercase letters indicate significant differences between among strains (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Presence-absence matrix summarizing plant-beneficial functions across the five strains.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8828630/v1/adaf4ab645c59a8d78025c71.png"},{"id":102833375,"identity":"d316c8d7-44ed-4d6e-8906-ffeb01d5e83f","added_by":"auto","created_at":"2026-02-17 10:28:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2838479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePairwise interactions among plant-beneficial strains and their consortia effects on ryegrass performance.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Pairwise interactions mediated by cell-free supernatants. For each strain (panel headings), bars show the change in bacterial abundance (ΔCFU/mL) when grown in TSB amended with supernatant from each donor strain (x-axis) relative to growth in TSB alone. Positive values indicate facility and negative values inhibition. Bars represent means ± standard error. Here, 12345 represents \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e and \u003cem\u003eM. makkahensis\u003c/em\u003e, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) Network summarizes facilitate and inhibit interactions among the five strains. (\u003cstrong\u003ec\u003c/strong\u003e) Ryegrass performance under all single- and multi-strain combinations in plate assays. Plant performance is a composite index integrating seedling height, fresh weight, and chlorophyll content. (\u003cstrong\u003ed\u003c/strong\u003e) Frequency distributions of ryegrass performance for all consortia that include each focal strain. (\u003cstrong\u003ee\u003c/strong\u003e) Individual contribution of each strain to plant performance in consortia. Significant effects are indicated by ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8828630/v1/1f9c79164a04f4bc22179604.png"},{"id":102833374,"identity":"dc39936b-ca57-42a2-a26a-2f723a9fd86d","added_by":"auto","created_at":"2026-02-17 10:28:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6041646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFour bacterial consortia enhance leaf chlorophyll and photosynthesis-related pathways in ryegrass. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Pot experiment evaluating four inoculation treatments—three rhizosphere-enriched consortia (1245, 124 and 12345) and single strain 1. Here, 12345 represents \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e and \u003cem\u003eM. makkahensis\u003c/em\u003e, respectively. (\u003cstrong\u003eb–d\u003c/strong\u003e) Effects of inoculation on total chlorophyll, chlorophyll a and chlorophyll b in ryegrass leaves. Bars represent means ± standard error. Different lowercase letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (\u003cstrong\u003ee–h\u003c/strong\u003e) KEGG pathways significantly enriched among upregulated genes in leaves inoculated with consortia 1245, 124, 12345 and 1 relative to the control. Bubble size indicates the number of genes assigned to each pathway. The rich factor is the ratio of differentially expressed genes to the total number of genes in a pathway, and the color indicates the Q-value (false discovery rate–adjusted \u003cem\u003ep\u003c/em\u003e-value).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8828630/v1/f806515f1ad021f3492646b6.png"},{"id":103049251,"identity":"d44db656-ed08-467c-8848-0f0931137089","added_by":"auto","created_at":"2026-02-20 07:38:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14986043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8828630/v1/07da0612-908c-4cad-80f3-ce5106c8f06f.pdf"},{"id":102833377,"identity":"a6b2bdb7-f9c8-477f-a25a-0e5bd64aac14","added_by":"auto","created_at":"2026-02-17 10:28:21","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":21443422,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8828630/v1/9b118b3651b9a6c18bfa9852.docx"}],"financialInterests":"","formattedTitle":"A driver bacterial strain and trait-complementary partners enhance photosynthesis and growth in ryegrass","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant species with contrasting life histories, dominant organs, and resource-use strategies tend to recruit microbial partners whose functions match their physiological requirements. Legumes serve as the canonical example: they form highly specific symbioses with nitrogen-fixing rhizobia, which is particularly advantageous in nitrogen-limited soils (Westhoek et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ashrafi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, many mycorrhizal trees and cereals rely on arbuscular or ectomycorrhizal fungi to expand root absorptive surfaces and mobilize limiting phosphorus, thereby helping to meet their phosphorus demand (Martin et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Non-mycorrhizal plants (e.g. Brassicaceae), which have lost key genes required to form mycorrhizal symbioses, instead enrich distinctive bacterial consortia that fulfil some of the functions typically provided by mycorrhizae\u0026mdash;contributing to nitrogen and phosphorus supply (Zhang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), producing phytohormones that regulate root growth (Zhang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shaffique et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and providing protection against soil-borne pathogens (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecies whose biomass and harvestable yield are concentrated in leaves form a distinct life-history type. In these leaf-dominated plants, including many forage grasses, performance is tightly linked to canopy photosynthesis and rapid replacement of defoliated tissue (Pavlů et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Perennial ryegrass (\u003cem\u003eLolium\u003c/em\u003e spp.) exemplifies this strategy: in temperate pasture systems it is repeatedly grazed or mown and must quickly rebuild green leaf area to maintain yield and nutritive value (Tubritt et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These characteristics make ryegrass particularly sensitive to processes that govern nutrient supply and photosynthetic capacity (Amri et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ihtisham et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Brito et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and thus an attractive system for studying how root-associated microbes support leaf growth. Previous work has characterized rhizosphere and endophytic microbiomes of ryegrass and identified plant growth-promoting microbes that can enhance biomass production or stress tolerance when inoculated (Ke et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Maimaitiyiming et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, we still lack field-based assessments of which beneficial bacteria ryegrass preferentially recruits to its rhizosphere, how these partners interact within consortia, and which functional traits enable them to support leaf growth and photosynthetic capacity.\u003c/p\u003e \u003cp\u003eHere, we used wild ryegrass growing in agricultural fields as a model to examine how root-recruited bacteria support leaf growth and photosynthetic performance. We first compared rhizosphere and bulk soils to identify bacterial taxa that are preferentially associated with ryegrass roots under field conditions. We then isolated these taxa and quantified a suite of seven plant-beneficial traits\u0026mdash;ACC deaminase activity, siderophore production, indole-3-acetic acid production, potassium solubilization, inorganic phosphate solubilization, organic phosphate mineralization and nitrogen fixation\u0026mdash;as well as their pairwise interactions. Finally, we constructed all single-strain and multi-strain communities to identify key driver strains within consortia and compared their effects on ryegrass growth, leaf greenness and transcriptional regulation. Building on the life-history framework outlined above, we hypothesized that leaf-dominated ryegrass preferentially recruits rhizosphere bacteria whose functional traits enhance leaf photosynthetic capacity, and that these root-recruited partners provide the greatest benefits when combined into trait-complementary consortia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRyegrass rhizosphere and bulk soil sampling\u003c/h2\u003e \u003cp\u003eField sampling was conducted in Dongxing Village, Gongzhuling, Changchun, Jilin Province, China (124\u0026deg;48\u0026prime;11\u0026Prime;E, 43\u0026deg;37\u0026prime;5\u0026Prime;N). The site has a temperate continental monsoon climate, with a mean annual temperature of 5.6℃ and mean annual precipitation of 594.8 mm. The soil is representative of thin-layer black soil in the region, where rotary tillage is the predominant management practice. Before the experiment, soil organic carbon was 11.79 g\u0026middot;kg⁻\u0026sup1;, and soil texture comprised 22.53% sand, 48.92% silt, and 28.55% clay.\u003c/p\u003e \u003cp\u003eRyegrass was established by manual sowing on May 8, 2022, in six plots (2 m \u0026times; 2 m) separated by 1m buffer zones. No crops were planted at the site prior to ryegrass cultivation. Soil sampling was performed on October 1, 2023. Whole ryegrass plants were carefully uprooted, and loosely attached soil was gently shaken off. Rhizosphere soil was then collected by brushing soil tightly adhering to the soils (within 2 mm of the root surface) into sterile containers using sterile brushes. In parallel, bulk soil was collected from the 0\u0026ndash;20 cm layer at locations away from the ryegrass root system.\u003c/p\u003e \u003cp\u003eRhizosphere and bulk soils were homogenized separately, transported to the laboratory on ice, and sieved through a 2 mm mesh to remove plant debris and stones. Each soil sample was split into two subsamples: one was stored at \u0026minus;\u0026thinsp;80\u0026deg;C for DNA extraction, and the other (fresh soil) was used immediately for bacterial isolation and cultivation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSoil DNA extraction, amplicon sequencing, and bioinformatics\u003c/h3\u003e\n\u003cp\u003eTotal DNA was extracted from 0.5 g of soil using the FastDNA\u0026reg; Spin Kit for Soil (MP Biomedicals, US) according to the manufacturer\u0026rsquo;s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using barcoded primers 341F (CCTACGGGNGGCWGCAG) and 785R (GACTACHVGGGTATCTAATCC (Lee et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Muyzer et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). PCR reactions contained 15 \u0026micro;L Phusion\u0026reg; High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.2 \u0026micro;M of each primer, and about 10 ng template DNA. The cycling program was 98℃ for 1 min, followed by 30 cycles of 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation at 72℃ for 30 s; followed by a final extension at 72℃ for 5 min. Amplicons were checked on a 2% agarose gel (mixed with 1\u0026times; loading buffer containing SYBR Green), pooled at equimolar concentrations, and purified using a Universal DNA Purification Kit (Tiangen, China; DP214).\u003c/p\u003e \u003cp\u003eSequencing libraries were generated using NEB Next\u0026reg; Ultra\u0026trade; II FS DNA PCR- Free Library Prep Kit (New England Biolabs, USA; E7430L) following the manufacturer\u0026rsquo;s recommendations, with index sequences added during library construction. Libraries were quantified using Qubit and real-time PCR and assessed for size distribution using a Bioanalyzer. Pooled libraries were sequenced on an Illumina platform using paired-end chemistry.\u003c/p\u003e \u003cp\u003eRaw paired-end reads were demultiplexed by unique barcodes, and barcode/primer sequences were removed. Paired reads were merged with FLASH (V1.2. 1 1, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ccb.jhu.edu/software/FLASH/\u003c/span\u003e\u003cspan address=\"http://ccb.jhu.edu/software/FLASH/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Magoč and Salzberg \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Quality filtering was performed using fastp v0.23.1 to obtain high-quality clean reads, and chimeric sequences were identified against the SILVA 16S reference database using the UCHIME algorithm and removed. Amplicon sequence variants (ASVs) were inferred in QIIME2 (v2020.06) using the DADA2 (default) or deblur denoising workflow. Taxonomic assignment of ASVs was performed in QIIME2 using the SILVA database.\u003c/p\u003e\n\u003ch3\u003eIsolation, cultivation, and identification of bacterial strains\u003c/h3\u003e\n\u003cp\u003eTo isolate cultivable bacteria, 1 g of fresh ryegrass rhizosphere soil was suspended in 9 mL sterile water, shaken at 200 rpm and 30℃ for 1 h, and allowed to settle for 10 min. The supernatant was serially diluted to 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e. Aliquots (100 \u0026micro;L) from the 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e dilutions were spread onto TSB agar plates in triplicate and incubated at 30℃ for 2\u0026ndash;7 days. Morphologically distinct colonies were picked and purified by repeated streaking. Pure isolates were then grown in 20 mL TSB broth at 30\u0026deg;C and 200 rpm for 1\u0026ndash;3 days, with cultures split for identification and long-term preservation.\u003c/p\u003e \u003cp\u003eFor strain identification, genomic DNA was extracted from 1 mL bacterial culture using the TIANamp Bacteria DNA Kit (Tiangen, China) following the manufacturer\u0026rsquo;s protocol. Nearly full-length 16S rRNA genes (V1\u0026thinsp;\u0026minus;\u0026thinsp;V9) were amplified using primers 27F (5\u0026prime;-AGAGTTTGATCCTGGCTC-3\u0026prime;) and 1492R (5\u0026prime;-CGGCTACCTTGTTACGACTT-3\u0026prime;) under the following conditions: 94\u0026deg;C for 5 min; 35 cycles of 94\u0026deg;C for 1 min, 56\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min; and a final extension at 72\u0026deg;C for 10 min. PCR products were sequenced by Beijing Tsingke Biotech Co., Ltd. (Beijing, China), and taxonomic identities were determined by BLAST searches against the NCBI database. For preservation, isolates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in 30% (v/v) glycerol. Based on amplicon sequencing and isolate identification, five indicator strains (\u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e, and \u003cem\u003eM. makkahensis\u003c/em\u003e) were selected for subsequent experiments.\u003c/p\u003e\n\u003ch3\u003ePot experiment with single-strain inoculation\u003c/h3\u003e\n\u003cp\u003eA pot experiment was conducted using non-sterilized field soil collected from the sampling site. Six treatments were established: inoculation with one of five individual bacterial strains (\u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e, and \u003cem\u003eM. makkahensis\u003c/em\u003e) or a non-inoculated control receiving sterile water only. For inoculated treatments, each strain was applied to achieve a final density of 10⁷ CFU g⁻\u0026sup1; dry soil. Each treatment included three independent replicate pots (11 \u0026times; 11 \u0026times; 10 cm), and each pot was sown with 1.5 g perennial ryegrass seeds. Ryegrass height was measured at 10, 20, and 30 days after sowing. Leaf chlorophyll content was determined on day 10 using the acetone\u0026ndash;ethanol extraction method as described by Ritchie (Ritchie \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAssays for plant growth-promoting traits of the five indicator strains\u003c/h3\u003e\n\u003cp\u003eWe evaluated seven plant growth-promoting traits of the seven indicator strains: ACC deaminase activity, siderophore production, indole-3-acetic acid (IAA) production, potassium solubilization, inorganic phosphate solubilization, organic phosphate mineralization, and nitrogen-fixing potential.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBacterial activation and preparation\u003c/h2\u003e \u003cp\u003eStrains preserved at -80℃ in glycerol stocks were revived by inoculating 1% (v/v) into 20 mL TSB broth and incubating at 30\u0026deg;C and 200 rpm until cultures reached OD₆₀₀ \u0026asymp; 0.8. These activated cultures were used for all assays described below.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eACC deaminase activity\u003c/h3\u003e\n\u003cp\u003eACC deaminase activity was assessed based on growth on ACC as the sole nitrogen source (Ali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Briefly, 50 \u0026micro;L of activated culture was inoculated into 5 mL DF minimal medium and incubated at 30\u0026deg;C and 200 rpm for 24 h. Subsequently, 40 \u0026micro;L of culture was transferred into 1 mL ADF medium containing ACC; DF medium without ACC served as the negative control. Cultures were incubated in 24-well plates for 48\u0026ndash;72 h, and growth was quantified by measuring OD₆₀₀. Strains showing growth in ACC-containing ADF medium were considered ACC deaminase-positive. All treatments were run in triplicate.\u003c/p\u003e\n\u003ch3\u003eSiderophore production\u003c/h3\u003e\n\u003cp\u003eSiderophore production was assessed on CAS agar plates (Murakami et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Activated cultures (10 \u0026micro;L) were spotted onto CAS plates (three replicates per strain) and incubated at 30\u0026deg;C for 3\u0026ndash;7 days. The diameter of the clear halo (D) and the colony diameter (d) were measured; strains with D/d\u0026thinsp;\u0026gt;\u0026thinsp;1 were scored as positive.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIAA production\u003c/h2\u003e \u003cp\u003eIAA production was quantified using King\u0026rsquo;s medium supplemented with tryptophan (0.2 g L⁻\u0026sup1;) and the Salkowski colorimetric assay (Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cultures were inoculated at 1% (v/v) and incubated at 30\u0026deg;C and 200 rpm for 3 days (three replicates per strain). Cultures were centrifuged at 12,000 rpm for 10 min, and 1 mL supernatant was mixed with an equal volume of Salkowski reagent in a 24-well plate. After incubation in the dark at room temperature for 30 min, absorbance was measured at 530 nm. IAA concentrations were calculated from an IAA standard curve; detectable IAA indicated a positive result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePotassium solubilization and phosphorus transformation\u003c/h2\u003e \u003cp\u003ePotassium solubilization, inorganic phosphate solubilization, and organic phosphate mineralization were evaluated using plate assays on potassium-solubilizing medium, PKO (inorganic P) medium, and Mongina (organic P) medium, respectively (Zhou et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Setiawati and Mutmainnah \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Activated cultures (10 \u0026micro;L) were spotted onto each medium (three replicates per strain) and incubated at 30\u0026deg;C for 3\u0026ndash;7 days. Halo (D) and colony (d) diameters were measured, and strains with D/d\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered positive for the corresponding trait.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNitrogen-fixing potential (acetylene reduction assay)\u003c/h2\u003e \u003cp\u003eNitrogen fixation potential was assessed using the acetylene reduction assay (ARA) (Montes-Luz et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Briefly, 1 mL activated culture was transferred into a 100 mL serum bottle, amended with 2 mL of 0.1 mol L⁻\u0026sup1; glucose, and sealed with a butyl rubber stopper and aluminum cap. A 5 mL headspace sample was withdrawn and replaced with 5 mL acetylene gas, and bottles were incubated at 28\u0026deg;C for 2 days. After incubation, 500 \u0026micro;L headspace gas was sampled and ethylene production was quantified by gas chromatography. ARA was calculated as:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\u003cp\u003ewhere K is the ratio of ethylene to acetylene peak height, T is absolute temperature, X is ambient temperature during measurement, Y is atmospheric pressure during measurement, Z is the injected acetylene volume (mL), W is sample mass (g) (or volume for liquid samples), and t is the incubation time after acetylene injection (h). Strains with ARA\u0026thinsp;\u0026gt;\u0026thinsp;0 were considered to have nitrogen-fixing potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePairwise interactions among indicator strains\u003c/h2\u003e \u003cp\u003eIndicator strains stored at \u0026minus;\u0026thinsp;80\u0026deg;C in glycerol were revived by inoculating 1% (v/v) into 20 mL TSB broth and incubating at 30\u0026deg;C with shaking (200 rpm) until cultures reached OD₆₀₀ \u0026asymp; 0.5. Each culture was then split into two fractions to prepare (i) washed cell suspensions and (ii) cell-free supernatants. For washed cell suspensions, one fraction was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in an equal volume of sterile water. This washing step was repeated twice to remove residual TSB, yielding a medium-free bacterial suspension. For cell-free supernatants, the second fraction was centrifuged at 8,000 rpm for 10 min; the supernatant was collected and passed through a 0.22 \u0026micro;m membrane filter to obtain sterile, cell-free filtrates.\u003c/p\u003e \u003cp\u003eTo quantify pairwise effects mediated by extracellular products, equal volumes of each strain\u0026rsquo;s washed cell suspension were mixed with (i) the sterile supernatant from each of the other four strains, and (ii) sterile TSB broth as a control. All mixtures were incubated at 30\u0026deg;C and 200 rpm for 4 h, after which cultures were serially diluted and plated to determine viable cell density (CFU). For each focal strain, interaction strength was calculated as the difference in CFU between growth in TSB supplemented with a heterologous sterile supernatant and growth in TSB alone; this \u0026ldquo;difference in bacterial concentration\u0026rdquo; was used to characterize pairwise interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePlate assays to test the effects of bacterial consortia on ryegrass performance\u003c/h2\u003e \u003cp\u003ePerennial ryegrass seeds were surface-sterilized by immersion in 70% ethanol for 1 min, followed by 5 min in a disinfectant solution containing 10% (v/v) sodium hypochlorite and 0.1% (w/v) SDS. Seeds were then rinsed ten times with sterile water, suspended in 0.15% agarose, and pre-incubated at 4\u0026deg;C in the dark for 3 days. Bacterial inoculants were prepared by scraping single colonies from TSB agar plates and resuspending them in sterile water. For single-strain treatments, suspensions were adjusted to a common OD₆₀₀ of 0.01. For multi-strain consortia, equal volumes of the component strains were combined and diluted to a final OD₆₀₀ of 0.01 (approximately 0.5 \u0026times; 10⁷ CFU mL⁻\u0026sup1;). Consortia were inoculated directly onto individual seeds by applying 2 \u0026micro;L of bacterial suspension per seed on 20 \u0026times; 20 cm TSB agar plates. Each plate contained 20 seeds, and each treatment was replicated three times. Plates were incubated for 14 days under controlled conditions (22\u0026deg;C, 60% relative humidity, 100 \u0026micro;E m⁻\u0026sup2; s⁻\u0026sup1; light intensity, and a 12 h light : 12 h dark photoperiod).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePot experiment with inoculation of optimal bacterial consortia and transcriptome analysis\u003c/h2\u003e \u003cp\u003ePot experiments were conducted using non-sterilized field soil collected from the sampling site. Five treatments were established: four optimal bacterial consortia (1245, 124, 12345, and 1) and a sterile-water control. For each inoculated treatment, the consortium was applied to achieve a final density of 10⁷ CFU g⁻\u0026sup1; dry soil. Each treatment included three independent replicate pots (11 \u0026times; 11 \u0026times; 10 cm), and each pot was sown with 1.5 g of perennial ryegrass seeds. Leaf chlorophyll content was measured 10 days after sowing, and plant tissues were collected for transcriptome sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quality control\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) and treated with RNase-free DNase I (Takara, Kusatsu, Japan). RNA degradation and contamination were checked on 1% agarose gels. RNA quantity and integrity were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and purity was assessed with a NanoDrop spectrophotometer (Thermo Scientific, DE, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLibrary construction and Illumina sequencing\u003c/h2\u003e \u003cp\u003eFor each sample, 1.5 \u0026micro;g of total RNA was used for library preparation with the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg; (New England Biolabs, USA), following the manufacturer\u0026rsquo;s protocol, with index codes added to label individual samples. Briefly, mRNA was enriched from total RNA using poly(T) oligo-attached magnetic beads and fragmented in NEBNext First Strand Synthesis Reaction Buffer (5\u0026times;) with divalent cations at elevated temperature. First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H\u0026minus;), followed by second-strand synthesis using DNA Polymerase I and RNase H. Double-stranded cDNA ends were repaired and blunted, 3\u0026prime; ends were adenylated, and NEBNext adaptors (hairpin-loop structure) were ligated. Libraries were size-selected (target insert size\u0026thinsp;~\u0026thinsp;200\u0026ndash;250 bp) using AMPure XP beads (Beckman Coulter, USA). USER enzyme (NEB, USA) treatment (37\u0026deg;C for 15 min, then 95\u0026deg;C for 5 min) was performed prior to PCR enrichment using Phusion High-Fidelity DNA polymerase with universal and index primers. Final libraries were purified with AMPure XP and evaluated on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NovaSeq 6000 platform (paired-end 150 bp) by Beijing Allwegene Technology Co., Ltd. (Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRead processing, mapping, and gene expression quantification\u003c/h2\u003e \u003cp\u003eRaw FASTQ reads were processed using in-house Perl scripts to remove adaptor contamination, poly-N reads, and low-quality reads, generating clean reads. Quality metrics including Q20, Q30, GC content, and sequence duplication levels were calculated, and all downstream analyses were based on high-quality clean reads. Clean reads were aligned to the reference genome using STAR. Only reads with perfect matches or a single mismatch were retained for subsequent analyses and annotation. Aligned BAM files were processed using Picard-tools v1.41 and SAMtools v0.1.18 to sort reads, remove PCR duplicates, and merge alignments for each sample. Gene-level read counts were generated with HTSeq v0.5.4p3, and gene expression was normalized as fragments per kilobase of transcript per million mapped reads (FPKM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDifferential expression and pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eDifferential expression was assessed by comparing each consortium treatment to the control (1245 vs control, 124 vs control, 12345 vs control, and 1 vs control) using DESeq (R package v1.10.1), which models read counts with a negative binomial distribution. P-values were adjusted for multiple testing using the Benjamini\u0026ndash;Hochberg method, and genes with an adjusted \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered differentially expressed. KEGG pathway enrichment of differentially expressed genes was performed using KOBAS (Mao et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eFor the field survey, bacterial α-diversity was quantified using the Shannon index. Community β-diversity was calculated based on Bray-Curtis dissimilarity and visualized by principle coordinates analysis (PCoA) to evaluate differences in bacterial community composition between ryegrass rhizosphere and bulk soils. PCoA was implemented in R using the \u003cem\u003evegan\u003c/em\u003e package (Oksanen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo identify bacterial genera that best discriminated between rhizosphere and bulk soil, we performed random forest analysis using the \u003cem\u003erandomForest\u003c/em\u003e package in R (RColorBrewer and Liaw \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Genera were ranked by mean decrease in accuracy, with higher values indicating greater importance for classification. Model performance and feature selection were assessed using 10,000 trees and the rfcv function, and cross-validation curves were visualized with matplot function.\u003c/p\u003e \u003cp\u003eWe employed the Shapiro-Wilk test to assess whether the data conformed to a normal distribution. For datasets that adhered to a normal distribution, we applied one-way ANOVA to analyze variance and employed LSD test for post hoc multiple comparisons. Conversely, for datasets that did not follow a normal distribution, we utilized the Kruskal-Wallis test, a non-parametric method, and conducted multiple comparisons using Dunn\u0026rsquo;s Test. In the context of pot and plate experiments, the treatment effects on variables such as ryegrass height, chlorophyll content, OD600 (ADF-DF), siderophore production (CAS halo ratio, D/d), IAA concentration, acetylene reduction activity (ARA), and plant performance were evaluated using these statistical approaches, ensuring a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eA composite plant performance index was calculated by min\u0026ndash;max standardizing seedling height, fresh weight, and chlorophyll content to a 0\u0026ndash;1 scale and then averaging the standardized values across the three traits.\u003c/p\u003e \u003cp\u003eFor plate assays, hierarchical partitioning was employed to quantify the independent contribution of each bacterial strain to variation in plant performance. Analysis were conducted in R using rdacca.hp with supporting functions from \u003cem\u003evegan\u003c/em\u003e package (Lai et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003ePerennial ryegrass recruits beneficial bacteria to facilitate photosynthesis\u003c/h2\u003e \u003cp\u003eWe compared bacterial communities in the ryegrass rhizosphere and adjacent bulk soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Bacterial α-diversity (Shannon index) was significantly lower in the rhizosphere than in bulk soil, indicating selective filtering of soil bacteria near roots (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Community composition also differed clearly between two compartments, with rhizosphere and bulk soil formed forming two well-separated clusters (Bray-Curtis, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.29, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb). At the phylum level, Actinobacteria were a dominant group in both the rhizosphere and bulk soil. Notably, their relative abundance was 10 percentage points higher in the rhizosphere than in bulk soil (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe identified 20 indicator genera that distinguished rhizosphere from bulk soil, including 9 enriched in the rhizosphere and 11 enriched in bulk soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb; Fig. S2). Ten-fold cross-validation with five repeats showed that model error stabilized with the 10 most relevant genera (Fig. S3). We therefore focused on the top 10 ranked genera, of which six were significantly enriched in the ryegrass rhizosphere. From these, we isolated representative strains from five genera: \u003cem\u003eArthrobacter\u003c/em\u003e (\u003cem\u003eA. pascens\u003c/em\u003e), \u003cem\u003eNocardioides\u003c/em\u003e (\u003cem\u003eN. lianchengensis\u003c/em\u003e), \u003cem\u003eMicrovirga\u003c/em\u003e (\u003cem\u003eM. makkahensis\u003c/em\u003e), \u003cem\u003eAeromicrobium\u003c/em\u003e (\u003cem\u003eA. tamlense\u003c/em\u003e), and \u003cem\u003eChryseobacterium\u003c/em\u003e (\u003cem\u003eC. indoltheticum\u003c/em\u003e). To test whether rhizosphere-enriched bacteria benefit ryegrass, we inoculated indicator isolates into natural soil planted with ryegrass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Compared with the control, inoculation with \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e, and \u003cem\u003eM. makkahensis\u003c/em\u003e significantly increased ryegrass height and the total chlorophyll content of ryegrass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef; Fig. S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eThe five indicator strains exhibit plant growth-promoting traits\u003c/h2\u003e \u003cp\u003eTo explore how the indicator bacteria may promote ryegrass growth, we screened seven plant growth-promoting functions in the five isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Three strains\u0026mdash;\u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e and \u003cem\u003eM. makkahensis\u003c/em\u003e\u0026mdash;showed ACC deaminase activity, with the strongest response in \u003cem\u003eA. tamlense\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Siderophore production was detected in \u003cem\u003eC. indoltheticum\u003c/em\u003e and \u003cem\u003eN. lianchengensis\u003c/em\u003e, with \u003cem\u003eC. indoltheticum\u003c/em\u003e exhibiting the larger halo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). IAA production was observed in \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e and \u003cem\u003eA. tamlense\u003c/em\u003e, and was highest in \u003cem\u003eA. pascens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In addition, all five strains reduced acetylene to ethylene, indicating nitrogen-fixing potential, with activity ranking \u003cem\u003eC. indoltheticum\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eN. lianchengensis\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eA. pascens\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eA. tamlense\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eM. makkahensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eInteractions among indicator bacteria shape combinatorial effects on ryegrass performance\u003c/h2\u003e \u003cp\u003ePairwise interaction assays revealed a largely facilitative network among four strains\u0026mdash;\u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e and \u003cem\u003eM. makkahensis\u003c/em\u003e\u0026mdash;which generally promoted each other\u0026rsquo;s growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In contrast, \u003cem\u003eA. tamlense\u003c/em\u003e showed consistent antagonistic effects on the other four strains, although its own growth could be supported by them (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Notably, \u003cem\u003eC. indoltheticum\u003c/em\u003e was most strongly stimulated by \u003cem\u003eA. pascens\u003c/em\u003e and \u003cem\u003eM. makkahensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next evaluated all possible strain combinations for their effects on ryegrass performance (A composite index integrating seedling height, fresh weight, and chlorophyll content.). Plant performance varied substantially across consortia, with consortia 1245 (\u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e, \u003cem\u003eM. makkahensis\u003c/em\u003e), 124 (\u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e), 12345 (\u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eC. indoltheticum\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, \u003cem\u003eN. lianchengensis\u003c/em\u003e, \u003cem\u003eM. makkahensis\u003c/em\u003e), and strain 1 alone (\u003cem\u003eA. pascens\u003c/em\u003e) showing relatively higher performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec; Fig. S5). Across combinations, consortia containing \u003cem\u003eA. pascens\u003c/em\u003e tended to outperform those built around the other strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Diversity effects were strain-dependent: performance increased with consortium richness when \u003cem\u003eC. indoltheticum\u003c/em\u003e or \u003cem\u003eM. makkahensis\u003c/em\u003e was included, whereas consortia containing \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e, or \u003cem\u003eN. lianchengensis\u003c/em\u003e showed a non-linear pattern, reaching a minimum at three-member consortia (Fig. S6). Variance decomposition and hierarchical partitioning identified \u003cem\u003eA. pascens\u003c/em\u003e, \u003cem\u003eA. tamlense\u003c/em\u003e and \u003cem\u003eC. indoltheticum\u003c/em\u003e as the strongest contributors to enhanced ryegrass performance, with \u003cem\u003eA. pascens\u003c/em\u003e as the primary driver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eBacterial consortia enhance photosynthesis-related traits and transcriptional signals\u003c/h2\u003e \u003cp\u003eWe next examined the mechanisms by which the optimal consortia (1245, 124, 12345, and strain 1) promote ryegrass growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Compared to the control, all four treatments significantly increased total chlorophyll, Chlorophyll \u003cem\u003ea\u003c/em\u003e and chlorophyll \u003cem\u003eb\u003c/em\u003e contents. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscriptomic profiling showed extensive transcriptional reprogramming in ryegrass leaves following inoculation, with thousands of genes up- or downregulated across treatments (Fig. S8). KEGG enrichment of upregulated genes consistently highlighted photosynthesis-related pathways, particularly photosynthesis-antenna proteins and photosynthesis, across all four consortia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026ndash;f). In contrast, downregulated genes were enriched in lipid-related pathways, with α-linolenic acid metabolism repeatedly suppressed across treatments (Fig. S9a\u0026ndash;d). Collectively, these results indicate that the optimal consortia promote ryegrass growth primarily by enhancing photosynthesis-associated processes while concomitantly repressing linolenic acid metabolism.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrates that the bacterial α-diversity in the rhizosphere of ryegrass is significantly lower than in the adjacent bulk soil, with the two communities forming distinctly clusters. This is consistent with the well-established rhizosphere effect, where plant roots recruit and shape microbial communities primarily through the release of soluble carbon sources and signaling molecules (Singh et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Panchal et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These environmental changes create selective pressures, leading to the enrichment of specific microbial groups while reducing overall diversity (Ling et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, Actinobacteria dominate both the rhizosphere and bulk soil, with a higher relative abundance observed in the rhizosphere. This finding aligns with previous studies that have shown Actinobacteria to be a dominant group in the rhizosphere of several plant species, including ryegrass (Lagos et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Borowik et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This enrichment of Actinobacteria can be attributed to several factors, including their ability to use complex substrates, their tolerance to drought and low-nutrient conditions, and their rich secondary metabolism and antagonistic potential (Boukhatem et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These characteristics allow them to thrive in the challenging rhizosphere environment, where competition for resources is high. Furthermore, our study identified \u003cem\u003eArthrobacter pascens\u003c/em\u003e, a strain within the Actinobacteria phylum, as a key contributor to plant performance in ryegrass rhizosphere. We found that \u003cem\u003eA. pascens\u003c/em\u003e significantly promoted plant growth, likely through its production of IAA, which has been shown in previous studies to stimulate root elongation and enhance overall plant growth (Tian et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Etesami and Glick \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This suggests that \u003cem\u003eA. pascens\u003c/em\u003e may serve as keystone bacteria in the ryegrass rhizosphere, directly influencing plant growth and enhancing community-level functional outputs by promoting other beneficial strains.\u003c/p\u003e \u003cp\u003eThe five representative strains from rhizosphere-enriched groups significantly enhanced ryegrass growth, particularly increasing plant height and chlorophyll content. These strains exhibited a range of plant growth-promoting traits, such as ACC deaminase activity, which modulates ethylene levels to promote root and shoot growth (Gamalero et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); IAA production, which promotes the elongation of plant cells and stimulates root growth (Mohite \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); and siderophore production, which improves iron bioavailability (Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), essential for chlorophyll formation (Therby-Vale et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, all five strains exhibited acetylene reduction activity, indicating nitrogen-fixing potential, which further supports increased chlorophyll content and overall plant growth (Muhammad et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We propose that the synergistic effects of bacterial-derived IAA and siderophores contribute to the increase in chlorophyll content and photosynthetic efficiency (Salazar-Iribe and De-la-Pe\u0026ntilde;a \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Brick et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, the potential contributions from nitrogen fixation and ACC deaminase regulation may further support leaf development and photosynthesis (Mu and Chen \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chandwani and Amaresan \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), leading to improved plant performance. These findings highlight the importance of these bacteria\u0026rsquo;s functional traits in regulating plant metabolic processes, which are essential for optimizing growth and productivity.\u003c/p\u003e \u003cp\u003eOur analysis of bacterial interactions revealed both cooperative and antagonistic behaviors that collectively influence plant performance. Except for \u003cem\u003eA. tamlense\u003c/em\u003e, the other four rhizosphere-enriched strains promoted each other\u0026rsquo;s growth, forming a mutually beneficial network. In contrast, \u003cem\u003eA. tamlense\u003c/em\u003e exhibited antagonistic effects on the other strains, though its growth was still supported by them. This asymmetric mutualism-antagonism network implies the importance of balancing positive and negative interactions to prevent the overgrowth of any single strain, ensuring community stability and ecological resilience (Lopes et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Further analysis of multi-strain consortia showed that combinations centered around \u003cem\u003eA. pascens\u003c/em\u003e resulted in the best plant performance, even with single-strain inoculation. However, multi-strain consortia exhibited a nonlinear response, with a performance dip at three-strain combinations (Fig. S6). This can be explained by a balance between functional complementarity and resource competition: when bacterial richness was low (fewer than four strains), competition limited plant benefits, but as richness increased, complementary functions outweighed the competitive costs, enhancing plant growth. This supports the concept of functional complementarity, where diverse microbial communities provide synergistic effects, particularly in complex environments (Sarsan et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Puente-S\u0026aacute;nchez et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, increasing community richness, including two other strains, led to improved plant performance, consistent with previous reports linking bacterial diversity to plant growth (Weidner et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Laforest-Lapointe et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe optimal bacterial consortia significantly enhanced chlorophyll content, particularly chlorophyll \u003cem\u003eb\u003c/em\u003e, reflecting an increase in the plant\u0026rsquo;s photosynthetic capacity. Transcriptomic analysis further confirmed that the upregulated genes were predominantly involved in the photosynthesis-antenna proteins and photosynthesis pathways, suggesting that bacterial inoculation enhances plant growth by improving light capture and electron transport. The increase in chlorophyll \u003cem\u003eb\u003c/em\u003e correlates with the expansion of light-harvesting complexes, optimizing light energy absorption (Lokstein et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, a consistent downregulation of α-linolenic acid metabolism was observed across all treatments. This pathway is a precursor for jasmonic acid (JA) and other oxylipins (Mosblech et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The inhibition of this pathway suggests a shift in the growth-defense trade-off: when beneficial microbes colonize, plants may downregulate JA-related pathways to reduce investment in defense, thereby reallocating resources towards photosynthesis and growth, ultimately achieving higher plant performance (He et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the critical role of \u003cem\u003eA. pascens\u003c/em\u003e in promoting ryegrass growth, particularly through IAA production. While \u003cem\u003eA. pascens\u003c/em\u003e has been identified as a keystone bacterium in the ryegrass rhizosphere, the exact mechanisms by which ryegrass recruits this beneficial microbe remain unclear and warrant further investigation. Furthermore, it is still unknown whether \u003cem\u003eA. pascens\u003c/em\u003e is widely enriched in the rhizosphere of other leaf-dominated plants, such as forage grasses. Alongside the role of individual bacteria, our findings suggest that the composition and number of strains in synthetic microbial consortia play a crucial role in optimizing plant performance. Identifying the optimal number of strains for maximum plant benefit is an intriguing and underexplored area that requires further investigation. Understanding how microbial richness and functional complementarity affect plant growth will be essential for improving microbial management strategies. This deeper understanding could lead to more targeted, effective applications of microbial consortia in sustainable agricultural practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTing Liu and Jiaguo Jiao were supported by the National Key R\u0026amp;D Program (2024YFD1501803 and 2023YFD1901401). Weichen Hou was supported by Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (Project No. KYCX25_0998). Libo Fu and Yingxue Wang were supported by the Earmarked fund for Modern Agroindustry Technology Research System-Green manure (CARS-22).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAli SZ, Sandhya V, Venkateswar Rao L (2014) Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp. 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Adv Mater Res 590:100\u0026ndash;105. https://doi.org\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/10.4028/www.scientific.net/AMR.590.100\u003c/span\u003e\u003cspan address=\"http:///10.4028/www.scientific.net/AMR.590.100\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Forage grasses, Rhizosphere bacteria, Bacterial interactions, Plant-microbe interaction, Plant performance","lastPublishedDoi":"10.21203/rs.3.rs-8828630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8828630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground and Aims\u003c/em\u003e Forage grasses whose yield is almost entirely leaf-based rely on high canopy photosynthesis and rapid leaf regrowth, yet how they recruit root-associated bacteria to support this aboveground performance remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethods\u003c/em\u003e Soil samples from ryegrass rhizospheres and bulk soils were analyzed through 16S rRNA sequencing and bacterial isolation, followed by pot and plate experiments to assess the plant growth-promoting effects and interactions of selected strains, along with their transcriptomic responses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e We identify a small set of root-recruited bacterial strains in wild ryegrass that consistently enhance plant growth and leaf greenness. Each strain individually increased biomass and chlorophyll content, but they differed in plant-beneficial functions such as hormone production and nutrient mobilization. When assembled into single- and multi-strain communities, their effects on plant performance were strong and non-additive:\u003cem\u003eArthrobacter pascens\u003c/em\u003e, the field-dominant rhizosphere strain of wild ryegrass, generated disproportionate gains in growth and chlorophyll, whereas other members acted as complementary helpers that further amplified plant responses in mixtures.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusion\u003c/em\u003e Our work illustrates how leaf‐dominated crops can harness naturally recruited microbial allies to enhance photosynthetic capacity and leaf production, providing insights for designing microbial inoculants for forage grasses.\u003c/p\u003e","manuscriptTitle":"A driver bacterial strain and trait-complementary partners enhance photosynthesis and growth in ryegrass","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 10:28:15","doi":"10.21203/rs.3.rs-8828630/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-03-25T04:02:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-12T08:47:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-12T08:26:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-02-11T01:48:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T01:07:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-02-09T04:39:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"931f5d87-d3b0-4dfe-b7eb-a7e2c2c983bd","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T00:35:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 10:28:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8828630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8828630","identity":"rs-8828630","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}