Bio-inspired core-shell microcapsules enhance plant salinity tolerance by improving stability and survival of non-spore bacteria

Abstract

Non-spore-forming bacteria can enhance crop salinity tolerance via various strategies, but poor viability during storage and field application limits their use. Inspired by the robust structure of spore dormancy, a core-shell microcapsule consisted of sodium alginate, poly (γ-glutamic acid), and chitosan (APC) is proposed. Using Pantoea alhagi NX-11 as a model, we found that APC encapsulation significantly enhanced bacterial survival during room-temperature storage compared to free cells or alginate beads. This protective effect was further confirmed using two other non-spore-forming strains. The survival mechanism was mainly attributed to the APC-induced metabolic dormancy via suppression of the TCA cycle and oxidative phosphorylation pathways. Furthermore, inoculation with APC-encapsulated NX-11 increased dry weight of rice plant by 24.2% under salt stress in greenhouses and increased grain yield by 15.8% in saline fields, attributing to the enhanced root colonization of NX-11. Overall, this bio-inspired encapsulation strategy provides an effective approach for developing robust microbial inoculants to improve crop resilience in saline soils.

Poor storage stability and soil survival limits agricultural application of non-spore forming bacteria. Here, the authors develop a hydrogel microcapsule which improves storage and soil survival, demonstrating application in improving plant salt tolerance under field conditions.

Introduction

Abiotic stress seriously affects crop growth and yield in modern agricultural production, among which salt stress is one of the most important factors^1,2. Approximately 20% of arable land worldwide is affected by salinization, with an annual increase rate of 10%. It is estimated that by 2050, over 50% of arable land worldwide will be affected by salinization^3,4. Under salt stress, plants accumulate excessive Na⁺ and Cl^-, which cause osmotic stress, ion stress, and inhibition of the synthesis of proteins. Moreover, the reactive oxygen species induced by salt stress can cause peroxidization of the lipids in the plant cell membrane^5,6. Hence, it is urgent to alleviate salt damage in plants to sustain a steady supply of food in the face of a growing human population.

Plant growth-promoting bacteria (PGPB), as soil-derived probiotics, can enhance plant salt tolerance by inducing the antioxidant system⁷, increasing the activity of antioxidant enzymes⁸, and promoting the accumulation of osmotic regulatory substances in plants^9,10. However, it is still challenging to develop high-quality microbial inoculants because most species of PGPB are susceptible to environmental stresses during storage and after inoculation to the soil¹¹. For example, certain microbial inoculants might not be able to compete with the native rhizosphere microbiome leading to low efficiency of colonization^12,13. As a result, some studies have reported only limited success of PGPB^14,15. This is more difficult for the non-spore forming bacteria that lack of spore dormancy structure, which are considered as the majority of species of PGPB¹⁶. Therefore, it is significant to develop high-quality microbial inoculant of non-spore forming bacteria to ensure long-term survival of inoculant and finally increase plant salt tolerance¹⁷. However, relevant technologies are needed to simultaneously facilitate long-term storage stability and effective rhizosphere colonization of non-spore-forming bacteria, and the underlying mechanisms are necessary to explore.

Oxygen and water activity are two critical stress factors affecting the stability of microbial inoculants^18,19. Consequently, most current research focuses on reducing the moisture content of microbial cells through dehydration and drying technologies, thereby inducing dormancy and extending shelf life^19,20. However, this approach is less effective for non-spore-forming bacteria, primarily due to sensitivity to moisture, which leads to an exponential decline in viable cell counts during the drying process^17,21. Instead, inducing microbial dormancy by controlling oxygen levels represents the most reliable strategy for non-spore-forming bacteria. Additionally, establishing protective micro-environment that protect microbial cells against competitive indigenous soil microflora can further facilitate application of microbial inoculants in soil environment²². However, an integrated approach combining the induction of microbial dormancy and the facilitation of rhizosphere colonization remains unexplored, representing a pivotal challenge in translating microbial technologies to sustainable agriculture.

Here, inspired by the dormancy structure of microbial spores, we developed core-shell microcapsules consisted of sodium alginate, poly (γ-glutamic acid), and chitosan (APC) to induce dormancy of microbial cells and extend the shelf life of microbial inoculant through inhibiting oxygen permeability (Fig. 1). Sodium alginate (SA), a crucial extracellular polymeric substance of Pseudomonas aeruginosa biofilms, can protect microbial cells from harsh storage conditions²³. Poly (γ-glutamic acid) (γ-PGA) is the key component of Bacillus subtilis biofilms with characteristics of enhancing the microbial inoculant viability under adverse conditions, and the plant stress tolerance^24–28. Here, we developed hydrogel core with dual-network of SA and γ-PGA to improve encapsulation efficiency of microcapsules, and establish a biofilm-like microenvironment for microbial cells, thereby facilitating their plant growth-promoting functions. Chitosan (CS) was chosen as cationic polymer to form spore-like structure microcapsules through electrostatic interaction. Pantoea alhagi NX-11, a non-spore-forming bacteria isolated from rhizosphere soil of rice in saline soil, is a PGPB with capacity of enhancing salt tolerance of rice²⁹. Here, we used NX-11 as a model non-spore forming bacteria to verify the cytoprotection of the core-shell microcapsules. Taguchi orthogonal array design was employed to determine the optimum conditions for preparation of core-shell structured microcapsules, and the resulting microstructure of microcapsules was then characterized. The mechanism of multilayer structure on viability of microbial cells during storage was investigated using proteomics. Furthermore, we investigated the effects of APC-encapsulated NX-11 on the rhizosphere bacterial community structure of rice, the survival of NX-11 in the rhizosphere, and plant growth under salt stress.

Fig. 1. Hydrogel microsphere with core-shell structure for enhanced viability of microbial inoculants for improving plant salinity tolerance.

a Preparation process of core-shell microcapsules. Sodium alginate (SA), microbial cells, and poly (γ-glutamic acid) (γ-PGA) formed hydrogel core with biofilm-like structure, and microparticles are encapsulated with chitosan (CS) throughelectrostatic interaction. b The dense core-shell structure (scale bar = 100 µm) can limit oxygen diffusion. During storage, bacteria entrapped in core-shell microcapsules (scale bar = 50 µm) grow into aggregate cell colonies (scale bar = 5 µm), due to inhibition of energy metabolism. The experiment was repeated three times independently with similar results. In bubble chart, the sizes of the bubbles indicate the number of differentially expressed proteins enriched in each signaling pathway and the color of the bubbles represents the p values (Fisher’s exact test). c Schematic drawing showing how encapsulated microbial inoculant promotes the growth of rice under salt stress. Direct inoculation of bacterial inoculant into the soil environment leads to a sudden decrease of viability due to abiotic environmental stresses and competition of native microflora. Microcapsules can provide a temporary barrier for bacterial inoculant, thus ensures a slowed release of the inoculant and promotion of colonization in plant rhizosphere.

Results

Optimization conditions for preparing core-shell microcapsules

A technique of one-factor-at-a-time (OFAT) experimental design was utilized to determine the suitable and reliable range of each process factor (Supplementary Fig. 1, see Appendix S1 for more details of OFAT) for preparing core-shell microcapsules. The encapsulation efficiency, defined as the percentage of viable bacterial cells successfully incorporated into the microcapsules relative to the total number of viable cells initially added, was selected as the response variable to optimize the preparation process. The encapsulation efficiency showed specific patterns with different components: Increasing SA/γ-PGA ratio from 1:1 to 2:1 improved encapsulation efficiency, but ratios above 4:1 reduced efficiency. CaCO₃ concentrations (0.5–1.5 wt%) enhanced encapsulation, while exceeding 2.0% decreased performance. Higher polymers/bacteria ratio (1:1 to 6:1) progressively increased encapsulation. CS concentration beyond 0.3 wt% caused sharp declines of encapsulation efficiency and viable cells (Supplementary Fig. 2). There was no remarkable change of encapsulation efficiency when other conditions varied within a specific range (oil-to-water phase ration, 3:1-5:1; concentration of acetic acid, 1500–2500 μL/L; emulsification time, 10–25 min; stirring speed, 250–750 rpm; concentration of Span 80, 2–5 wt%).

Taguchi orthogonal array design based on the results of OFAT experiments was applied to determine the optimum conditions and the relative importance of different factors for microcapsules preparation (see Appendix S2 for more details of Taguchi orthogonal array design). Encapsulation efficiency was used as an optimization index for orthogonal array analysis. The L₁₆ (4⁵) orthogonal model is shown in the Supplementary Table 1. The optimum conditions for encapsulation efficiency of microcapsules were as follows: SA/γ-PGA ratio of 4:1, CaCO₃ concentration of 1.5%, polymers/bacteria ratio of 6:1, and CS concentration of 0.3%. The relative importance of different factors was in the order: ratio of polymers/bacteria > concentration of CS > concentration of CaCO₃ > ratio of SA/γ-PGA (Supplementary Table 2). Ratio of polymers to bacteria, concentration of CS as well as concentration of CaCO₃ significantly influenced the encapsulation efficiency of microcapsules (Supplementary Table 3). The microcapsules prepared under optimum technological conditions were shown in Supplementary Table 4.

Characterization of core-shell structure formation

In the infrared spectrum of microcapsules prepared under optimum technological conditions (Fig. 2a, b), the disappearance of the amino absorption peak at 1652 cm^-1 in chitosan, the carboxyl absorption peak at 1640 cm^-1 in γ-PGA, and the carboxyl absorption peak at 1602 cm^-1 in sodium alginate indicated the formation of chemical bonds between amino and carboxyl groups through electrostatic interactions³⁰. The stretching vibrations of the O-H in sodium alginate and γ-PGA led to broad absorption bands at 3321 cm⁻¹ and 3276 cm⁻¹ respectivley³¹. The corresponding absorption band in microcapsules shifted to 3232 cm⁻¹, which indicated the presence of hydrogen bonds between sodium alginate and γ-PGA³². The interaction between CS, SA, and γ-PGA was verified through XRD characterization (Fig. 2c). CS exhibited two strong absorption peaks at 11.69 ° and 20.33 °, which related to the hydrated and anhydrous crystals, respectively³³. SA exhibited two broad absorption peaks at 13.46 ° and 21.82 °, due to its amorphous structure. γ-PGA only exhibited one broad absorption peak at 23.64 °, which indicated its amorphous structure³⁴. In contrast, the XRD patterns of microcapsules showed an amorphous morphology, which suggested that electrostatic interaction broke the hydrogen bonding between amino and carboxyl groups. SA and CS exhibited similar thermal behavior, and lost half of weight at 261.06 °C and 261.36 °C (Fig. 2d). In addition, APC lost half weight at 340.12 °C, and exhibited the lowest weight loss (62.04%), which indicated the structure of microcapsules can improve the thermal stability.

Fig. 2. Characterization of core-shell structure formation.

a Images of core-shell structure microcapsules. The experiment was repeated three times with similar results and one representative image is shown. b FTIR spectra of the raw biomaterials and microcapsules. c X-ray diffraction (XRD) patterns of raw biomaterials and microcapsules. d Thermal gravimetric analysis of raw biomaterials and microcapsules. e X-ray photoelectron spectroscopy (XPS) wide scan of raw biomaterials and microcapsules. XPS N 1 s narrow scans with the curve fit of γ-PGA (f), CS (g), and microcapsules (h). SA sodium alginate, γ-PGA poly (γ-glutamic acid), CS chitosan, APC core-shell microcapsule consisted of sodium alginate, poly(γ-glutamic acid), and chitosan.

XPS analyses were performed to verify the potential interaction of amino and carboxyl groups in the obtained biocomposite and to confirm the proposed adsorption mechanism by FTIR and XRD characterization. The signal corresponding to the binding energy of N 1 s was observed in γ-PGA, CS and APC, while no peak of N 1 s was found in the XPS spectra of SA, indicating the surfcace of microcapsules were consisted of SA, γ-PGA and CS (Fig. 2e). The presence of the signal corresponding to the binding energy of Ca 2p likely contributed to the formation of gel achieved by ionic crosslinking. N 1 s high-resolution spectrum of γ-PGA and CS revealed two binding energy signals at 399.2 and 401.3 eV (Fig. 2f, g), corresponding to the amine and amide groups, respectively. In addition, the spectrum of APC revealed another peak at 402.0 eV (Fig. 2h), which was considered as protonated amine^33,35. The presence of protonated amine on the surface of microcapsules suggested that amine could interact through static electricity and thus formed composite coatings on surface of microcapsules. FTIR, XRD, and XPS analyses collectively demonstrate the successful formation of a core-shell structure in the APC microcapsules, characterized by SA/γ-PGA hydrogel core encapsulated within CS shell via electrostatic interactions.

Characterization of microcapsules with different structure

Microcapsules with different structure (A, sodium alginate microcapsules; AP, sodium alginate/poly (γ-glutamic acid) microcapsules; APC, sodium alginate/poly (γ-glutamic acid)/chitosan core-shell microcapsules) were fabricated to determine the effect of core-shell structure on microcapsules (Fig. 3a–d). The dynamic rheological properties of hydrogel were illustrated in Fig. 3e. In the entire frequency range, the storage modulus (G’) of all samples exhibited higher than loss modulus (G”), confirming a typical solid-like rheological behavior³⁶. Moreover, the G’ was almost an order of magniude higher than the G”, indicating a strong network structure. The addition of CS significantly increased the values of G’ and G”, indicating that the formation of composite coatings on surface of microcapsules strengthened the structure of microcapsules. In addition, values of G’ and G” gradually increased with the increasing frequency in all samples, suggesting a frequency dependence.

Fig. 3. Characterization of microcapsules with different structure.

a Fabrication of SA microcapsules (A). b Fabrication of SA-γ-PGA microcapsules (AP). c Fabrication of sodium alginate/poly (γ-glutamic acid)/chitosan core-shell microcapsules (APC). d Optical images of APC microcapsules. Scale bar = 200 µm. e Rheology characterization of hydrogel with different structure. f Scanning electron microscopy images of microcapsules. Scale bar = 50 µm. Diameter-size distribution of A (g), AP (h), and APC (i), 100 microspheres per condition. The adsorption-desorption isotherm of nitrogen (j) and the pore size distribution (k) of the microcapsules. l Degradation of microcapsules in soil at room temperature. In (d,f), the experiment was repeated three times independently with similar results. In l, data are presented as mean values ± SD (n = 3 biological replicates).

The morphologies of microcapsules were examined by SEM, and we found that the microcapsules were made dense, and formed into nearly spherical shape due to the addition of γ-PGA and CS (Fig. 3f). Moreover, compared to AP, APC exhibited denser surface and increased volume, indicating that surface of microcapsules was successfully encapsulated by CS coating. The average diameter of A microcapsules was 324.22 µm, while AP microcapsules presented a mean diameter of 207.07 µm (Fig. 3g–i). All microcapsules (A, AP, and APC) were fabricated under identical preparation conditions before characterization (see more details of preparation conditions on Supplementary Table 4). These results also revealed that the incorporation of γ-PGA contributed to the narrower distribution of size and the denser structure of AP microcapsules. After encapsulated with CS, the mean diameter of microcapsules increased to 234.95 µm due to formation of core-shell structure. The specific surface area, pore volume, and pore size of the microcapsules were determined through nitrogen adsorption-desorption isotherms (Fig. 3j, k). We found that compared with sodium alginate microcapsules, the specific surface area and pore size of AP microcapsules decreased (Supplementary Table 5). These results indicated that the addition of γ-PGA formed the denser structure of microcapsules. Furthermore, compared with AP microcapsules, the specific surface area, the pore volume, and the diameter of microcapsules with core-shell structure decreased by 60.47%, 60.06%, and 28.27%, respectively. The electrostatic interaction between amino and hydroxyl groups likely filled the pore in gel network, which further reduced the release rate of encapsulated microbial agents. To determine whether the microcapsules in our study can be easily degraded, we added A, AP, and APC microcapsules to the soil. We found that SA bead exhibited a 61.1% weight loss after incubation for 28 days (Fig. 3l). The rate of weight loss was slightly increased with addition of γ-PGA, which likely due to that γ-PGA can be easily degraded by soil microbes. In contrast, APC exhibited only 44.7% weight loss after 28 days in soil, which may be due to the complex gel structure of the core-shell microcapsules. A lack of degradation of microcapsules limits the release of encapsulated PGPB and further soil colonizaiton, whereas rapid degradation limits the protective function of microcapsules, as a result, a moderate biodegradation rate of microcapsules is crucial for bacterial soil colonization. The degradation rate of 44.7% after 28 days in soil suggests that the APC microcapsule can provide protection for PGPB and a sustained release of the encapsulated bacteria into the soil.

Viability of microbial inoculant during storage

The cells encapsulated in A microcapsules exhibited a planktonic state. In contrast, bacteria in APC microcapsules were tightly associated together, growing into aggregate cell colonies (Fig. 4a). To evaluate the effect of microcapsules on bacterial viability during long-term storage, a comparative study on the storage stability of the free cells and encapsulated microbial inoculant within microcapsules was conducted at 25 °C for 12 weeks. The initial viable cell count was standardized to approximately 10⁹ CFU/g across all treatment groups. We found that the viability of free NX-11 decreased to 2.95 log CFU/g, while NX-11 in A, AP and APC microcapsules exhibited viability values of 6.65, 7.32 and 8.43 log CFU/g, respectively, at the end of the experiment (Fig. 4b). In addition, microcapsules decreased reactive oxygen species (ROS) generation in inoculants during storage (Supplementary Fig. 3, see Appendix S3 for more details of ROS assay). Similarly, free Lysobacter enzymogenes OH11 and Pseudomonas nitroreducens L16 decreased to 8.01 and 4.95 log CFU/g after 8-week storage, while the viability of Lysobacter enzymogenes OH11 and Pseudomonas nitroreducens L16 entrapped within APC showed 9.04 and 6.94 log CFU/g, respectively (Fig. 4c, d). The release of bacteria from APC was slower than from A and AP, which may attributed to the denser microstructure of the core-shell system (Supplementary Fig. 4a). To analyze the release mechanism, we fitted the data to kinetic models according to the methods described by previous studies^37,38 (see Appendix S4 for more details of release kinetics). The release process was best described by the Ritger-Peppas model based on the correlation coefficient (R²) analysis (Supplementary Fig. 4b–d). The calculated diffusional exponent (n) exceeded 0.85 for all microcapsules, identifying Case-II transport as the dominant mechanism. This indicated that the release of bacteria from microcapsules is primarily controlled by the corrosion and dissolution of the polymer skeleton.

Fig. 4. Viability of microbial inoculant during storage.

a SEM photographs of cells entrapped in microcapsules after 12-week storage. Scale bar = 5 µm. The experiment was repeated three times independently with similar results. b Viability of free Pantoea alhagi NX-11 and encapsulated Pantoea alhagi NX-11 during storage. CK, free P. alhagi NX-11; A-NX, P. alhagi NX-11 entrapped in sodium alginate microcapsules; AP-NX, P. alhagi NX-11 entrapped in sodium alginate/poly (γ-glutamic acid) microcapsules; APC-NX, P. alhagi NX-11 entrapped in sodium alginate/poly (γ-glutamic acid)/chitosan microcapsules. c Viability of free and core shell microcapsules-encapsulated Lysobacter enzymogenes OH11 during storage. d Viability of free and core shell microcapsules-encapsulated Pseudomonas nitroreducens L16 within during storage. In (b–d), data are presented as mean values ± SD (n = 3 biological replicates). e Principal component analysis (PCA) of the proteomics data. f Volcano plots of the protein expression profiles between the CK-12W (free P. alhagi NX-11 after 12-week storage) and CK-0W treatments (fresh P. alhagi NX-11). g Volcano plots of the protein expression profiles between the APC-12W (P. alhagi NX-11 entrapped in APC microcapsules after 12-week storage) vs CK-12W treatments. Red, blue, and gray dots represent significantly up-regulated, down-regulated, and non-significantly changed proteins. h Enrichment bubble chart of the KEGG pathways of down-regulated DEPs between APC-12W and CK-12W. i Enrichment bubble chart of the KEGG pathways of up-regulated DEPs between CK-12W and CK-0W. The sizes of the bubbles indicate the number of differentially expressed proteins enriched in each signaling pathway and the color of the bubbles represents the p values. Biochemical metabolic pathways affected by encapsulation during storage, including oxidative phosphorylation and TCA cycle (j). The different colors indicate proteins expression levels based on the log₂ fold change values. Blue and red colors represents down and up regulated proteins, respectively. In panels f-g, the p values of two-sided unpaired t-test are indicated. In (h–I), the p values of Fisher’s exact test are indicated.

To investigate the mechanisms involved in the long-term storage of microbial inoculant in APC, the differentially expressed proteins (DEPs) of the strains were further analyzed using data-independent acquisition (DIA) analysis. Clear variations in protein composition were observed between inoculants in A, AP and APC microcapsules (Fig. 4e). 1373 DEPs between the free inoculant at the start of the storage experiment (CK-0W) and after 12-week storage (CK-12W), including 593 up-regulated and 780 down-regulated (Fold change > 2, p < 0.05, Fig. 4f). 1217 up-regulated proteins and 249 down-regulated proteins were found between the free NX-11 after 12-week storage (CK-12W) and APC-encapsulated NX-11 after 12-week storage (APC-12W) (Fig. 4g). The KEGG pathway enrichment analysis showed that oxidative phosphorylation was significantly down-regulated in the APC-12W treatment compared to CK-12W treatment (Fig. 4h).In contrast, oxidative phosphorylation was up-regulated in the CK-12W treatment (Fig. 4i), compared to the CK-0W treatment. Additionally, compared to the CK-0W treatment, sulfur metabolism was down-regulated in the APC-12W treatment (Supplementary Fig. 5), but compared to the CK-12W treatment, sulfur metabolism was up-regulated in the APC-12W treatment (Supplementary Fig. 6).

Proteomic analysis revealed that APC encapsulation induced a dormancy-like state of NX-11 during storage, characterized by suppression of energy associated pathways, such as tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathways. Compared to CK-12W, APC-12W revealed a significant down-regulation of proteins associated with TCA cycle, including citrate synthase (B1H58_01115), isocitrate dehydrogenase (B1H58_19555), and fumarate hydratase (B1H58_17845), were markedly suppressed (Fig. 4j). This suppression likely limits the generation of NADH, and consequently lead to down-regulation of key complexes in the oxidative phosphorylation pathway, including NADH dehydrogenase (NuoB, Nuol), succinate dehydrogenase (SdhA, SdhB, SdhC), and ATP synthase (B1H58_07660, B1H58_07645, and B1H58_07670). In contrast, the CK-12W showed an up-regulation of these pathways compared to CK-0W. These results demonstrated that APC microcapsules enhance storage stability by inhibiting energy metabolism.

Effects of encapsulated microbial inoculant on plant growth under salt stress

Inoculation with APC-encapsulated NX-11 significantly increased the shoot length and dry weight of rice seedlings compared to uninoculated plants under salt stress (Fig. 5a–d, Supplementary Fig. 7a, see Supplementary Table 6 for physiochemical characteristics of the soil). Importantly, the dry weight of rice seedlings inoculated with APC-encapsulated NX-11 was increased by 14.61% compared to the ones inoculated with free NX-11 and increased by 50.88% compared to the ones inoculated with empty microcapsules. Moreover, the inoculation with APC-encapsulated NX-11 significantly increased soluble protein, soluble sugar, and proline contents in rice than the inoculation with free NX-11 (Supplementary Fig. 7b–d). APC-encapsulated NX-11 significantly increased the activities of SOD, CAT, and POD of rice, while decreased the content of H₂O₂ and MDA compared to empty microcapsules and free NX-11 treatments (Fig. 5e–g and Supplementary Fig. 8a, b). Notably, the application of empty microcapsulse had no significant effect on the content of H₂O₂ and MDA compared to uninoculated plants under salt stress. The inoculation of APC-encapsulated NX-11 also increased the content of ABA in rice compared to empty microcapsules and free NX-11 treatments, while had no effect on IAA content (Supplementary Fig. 8c, d). These results suggested that APC encapsulation enhanced the efficiency of NX-11 in promoting the growth of rice under salt stress via alleviating the oxidative stress and accumulating osmotic adjustment substances. APC-encapsulated NX-11 showed higher efficienct in alleviating oxidative stress and promoting the growth of rice under salt stress compared to NX-11 entrapped in sodium alginate microcapsules (Supplementary Fig. 9), indicating the advancement of bio-inspired core-shell microcapsules.

Fig. 5. Effects of encapsulated microbial inoculant on growth of different crops under salt stress.

a Experimental design of the greenhouse experiments. Growth status (b), shoot length (c), dry weight (d), SOD activity (e), CAT activity (f), and POD activity (g) of rice seedlings grown in greenhouse under different treatments (n = 5 biological replicates). Growth status of tomato (h), maize (i), and lettuce (j) under different treatments. k Growth status of rice in saline field. 1000-grain weight (l) and yield (m) of rice grown in saline field conditions under different treatments. Each treatment in the field experiments have three independent plots (n = 3 biological replicates; 6.0 m × 4.0 m) and 50 plants from five sites in each plot were collected. C, crops without any treatment; S, crops without inoculant under salt stress; NX-S, crops inoculated with free P. alhagi NX-11 under salt stress; M-S, crops inoculated with empty APC microcapsules under salt stress; APC-S, crops inoculated with P. alhagi NX-11 entrapped in APC microcapsules under salt stress. Data in this figure are presented as mean values ± SD. Different letters represent significant differences among the treatments (one-way ANOVA with correction by Tukey’s HSD test, p < 0.05, p values are shown in source data).

The enhanced promoting efficiency of APC-encapsulated NX-11 was also observed on tomato, maize and lettuce under salt stress through enhancing accumulation of osmoprotectants and activities of antioxidant enzymes (Fig. 5h–j, Supplementary Figs. 10–12). Moreover, the field experiments were conducted in saline field to further investigate the effect of microencapsulated NX-11 on plant growth under salt stress (see Appendix S6 and Supplementary Table 7 for more details of field experiments). The inoculation of encapsulated NX-11 increased shoot length and dry weight of rice seedlings (Fig. 5k, Supplementary Fig. 13). Compared to free NX-11 treatment, inoculation with encapsulated NX-11 increased 1000-grain weight and yield by 3.84% and 15.78% (Fig. 5l, m). These results suggested core-shell microcapsules can enhance growth-promoting effect on different crops, and exhibited practical applicability.

Effect of encapsulated inoculant on rhizosphere microbial communities and its soil survival under salt stress

Salt stress significantly decreased the Shannon index of rhizosphere bacterial community (Fig. 6a), and the inoculation of NX-11, empty microcapsules, and APC-encapsulated NX-11 had no significant effects on Shannon index compared to the salt treatment. Principal coordinate analysis (PCoA) based on Bray–Curtis distance metrics revealed that bacterial community structures under salt stress were clearly distinguishable from the non-salt treatment (Fig. 6b). The inoculation of free NX-11 or APC-encapsulated NX-11 clearly changed the structure of the rhizosphere bacterial community compared to the salt treatment. Proteobacteria, Bacteroidota, Firmicutes, Acidobacteriota, and Actinobacteriota were the top 5 bacterial phylum of all treatments (Fig. 6c), which accounted for > 80% of the total sequences, while no significant variations existed in the relative abundance of these phyla between the treatments. However, we found clear variations in the relative abundance of top genera between the five treatments resulting in clear 5 clusters (Fig. 6d). The LEfSe analysis showed that Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Sphingopyxis, Caenispirillum, Sphingomonas, Pedobacter, Pantoea, and Devosia were significantly enriched (LDA Score > 3.2, p < 0.05) in rhizosphere of rice inoculated with encapsulated NX-11 (Fig. 6e). Importantly, we found that NX-11 was uniquely detected in the rice rhizosphere with the inoculation of NX-11 entrapped in APC microcapsules after matching the full-length 16 s rRNA gene sequences of the NX-11 with the ASVs (Fig. 6f).

Fig. 6. Effect of NX-11 encapsulated with APC microcapsules on bacterial community composition in rice rhizosphere soil under salt stress.

a Shannon index representing bacterial alpha diversity. b Principal coordinates analysis of beta diversity based on Bray-Curtis distances, c Relative abundance (%) of dominant bacterial phylum. d Heatmap displaying the relative abundance of the top 80 genus. e LDA analysis to identify the distinct bacterial genera in rice rhizosphere between treatments. Only the taxa with LDA scores > 3.2 are shown. f Relative abundance of NX-11 (ASV2786) among different treatments. g LSCM images of rice roots inoculated with free GFP-NX-11 (NX) or GFP-NX-11 encapsulated in APC. The experiment was repeated three times independently with similar results. h Effects of APC microcapsules on the colonizaing population of on the roots of rice. NX, free NX-11; APC-NX, NX-11 encapsulated in APC microcapsules. For box plots in this figure, the center line indicates the median, tops and bottoms of boxes indicate the upper and lower quartiles (25 and 75th percentiles), and the whiskers extend to the minimum and maximum data points (n = 5 biological replicates). In h, data are presented as mean values ± SD (n = 3 biological replicates). In (a,f), different letters represent significant differences among the treatments (one-way ANOVA with correction by Tukey’s HSD test, p < 0.05, p values are shown in source data). In h, statistically significant differences between the means are compared using two-tailed Student’s t-test.

To verify promoted colonization of encapsulated NX-11 on root, we used NX-11 tagged with green fluorescent protein (GFP) and laser scanning confocal microscope (LSCM) to visualize the colonization of this strain on rice roots. The fluorescence images of rice root showed that the colonization rate of APC-encapsulated NX-11 was higher than that of free NX-11 (Fig. 6g). Plate counting analysis revealed that APC microcapsule increased the abundance of NX-11 on rice roots by 342.32% compared to the free NX-11 treatment (Fig. 6h). Moreover, there was no significant difference in viability between free NX-11 and APC-encapsulated NX-11 under different NaCl concentrations in vitro (Supplementary Fig. 14). No significant difference in the survival of NX-11 between in non-saline and saline soil further proved that the enhanced colonization of NX-11 in rice rhizosphere by APC microcapsules were not attributed to the protective effects of microcapsules against salt (Supplementary Fig. 15). Critically, compared to free NX-11 in sterilized soil (6.87 log CFU/g), the viability of free NX-11 declined sharply to 4.82 log CFU/g in non-sterile soil applied with salt, while the APC-encapsulated NX-11 maintained a significantly higher population of 6.76 log CFU/g. Moreover, APC-encapsulation significantly alleviated the decrease of NX-11 in both rhizosphere and root of rice under salt stress (Supplementary Fig. 16). These results indicated that soil microbiome strongly suppressed the survival of NX-11 in soil and core-shell microcapsules can provide a biofilm-like protective microenvironment for NX-11 against soil native microflora, which further lead to higher abundance of NX-11 in the rhizosphere. It should be noted that APC-encapsulation exhibited a higher population of NX-11 in natural soil applied with salt than alginate microcapsules (A) (Supplementary Fig. 17).

Discussion

Soil salinity is one of the most devastating stresses causing major reduction in cultivated land area and crop productivity and quality¹. PGPBs play key roles in improving the tolerance of crops to salt stress, representing promising tools to alleviate salt stress in various crops and increase agricultural productivity in saline soils^3,9,20,39. However, PGPBs, especially non-spore-forming bacteria, are sensitive to deleterious factors during storage and have difficulties in sustaining activity under soil microbiome environment. In this study, we developed core-shell microcapsules to extend the shelf life of microbial inoculant, and explored the potential of core-shell microcapsules-encapsulated inoculant to enhance tolerance of rice seedlings to salt stress.

The microcapusles formed by internal gelation method of emulsification technique exhibits a more homogeneous gel structure due to homogeneously dispersed metal salt and controlled release of metal ions in gelling solution, which can avoid the disruption of emulsion equilibrium and irregular shape of microcapsules led by direct addition of crosslinking agent⁴⁰. Therefore, internal gelation method was applied to produce core-shell structured microcapsules in this study. According to the results of OFAT and Taguchi orthogonal array design, ratio of polymers to bacteria, concentration of CS as well as concentration of CaCO₃ significantly influenced the encapsulation efficiency of microcapsules. The encapsulation efficiency of microcapsules was observed decreased sharply with the increase of ratio of polymers to bacteria. This result indicated that high concentration of bacteria may limit cross-link of polymers. The low concentration of calcium carbonate leads to insufficient ions for generating a densely electrostatically cross-linked gel, whereas, similar to previous finding⁴¹, we found that relatively high Ca²⁺ caused low encapsulation efficiency due to disruption of cell. The low encapsulation efficiency caused by increased concentration of chitosan likely due to the antimicrobial activity of chitosan.

SA is one of the most commonly used embedding materials, and SA microcapsules have a great potential in enhancing bacteria survivability⁴². However, porous gel structures and poor mechanical strength of SA microcapsules may result in an undesired and premature leakage of cells and insufficient protective effect⁴¹. The addition of γ-PGA reduced the pore size of microcapsules and alleviated the loss of bacterial viability during storage due to the formation of dual-network microcapsules. We designed the core-shell structure established by electrostatic interactions between CS and dual-network hydrogel core to further strengthen the structure of microcapsules, and reduce degradation rate in soil environment. This core-shell structure can provide protection for PGPB due to its denser structure and biofilm-like microenvironment and achieve a sustained release of the encapsulated bacteria into the soil.

The TCA cycle is crucial for bacteria to synthesize fatty acids, carbohydrates, and amino acids, as well as for the production of NADH, a key product of the cycle and a primary precursor for the oxidative phosphorylation pathway^43,44. It has been reported that a decrease in the activity of citrate synthase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase can directly lead to reduced efficiency of the TCA cycle^45,46. This down-regulation could also diminish the oxidative phosphorylation pathway due to the decreased availability of NADH, which is primarily generated by isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase within the TCA cycle⁴⁷. Additionally, the down-regulation of dihydrolipoyl dehydrogenase (B1H58_03375), a subunit of the pyruvate dehydrogenase complex for converting pyruvate to acetyl-CoA may limit acetyl-CoA supply and further restrict citrate synthesis and TCA cycle initiation⁴⁸. As the final step of aerobic respiration, oxidative phosphorylation is essential for energy conversion from organic compounds and ATP production necessary for cellular metabolism and growth⁴⁹. The down-regulation of proteins associated with Complex I could result from decreased NADH levels caused by inhibition of TCA cycle, whereas the reduced expression of Complex III and IV components could be linked to impaired electron flow. Moreover, succinate dehydrogenase is integral to both the TCA cycle and the oxidative phosphorylation pathway for controlling rates of respiration⁵⁰. The down-regulation of these proteins suggested that the APC microcapsules could inhibit energy metabolism during storage. Moreover, SdhC is the key gene in the early ROS release process of bacteria⁵¹, and CydA can reduce hydrogen peroxide to water for preventing the formation of ROS⁵². These results also indicated that encapsulation reduce generation of ROS during bacteria storage.

The inoculation of PGPBs can promote the accumulation of osmoprotectants (such as soluble protein, soluble sugar, proline) and increased the antioxidant enzyme (SOD, CAT, and POD) activity to mitigate salt stress^53–55. Consistent with these findings, rice plants inoculated with free NX-11 exhibited increased levels of these osmotic adjustment substances. The inoculation of NX-11 also can increase activities of SOD, CAT and POD in plants to reduce the ROS accumulation and alleviate the damage caused by salt stress to rice seedlings. The core-shell microcapsules are composed of sodium alginate, poly (γ-glutamic acid) (γ-PGA), and chitosan. Previous studies have revealed that sodium alginate and chitosan alone exhibited little effect on growth of plant under salt stress^56,57, while γ-PGA was determined to enhance tolerance of plant against salt stress through enhancing activities of antioxidant enzyme by our pervious study⁵⁸. Therefore, the growth-promoting effect observed in the M-S treatment is likely attribute to the presence of γ-PGA within the microcapsule formulation. The promoting growth parameters observed in rice seedlings inoculated with APC-encapsulated NX-11 likely resulted from higher content of osmoprotectants and activities of antioxidant enzymes. Moreover, we found all the plants treated with APC-encapsulated NX-11, including tomato, maize, and lettuce, exhibited enhanced accumulation of osmoprotectants (soluble protein and proline) and activities of antioxidant enzymes (SOD, CAT, and POD) compared to free NX-11 treatment. Therefore, we attribute the improved plant growth to the ability of the APC microcapsules to enhance the survival of NX-11 in the soil, thereby increasing the function of NX-11 in the rhizosphere.

PGPBs can settle in the rhizosphere of plants, protecting them from many abiotic stressors, such as salinity, drought, and high temperatures⁵⁹. Furthermore, inoculation of PGPB can change the structure of the microbial community in the plant rhizosphere to alleviate salt stress⁶⁰. In this study, we found that the inoculation of APC-encapsulated NX-11 significantly increased the growth parameters of rice under salt stress and clearly changed the rhizosphere microbial community composition of rice, suggesting that the alleviation of salt damage to the rice seedlings was likely associated with the changes of microbial community. The inoculation of encapsulated NX-11 promoted the enrichment of potential nitrogen-fixing bacteria, including Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium^61,62, Devosia⁶³, and Sphingomonas⁶⁴. These findings suggested that APC-encapsulated NX-11 likely facilitated recruitment of beneficial bacteria, thereby enhancing rice seedling salt tolerance. Importantly, the enrichment of genus Pantoea and ASV2786 (100% sequences similarity to P. alhagi NX-11) was found in rhizosphere of rice inoculated with APC-encapsulated NX-11, whereas ASV2786 was never found in the rhizosphere of rice treated with non-encapsulated NX-11. APC-encapsulation increased the abundance of NX-11 in bulk soil, in rhizosphere soil of rice, and in roots of rice compared to free NX-11 inoculation. Furthermore, compared to free NX-11 in sterilized soil, the viability of free NX-11 declined sharply in natural soil applied with salt, while the APC-encapsulated NX-11 maintained a significantly higher population. These findings indicated that the APC microcapsules effectively protect NX-11 from competitive native microflora, further lead to higher abundance of NX-11 in the rhizosphere. In this study, we focused on developing bio-inspired core-shell microcapsules to enhance the survival of microbial inoculants during storage and soil application, and testing the effect of the core-shell microcapsules on alleviating salinity stress of plants. Further studies are needed to explore the effect of APC-encapsulated NX-11 on regulatory networks and metabolic pathways of rice using transcriptomics, metabolomics, and salt-tolerant mutants of plants.

Methods

Materials

γ-PGA (Mw = 2000 kDa) was obtained from Nanjing Shineking Biotech Co., Ltd. (Nanjing, China). Sodium alginate (SA), chitosan (CS), paraffin oil, Span80, calcium carbonate, and calcium carbonate were purchased from Meyer Biochemical Co., Ltd. (Shanghai, China). Lysogeny broth (LB), and agar powder were obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The kits used for enzyme-linked immunosorbent assay (ELISA) were purchased from Beijing Mreda Technology Co., Ltd. The kits used for determination of seedling growth parameters were purchased from Suzhou Comin Biotechnology Co., Ltd.

Preparation of microcapsules

The core-shell microcapsules were prepared via emulsification. SA, γ-PGA and calcium carbonate were added into 50 mL deionized water and stirred at 400 r/min for 6 h. The total mass of SA and γ-PGA was fixed at 2.5% (wt/vol). The cutured cells (10⁹ CFU/mL) were mixed with the sterile SA-γ-PGA solution to obtain the aqueous dispersed phase. Subsequently, 50 mL mixture solution was added into paraffin oil containing Span80 and stirred thoroughly. Acetic acid was then added into emulsion and stirred continuously for 30 min to harden. An chitosan aqueous was prepared by dissolving chitosan in 50 mL of 1% acetic acid solution and added into emulsion under gentle stirring for 30 min to form coating on the surface of microcapsules. Finally, the reaction mixture was centrifuged for 10 min at 6000 × g and washed repeatedly with deionized water to get the microcapsules in precipitate. These obtained microcapsules were stored at 4 °C for future use, which was denoted as APC. More details of optimization conditions for preparing core-shell microcapsules was illustrated in Appendix S1 and Supplementary Table 4.

Characterization of microcapsules

The chemical structure of materials and microcapsules were characterized by Fourier transform infrared (FTIR, Agilent Cary660, USA). The FTIR analysis of samples was carried out using the KBr pellet method, and FTIR spectra were obtained with a scan range of 4000 to 500 cm^-1. SA, γ-PGA, CS and microcapsules were characterized by X-ray diffraction (XRD) to determine the crystallinity of materials and microcapsules. Diffractograms were recorded from 10° to 50° at an angular speed of 1° /min on an X-ray diffractometer (XRD, D8 Advance, Bruker, Germany). Thermal degradation processes of materials and microcapsules were investigated using a thermogravimetry differential scanning calorimetry thermal analyzer (NETZSCH STA 449F3, Germany). The samples were heated from 25 °C to 500 °C under an N₂ atmosphere, with a heating rate of 10 °C/min. Elemental composition analysis was conducted using X-ray photoelectron spectroscopy (XPS, Thermo escalab 250Xi, Thermo fisher, USA) with Al‒Kα radiation as the source (hv = 1486.6 eV). Survey spectra were recorded for 0-1350 eV binding energy range. After the spectra were obtained, the areas of C 1 s, N 1 s, and O 1 s were determined using AVANTAGE V5 software.

To ensure the properties of different microcapsule comparable, the fabrication of all microcapsule types (A, AP, and APC) was performed under identical conditions. The total mass of polymers (SA or SA/γ-PGA) was fixed at 2.5%. All emulsification steps were conducted based on optimum conditions (see more details on Supplementary Table 4). Hydrogels with the same content of microcapsules were prepared for the characterization of rheological properties following a previously published methodology as reported by Feng et al¹³. Rheological characterization of the hydrogel was performed using parallel plates geometry (20 mm diameter, 1 mm gap) mounted on a Discovery Hybrid Rheometer (HR-10; TA Instruments, New Castle, DE, USA). The morphology of microcapsules with different composition were performed by scanning electron microscopy (SEM, Hitachi SU8010, Japan). The samples anchored onto an SEM sample holder using double-sided adhesive disks and gold plated after lyophilization. 100 randomly selected freshly prepared microcapsules were captured by an optical microscope (Olympus CX31, Japan), and then diameter size of the microcapsules was measured by the ImageJ software (USA).The low temperature N₂ adsorption-desorption was measured using a ASAP2020M + C (Micromeritics, USA) at −196 °C. Freeze-dried microcapsules were degassed under decreased pressure for 20 h at 40 °C prior to measurement. The Brunauer–Emmet–Teller (BET) method was applied to determine the specific surface area of microcapsules, and mean pore diameter was estimated by the Barrett–Joyner–Halendia (BJH) method.

The soil burial method was applied to investigate the biodegradability of microcapsules. 250 g of soil was placed into hermetic containers. 1 g of air-dried microcapsules was put in the 100 µm mesh nylon tea bags and buried 5 cm below the soil surface. The weight of dry samples and tea bags were determined at the beginning of the experiment. Distilled water was added to maintain 30% moisture content during the experiment. The weight of samples was determined after washing and air drying until no changes in weight on days 1, 3, 7, 14, 21 and 28. The biodegradability of microcapsules were calculated by the following equation:

$$\mathrm{Biodegradability}=\frac{W_i-W_d}{W_i}\times 100\%$$ 1

where W_i and W_d were the weight of microcapsules before and after degradation.

Viability of bacteria during storage

The storage stability of the free and encapsulated bacteria was examined a previously described method⁶⁵, with some modifications. For free bacterial cells treatment, 1 g of cultured suspension was centrifuged (6000 × g, 10 min) and resuspended in 2 mL sterile 0.9% sodium chloride solution. An equivalent mass of microcapsules (1 g) was suspended in sterile 0.9% saline to match the volumetric parameters of free-cell suspensions. The initial viable cell count was maintained with a measurement variation of less than 0.3 log CFU/g across treatment groups. All samples were stored at 25 °C for 12 weeks. To minimize the risks of contamination due to the repeated sampling, 36 replicate tubes per group were prepared, and triplicate samples were destructively sampled weekly. To quantify the viability of bacteria, each tube of microcapsules was disintegrated in 8 mL sterile 2% sodium citrate solution under vortex mixing for 10 min and further measured by serial dilution plating on on LB agar. The microcapsules after storage were lyophilizated to observe the morphology of encapsulated bacteria using SEM. To demonstrate the wider applicability of the APC microcapsules, two additional non-spore-forming bacteria species (Lysobacter enzymogenes OH11 and Pseudomonas nitroreducens L16) were encapsulated and tested their viability in 8-week period using the same method as described above.

Proteome analysis of bacteria after storage

All NX-11 samples - fresh (CK-0W), 12-week stored (CK-12W), and APC-microencapsulated stored (APC-12W) were maintained at −80 °C until processing. Proteomic analyses were conducted using materials and services provided by Bioyi Biotechnology Co., Ltd. (Wuhan, China). To extract protein, frozen samples were homogenized to a fine powder in liquid nitrogen using a mortar and pestle, then suspended in lysis buffer (8 M urea, 1% protease inhibitor cocktail) followed by ultrasonic disruption. After centrifugation (12,000 × g, 10 min, 4 °C), supernatants were subjected to sequential reduction (10 mM DTT, 37 °C, 1 h) and alkylation (40 mM iodoacetamide, dark, 30 min). Protein concentration was determined by Bradford assay. For tryptic digestion, samples were diluted with Tris-HCl to <2 M urea concentration, then incubated with sequencing-grade trypsin (1:50 w/w) at 37 °C overnight. Peptides were purified by desalting after centrifugation (12,000 × g, 15 min) and lyophilized. Peptides were separated on a Vanquish Neo UHPLC system (Thermo Scientific) using a PepMap Neo C18 trap column (5 µm, 300 µm × 5 mm). Mobile phases were Buffer A (0.1% formic acid) and Buffer B (0.1% formic acid in 80% acetonitrile). After separation, peptides were analyzed using an Orbitrap Astral mass spectrometer (Thermo Scientific) in data-independent acquisition (DIA) mode. Full-scan MS1 spectra (380–980 m/z) were acquired at 120,000 resolution (automatic gain control target 300%, maximum injection time 3 ms). For MS2, precursor ions were fragmented using higher-energy collisional dissociation (HCD) with 30,000 resolution (automatic gain control target 500%, maximum injection time 3 ms). Raw DIA-MS data were processed using DIA-NN software (version 1.9) in library-free mode. Post-translational modifications included carbamidomethylation (C) as a fixed modification, oxidation (M) and acetylation (protein N-terminal) as variable modifications. The protein and peptide search results were screened with a 1% false discovery rate. The fold change (FC) between groups was calculated as the average expression rate. DEPs were screened based on criteria of log₂ FC > 1 or log₂ FC < -1, with a level of significance set at p < 0.05 (Student’s t test). KEGG pathway analyses was conducted using the clusterProfiler package in R (version 3.6).

Greenhouse experiment

Rice seeds (Oryza sativa subsp. japonica “Nipponbare”) were surface sterilized with 75% ethanol for 2 min and 2.5% sodium hypochlorite for 20 min, and then rinsed four times with sterile water. Surface-sterilized seeds were then placed on petri dish covered with sterilzed moist filter paper and germinated at 30 °C for 7 days. Every 6 germinated seeds with the same growth status (the root length was about 5 cm) were transplanted into each pots containing 600 g of field soil (see Supplementary Table 6 for physiochemical characteristics of the soils). Before sowing, 350 mL water was used to completely saturate the soil in each pot. After 14 days, the plant and soil samples were collected to measure physiological parameters of rice seedlings and rhizosphere microbial community composition.

Determination of physiological parameters of rice seedlings

The dry weight of plant shoots and roots were measured after heating for 30 min at 105 °C and drying at 60 °C. Soluble sugar content was determined following the method of Watanabe et al. with minor modifications⁶⁶. The rice seedlings (0.2 g of fresh weight) were homogenized in 10 mL of 80% (v/v) ethanol and centrifuged at 10,000 × g for 10 min. The collected supernatant (1 mL) was mixed with anthrone reagent (3 mL) and heated at 100 °C for 10 min. The absorbance was measured at 620 nm after cooling. Soluble protein content was measured using the method described by Bradford⁶⁷. The fresh seedlings were grinded in liquid nitrogen, and then homogenized with 10% (w/v) trichloroacetic acid. After centrifugation at 4000 × g for 15 min, the supernatant was collected and then reacted with Coomassie Brilliant Blue G-250 for 5 min. The mixture was measured at 595 nm using a spectrophotometer. The content of proline in rice seedlings was determined using the method descrbied by Abid et al. with some modifications⁶⁸. Rice seedlings were grinded in liquid nitrogen and then extracted with 3% aqueous sulfosalicylic acid. The mixture was centrifuged at 10,000 × g at 4 °C for 10 min to collect the supernatant. The supernatant was mixed with ninhydrin and glacial acetic acid, incubated in boiling water for 30 min, and then extracted with methylbenzene after cooled. The absorbance of the extract was measured at a wavelength of 520 nm. The catalase (CAT) activity, peroxidase (POD) activity, superoxide dismutase (SOD) activity, H₂O₂ content and malondialdehyde (MDA) content were evaluated using assay kits (Comin Biotechnology Co., Ltd, Suzhou, China) according to the manufacturer’s instructions. The abscisic acid (ABA) and indole-3-acetic acid (IAA) contents were determined by the enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Beijing Mreda Technology Co., Ltd, China). Statistical significance was determined using analysis of variance (one-way ANOVA with correction by Tukey’s HSD test, p < 0.05) using SPSS (v 26).

Rhzosphere microbial community composition

To collect rhizosohere soil of rice, roots of each plant were gently shaken to remove the excess soil, and the residual soil attached to the root was collected, which was considered as rhizosphere soil. Total genomic DNA from rhizosphere soil samples was extracted by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). Primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′) were used for the polymerase chain reaction (PCR) to target the specified regions (V3-V4) of the bacterial 16S rRNA. After amplification, the product was purified and quantified using fluorescence quantification with a Quant-iT PicoGreen dsDNA Assay Kit, and the Miseq library was constructed and sequenced using Illumina MiSeq platform (Personal Biotechnology Co., Ltd., Shanghai, China). QIIME 2 version 2019.4 pipeline (https://docs.qiime2.org/2019.4/tutorials/) was used for biological information analysis. Sequences were then quality filtered, denoised, merged and chimera removed using the DADA2 plugin. The obtained sequences were clusterd into amplicon sequence variants (ASVs) at a 100% similarityand taxonomic assignments of the ASVs were obtained using SILVA 132 database. To match the full-length 16 s rRNA gene sequences of Pantoea alhagi NX-11 with the ASV sequences obtained from high throughput sequencing, we trimmed the full-length 16S rRNA gene sequences of the isolates to the same region of the high-throughput sequencing data using UPARSE. Sequences were then mapped to the ASV sequences at the threshold of 100% similarity, score of 430, and e value of 1e^-220 using UPARSE.

Root colonization assay

Surface-sterilized seeds were sown and grown in 1/2 MS solid medium for 5 days. Germinated seeds were transplanted to plastic pots containing 50 g sterilized vermiculites. Before transplanting, 50 mL sterilized 1/4 MS nutrient solution contained was used to completely saturate the soil in each pot. All the pots were weighed and replenished with sterile deionized water three times per week to keep the moisture constant. To visualize the distribution of the bacteria on rice roots, NX-11 was modified with green fluorescent protein (GFP-NX-11). After 5 days of incubation, 0.1 mL suspension of GFP-NX-11 or 0.1 g APC microcapsules containing GFP-NX-11 (10⁸ CFU) was added to the root surrounding. After another 7 days of incubation, roots of seedling were taken out and rinsed with sterilized water. 1 cm root ripening zone for each sample was then collected and quickly stored in the sterile tube for subsequent analysis by Laser Scanning Confocal Microscope (LSM 980, ZEISS, Germany) and plate counting.

Author contributions

K.Y. and Y.G. performed the experiments, analyzed the data, and wrote the original draft; C.T., C.W., and Z.Y. performed the experiments;. L.S., P.L., Z.X., X.F., and B.T.reviewed and edited the manuscript; W.B., R.W., and H.X. developed the ideas and supervised the project.

Peer review

Peer review information

Nature Communications thanks Ruifu Zhang and the other anonymous reviewer for their contribution to the peer review of this work. [A peer review file is available.]

Data availability

The proteomics data are publicly accessible at iProX (https://www.iprox.cn/page/home.html) with the dataset identifier PXD069461. The raw sequencing data have been deposited in the NCBI SRA under the accession number PRJNA1345549. Source data are provided with this paper, and are available for Figs. 2–5, Supplementary Fig. 1, and Supplementary Fig. 4-17. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-72523-4.