P6, N20, and N21 were selected as the experimental strains for this study based on their growth-promoting capabilities, including IAA production, nitrogen fixation, and phosphorus solubilization. These strains were isolated from the desert shrub R. soongorica rhizosphere soil of Qingtu Lake in the Shiyang River Basin, Northwest China by Li et al. (2020). They were stored in Luria-Bertani(1% tryptone, 0.5 yeast extract, 1% NaCl, LB) broth containing 25% glycerol at −80°C. The bacterial inoculums were prepared by growing every strain in LB at 28°C for 48 h. Each bacterial culture was harvested and diluted with sterile physiological saline (0.9% NaCl solution) to a final OD of 1 at 600 nm (~ 10⁹ CFU mL⁻¹).

For this experiment, utilized R. soongorica seeds were collected from Qingtu Lake located in Minqin County in 2021 and preserved in a dry refrigerator maintained at 0-4°C for an extended period. Prior to planting, the seeds underwent sterilization using a 1% sodium hypochlorite solution for half an hour, followed by five rinses with sterile water. In April 2023, these sterile seeds were sown in trays. The seedlings were transplanted into plastic flower pots with a diameter of 24.8 cm and a height of 28 cm, with one plant per pot, when they reached a height of 5 cm. The plastic flower pots contained 5 kg of a substrate mixture of clay, sand, and humus (1:2:1), soil organic matter(SOM): 28.84 g·kg^-1, total carbon: 37.81 g·kg^-1, total nitrogen: 0.98 g·kg^-1, (TP): 0.30 g·kg^-1, available phosphorus (AP): 14.71: mg·kg^-1, total potassium (TK): 3.57 g·kg^-1, available potassium (AK): 105.75 mg·kg^-1, and electrical conductivity (EC): 0.32 mS·cm⁻¹. R. soongorica seedlings grew under natural conditions at the experimental site of Gansu Agricultural University, located in Anning District, Lanzhou City, Gansu Province (36°5′N, 103°42′E), which is in a mid-temperate climate zone with distinct inland climate characteristics, clear seasonal changes, ample sunlight, and dry weather.

Three strains were activated on the LB solid medium and incubated at 30°C for 48 hours. Gram staining was performed according to the instructions of the Biosharp Gram Staining Kit. The physiological and biochemical properties of the strains were determined by referring to Bergey’s Bacteria Identification Manual and Manual of Identification of Common Bacterial Systems (Buchanan and Gibbons, 1984; Dong and Cai, 2001). Three strains were inoculated on LB solid medium containing 4%, 6%, 8%, 10%, and 12% NaCl to test the NaCl tolerance of strains.

The abilities of the strains to produce IAA were evaluated using a colorimetric assay (Bao et al., 2024; Kumar and Audipudi, 2015). According to Nautiyal (1999) and Amri et al. (2023), the abilities of the strains to solubilize organic and inorganic phosphorus were tested and were expressed by the size of the phosphorus dissolving circle around the colony. The strains were inoculated onto Ashby medium and considered to have nitrogen-fixing ability if they could still grow on Ashby medium after three successive transfers (Bao et al., 2024). All the above tests were repeated three times.

The strains were activated with LB solid medium, and incubated at 30°C for 24 hours. Use the bacterial DNA extraction kit to extract Bacterial DNA. The 16S rDNA sequences of the strains were amplified with primers 27F (5’-AGAGTTTGATCMTGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGATT-3’). Then, PCR products were sent to Guangdong Magigene Biotechnology Co., Ltd. In Guangzhou, China for Sanger sequencing. Finally, the 16S rRNA gene sequences were analyzed using the EzTaxon (https://www.ezbiocloud.net/apps) online database. A phylogenetic tree was constructed using the Maximum Likelihood Estimate method in MEGA 11.0 software, calculating evolutionary distances with the General Time Reversible (GTR) model, with bootstrap testing repeated 500 times to determine the taxonomic status of the isolated strains (Bao et al., 2024).

The test set up control (CK, without NaCl and strains), single salt treatment (S, only NaCl added), separate treatment of P6, N20, and N21 bacteria (P6, N20, N21), and co-treatment of salt and bacteria (P6+S, N20+S, N21+S). Each treatment had 6 replicates, 3 replicates for biomass determination, and 3 replicates for C and N content determination. According to reports, a low concentration (100 mM) of NaCl can stimulate the growth of R. soongorica, whereas higher concentrations (400 and 500 mM) significantly hinder its development (Yan et al., 2022b). Furthermore, exposure to 500 mM NaCl stress can result in the death of individual R. soongorica seedlings (Yan et al., 2022b). Consequently, a concentration of 400 mM NaCl was chosen for treating the seedlings in this study. To avoid osmotic shock, 500 mL of NaCl solution was poured into each basin per day for 3 consecutive days, with a total amount of 1500 mL, and the control were treated with the same amount of water. Trays were placed at the base of the pots to collect any excess water. After watering, the oozing water was poured into the pots again to prevent salt loss. The roots of the plants were inoculated with 100 mL of inoculums by pouring, and the control was inoculated with an equal amount of sterile saline.

Samples were taken after the seedlings were treated for 56 days. Before sampling, Photographs were taken with a digital camera, and height and basal diameter were measured for all R. soongorica seedlings; Then samples, and collect the plants and the aboveground and underground parts respectively, were back to the laboratory with a low-temperature sampling box, clean them with pure water, and then absorb the surface water with filter paper, and weigh the fresh weight respectively. Separate the roots, stems, and leaves, blanch them at 105 °C for 30 min, and dry them at 80 °C to constant weight. Then, use a digital balance with a progress of 0.0001g to measure the biomass of roots, stems, and leaves. The root length and surface area were determined using the LA-S root analysis system (WSEEN, China).

Crush the dried samples and pass them through a 100-mesh sieve for standby. The contents of N and C in roots stems, and leaves were measured by the Carbon and Nitrogen analyzer (Primacs^SNC-100, Skalar Co., the Netherland).

All experiments were designed with three replicates and analyzed using IBM SPSS Statistics 26.0 software for one-way analysis of variance (ANOVA). Duncan’s minimum significant difference method was used to assess the significance of differences among treatments, and the significance level was set at 0.05. The graphs were created using Origin 2021 and Adobe Illustrator 2022 software. Pearson correlation coefficient (r) and linear regression were used to analyze the correlation between the biomass allocation and the C, N content of roots stems, and leaves.

The main physiological and biochemical characteristics and growth-promoting functions of strains P6, N20, and N21 are shown in Table 1 . Strains P6, N20, and N21 are all gram-negative bacteria. It is positive under the Voges-Proskaue test, Indole test, and, Catalase test of these strains, but negative for Methyl red test, Starch hydrolysis test, and Gelatin hydrolysis test. In addition, their glucose fermentation tests are F type. Strains P6, N20, and N21 can grow in the highest NaCl concentration at 8%. Strains P6, N20, and N21 have nitrogen fixation ability, produce growth elements, and dissolve organic phosphorus and inorganic phosphorus.

The 16S rDNA sequencing results of strains P6, N20, and N21 were compared and analyzed. The phylogenetic tree based on the 16S rRNA gene sequences, as shown in Figure 1 , indicated that the dominant strains P2, N20, and N21 belong to Enterobacter, which was consistent with morphological identification results. In addition, the gene fragment of strain P6 was 1374 bp in length, which was the same branch as Enterobacter hormaechei subsp. Hoffmannii EN-114^T and had a similarity of 99.78%. The gene sequence length of strain N20 was 1374 bp, belonging to the same branch as E. hormaechei subsp. Hoffmannii EN-114^T, with a similarity of 99.56%. The gene fragment of strain N21 was 1371 bp, belonging to the same branch as Enterobacter asburiae JCM 6051^T, with a similarity of 99.20%. So, combining morphological and molecular biological characteristics, P6, N20, and N21 could be preliminarily determined as E. hormaechei, E. hormaechei, E. asburiae, respectively. The 16S rDNA sequences of P6, N20, and N21 strains have been deposited in the NCBI database with GenBank accession numbers PP998345, PP998346, and PP998347, respectively.

Inoculation of three strains of Enterobacter has a significant impact on the growth and development of R. soongorica and salt tolerance ( Figure 2 ). When inoculated with strains P6 and N21, the basal diameters of R. soongorica seedlings significantly increased by 21.26% and 33.46% (P<0.05) compared to the control (CK), the total root length increased by 23.26% and 21.52%. When inoculated with strains N20 and N21, the root surface area of R. soongorica seedlings significantly increased by 38.74% and 48.71% (P<0.05) compared with the control (CK). Under NaCl stress, the plant height, basal diameter, and root surface area of R. soongorica seedlings were significantly reduced, but when inoculated with P6, N20, and N21 strains, the plant height of R. soongorica seedlings significantly increased by 39.41%, 19.54% and 32.08% respectively (P<0.05) compared with NaCl treatment (S); the basal diameter significantly increased by 65.02%, 50.74% and 74.88% respectively (P<0.05), and the root surface area significantly increased by 147.97%, 124.49% and 109.98% respectively (P<0.05) compared with NaCl treatment (S). Moreover, inoculation with strains P6 and N21, the total root length of R. soongorica seedlings significantly increased by 46.34% and 25.81% (P<0.05) compared with NaCl treatment (S). Thus, inoculation with strains P6, N20, and N21 increased the basal diameters, total root length, and root surface area of R. soongorica seedlings, and the effects were more obvious under NaCl stress.

Inoculated with Enterobacter increased the dry weight of roots, stems, and leaves of R. soongorica seedlings, both with and without NaCl stress (see Figure 3 ). Compared with the control (CK), the root dry weight of R. soongorica seedlings inoculated with strains P6, N20, and N21 increased by 54.03%, 45.40%, and 58.97% respectively (P<0.05); inoculation with strains P6 and N21, the stem dry weight significantly increased by 19.38% and 16.90% (P<0.05); inoculation with strains N20 and N21, the root dry weight significantly increased by 34.24% and 36.34% (P<0.05). Under NaCl stress, the dry weight of roots, stems and leaves of R. soongorica seedlings significantly decreased by 28.73%, 62.83%, and 64.91% (P<0.05) compared with the control (CK), and the root-shoot ratio significantly increased by 99.20% (P<0.05). Inoculated with strains P6, N20, and N21, the roots dry weight of R. soongorica seedlings under NaCl stress significantly increased by 99.88%, 85.55%, 141.76% (P<0.05), the stems dry weight significantly increased by 94.60%, 68.33% and 124.59% (P<0.05), and leaves dry weight significantly increased by 143.58%, 103.88% and 190.56% (P<0.05) compared with NaCl treatment (S). However, there was no significant difference in the root-shoot ratio of R. soongorica seedlings. The results showed that strains P6, N20, and N21 could effectively improve the growth and enhance salt tolerance of R. soongorica seedlings by regulating biomasses of roots, stems, and leaves. Thus, Enterobacter P6, N20, and N21 have the potential as a microbial inoculant that can protect plants under salt-stress conditions.

NaCl treatment and inoculation with strains P6, N20, and N21 had significant effects on C, N content, and C: N ratio in various organs of R. soongorica seedlings (see Figure 4 ). The C contents in leaves and stems of R. soongorica seedlings were higher than that in roots, and the N contents in roots were higher than that in stems and leaves. Inoculated with strains P6, N20, and N21, the N contents in roots and leaves, and the C contents in leaves of R. soongorica seedlings increased. Among them, the N contents in roots of seedlings were significantly increased by 20.59%, 14.19%, and 8.33% (P<0.05), and the C contents in leaves increased by 2.78%, 3.41%, and 2.55% (P<0.05), compared with the control (CK). Additionally, the C: N ratio in the roots of seedlings decreased by 16.64%, 12.74%, and 7.41% (P<0.05). Under NaCl stress, the N in roots stems and leaves of R. soongorica seedlings increased by 59.52%, 25.07%, and 22.04%, and the C in the roots, stems and leaves decreased by 10.46%, 3.12%, and 3.88%, respectively, compared with the control (CK). Additionally, the C: N ratio of root, stem, and leaf decreased by 43.85%, 22.51%, and 21.24% respectively (P<0.05). However, after inoculation with P6, N20, and N21 strains, these change trends of the C, N contents, and C: N ratio in roots, stems, and leaves of R. soongorica seedlings under NaCl stress were reversed. When inoculated with P6, N20 and N21 strains, compared with NaCl treatment (S), the N contents in roots, stems, and leaves of R. soongorica under NaCl stress significantly decreased (P<0.05), the C contents in roots, stems, and leaves of seedlings significantly increased (P<0.05), the C: N ratio in roots, stems, and leaves of seedlings significantly increased(P<0.05). The effects of NaCl stress on the contents of the C, N, and the ratio of C: N in the roots, stems and leaves of R. soongorica seedlings were very significant, inoculated with Enterobacter P6, N20, and N21 could weaken the effects of salt stress. These Indicate that Enterobacter P6, N20, and N21 can promote plant growth and alleviate salt stress by regulating the distribution of nutrients distribution of R. soongorica seedlings.

Correlation analysis showed that there is a strong association between biomass and the C, N contents, and C:N ratio in different organs of R. soongorica ( Figure 5 ). The stem biomass was significantly positively correlated with the C contents of roots, stems, and leaves (r= 0.87; r= 0.70; r= 0.82), and negatively correlated with the N contents of roots, stems, and leaves (r= -0.81; r= -0.82; r= -0.70). The leaf biomass was significantly positively correlated with the C contents of rhizome, leaf, and stem (r= 0.79; r= 0.74; r= 0.85), and negatively correlated with the N contents of rhizome, leaf, and stem (r= -0.73); r= -0.74;r = -0.67). The C: N ratio of roots, stems, and leaves was significantly positively correlated with C contents of roots, stems, and leaves (r= 0.93; r= 0.71; r= 0.80), and significantly negatively correlated with N contents of roots, stems, and leaves (r= -0.99; r = -0.99;r =- 0.99). The root-to-shoot ratio was positively correlated with the N contents of roots, stems, and leaves (r= 0.88; r= 0.84; r= 0.69), and negatively correlated with the C contents of roots, stems, and leaves (r=-0.87; r =-0.62;r =-0.53). In conclusion, there is a close relationship between the biomass of roots, stems, and leaves and their C, N content and C: N ratio.

Enterobacter is widely recognized for its potential to improve the soil environment and promote crop growth (Wang et al., 2022a). It is one of the widely studied PGPRs, which can increase the availability of soil nutrients (Roslan et al., 2022), regulate soil enzyme activities (Ji et al., 2024), facilitate biofilm formation (Li et al., 2022a), bolster resilience (Chen et al., 2024; Ji et al., 2020), and exert biological control (Wang et al., 2024) to promote the good growth of plants under stress. In this study, the P6 and N20 strains were identified as E. hormaechei, and the N21 strain was identified as E. asburiae. They can not only produce IAA but also solve phosphorus and fix nitrogen, which is the characteristic of ideal PGPR(see Table 1 ). Especially in the barren desert environment, they play a key role in promoting the growth of desert plants and enhancing their stress resistance. Studies have demonstrated that Enterobacter cloacae effectively enhances the growth of cucumber seedlings (Fan et al., 2024), cotton (Yue et al., 2023), and wheat (Ji et al., 2020), when grown under saline-alkali conditions. E. hormaechei can improve plant physiology and yield through soil phosphate (P) and potassium (K) amelioration (Ranawat et al., 2021a, 2021b; Roslan et al., 2022; Shahzad et al., 2020). E. hormaechei DS02Eh01 has the ability to promote plant growth and biofilm formation and has the potential for environmental ecological restoration (Li et al., 2022a). E. asburiae enhances salt tolerance and increases yield in rice (Ning et al., 2024), Chenopodium quinoa (Mahdi et al., 2020), and Medicago sativa (Li et al., 2024). It can be seen that Enterobacter plays an important role in promoting the healthy growth of plants. We also obtained similar results through pot experiments. Under NaCl stress, the height, basal diameter, and root surface area of R. soongorica seedlings were significantly reduced compared with the control (CK). However, inoculation with strains P6, N20, and N21, the seedling height, basal diameter, and root surface area of R. soongorica seedlings under NaCl stress were significantly higher than those of NaCl-treated seedlings. The results showed that inoculation with Enterobacter could promote the growth and enhance salt tolerance of R. soongorica seedlings.

Plant organs serve distinct biological functions: leaves fix new carbohydrates, roots absorb water and nutrients and stems offer support and transportation pathways. The rational allocation of biomass among these organs is crucial in driving resource acquisition and facilitating growth (Poorter et al., 2011). At present, many studies on biomass allocation have been widely reported. Biomass allocation is affected not only by plant species and individual development but also by environmental conditions (Rutishauser et al., 2019). The study revealed that under low light and CO2 conditions, the proportion of biomass allocated to aboveground parts of plants was higher than that of roots (Guo et al., 2016). When plants are exposed to water and nutrient stress, more biomass is allocated to the plant root system (Grechi et al., 2007). This study found that under NaCl stress, the biomass of roots, stems and leaves of R. soongorica seedlings was significantly reduced compared with the control (CK), but the root-to-shoot ratio was significantly increased by 99.20%. Salt stress negatively affects the biomass in roots, stems, and leaves of R. soongorica. However, R. soongorica seedlings allocate more resources to roots, increase the proportion of root biomass, and increase the root surface area to enhance nutrient absorption. Under drought conditions, more biomass will be allocated to the roots of five tropical tree species, which is consistent with our research results (Asefa et al., 2022). This shows that plants respond to environmental changes by adjusting the allocation of biomass to different organs, prioritize the allocation of biomass to organs that have access to more limiting resources in order to promote growth (Guo et al., 2016), which is consistent with the optimal partitioning theory.

Microbial inoculants can resist biotic and abiotic stresses by regulating the biomass allocation. Inoculation of Enterobacter cloacae QZS3 significantly regulated Sorghum bicolor biomass and promoted plant growth under cadmium stress (Chen et al., 2024). PGPR and AMF inoculation can largely or at least partially offset the detrimental effects of drought on biomass production, water relations, and nutrient dynamics of Myrtus communis seedlings (Azizi et al., 2022). In our study, inoculation with strains P6, N20, and N21 can increase the biomass of roots, stems, and leaves of R. soongorica seedlings, both with and without NaCl stress. Notably, the greatest biomass increase was observed in the roots, while the smallest increase was seen in the stems. Similar results were reported that the dry weight significantly increased in the shoot and root of mustard under saline conditions after inoculation of salt-tolerant Pseudomonas JMM15 and Pseudomonas aeruginosa HMM57 strains (Phour and Sindhu, 2020). PGPR can stimulate root development, form lateral roots and adventitious roots, and promote plant growth by producing exogenous IAA and other plant growth hormones (Li et al., 2022b). We selected three strains of Enterobacter, all produced IAA, which may be one of the reasons why the root biomass and root surface area of R. soongorica seedlings increased significantly compared with the control. Inoculated with strains P6, N20, and N21, the root-to-shoot ratio of R. soongorica seedlings significantly increased. This indicates that the proportion of root biomass allocation increased, ensuring that plants can obtain more nutrients under salt stress. Our research results were consistent with those of Ma and Wang (2020) they found that the root growth rate was faster than that of aboveground in desert plant Salsola passerina. The results indicated that R. soongorica seedlings responded to salt stress by adapting their biomass allocation as an adaptive strategy. Inoculation with Enterobacter could increase the biomass of roots, stems, and leaves of R. soongorica seedlings, adjust the biomass allocation to promote growth and enhance salt stress tolerance.

Carbon (C) and nitrogen (N) are key elements for plant growth and development (Chen and Chen, 2021). C provides the structural basis of plants and is the largest component of plant biomass, accounting for 50% of plant dry weight (Ågren, 2008). N is a structural component of amino acids and a component of all enzymes and is involved in many physiological processes (Seepaul et al., 2019). C and N assimilation are two of the most important physiological processes associated with plant growth and productivity (Otori et al., 2017). A pot experiment was conducted to study the effects of NaCl stress and PGPR inoculums on C and N contents in different organs of R. soongorica seedlings. The results showed that under both salt stress and normal conditions, the N contents in roots and leaves of R. soongorica seedlings were higher than that in stems, and the C contents in leaves and stems of R. soongorica seedlings were higher than that in roots. Plant organs collaborate to accomplish various physiological and biochemical reactions as an integrated system (Zhang et al., 2018). The root and leaf exhibit a source-sink relationship, where the leaf serves as the source of C. The fixed C in the leaf is preferentially allocated to the organ closest to the nutrients, prior to being transported to other organs (Li et al., 2022c). Salt stress usually inhibits nitrogen and carbon metabolism in plants. The significant decrease of C content in the roots, stems and leaves of R. soongorica seedlings may be attributed to salt stress inhibiting plant photosynthesis and reducing carbon fixation (Rahnama et al., 2010). In addition, plants must expend extra energy to overcome salt stress (Chaves et al., 2009). However, the N contents were significantly higher than that of the control in roots, stems, and leaves of R. soongorica under salt stress. One reason is the accumulation of non-protein nitrogen in saline plants under salt stress (Pessarakli et al., 2012), that these non-protein forms of nitrogen play a crucial role in osmoprotection and osmoregulation (Xie et al., 2022). Another reason may be that the increase in enzyme amount per leaf area leads to increased N content of R. soongorica under salt stress, but, the increase of enzymes involved in photosynthesis is not enough to compensate for the decrease in enzyme activity (Wang et al., 2022b). Wang et al. (2015) observed 18 desert halophytes and found that the leaf N content of desert halophytes was higher than that of desert grassland plants, this is consistent with our results. Therefore, increasing leaf nitrogen content should be a common strategy for desert saline plants.

Inoculation of rhizosphere microorganisms can change the contents of C and N in plants, help overcome biotic and abiotic stress, as well as improve growth and nutrition in plants (Mariotte et al., 2017). For example, AMF plays a key role in nutrient uptake in the soil-plant continuum, increasing plant tolerance to drought (Xiao et al., 2023). Inoculated with PGPR, Sulla carnosa showed different C: N:P stoichiometry under salt stress, which indicated that PGPR could improve plant tolerance to salt stress through nutrient allocation mode (Hidri et al., 2019). Our study proved this point, inoculated with strains P6, N20, and N21 and the N content in the root of R. soongorica increased significantly compared with the control (CK), and the C content in the leaf increased significantly. When inoculated with P6, N20, and N21 strains, compared with NaCl treatment (S), the N contents in roots, stems, and leaves of R. soongorica under NaCl stress significantly decreased, the C contents in roots, stems, and leaves of seedlings significantly increased. Similar results were reported previously that inoculation AMF the N and P contents significantly increased in Cinnamomum migao seedlings, with the C contents in leaves and stems of plants higher than in control plants (Xiao et al., 2023). Therefore, it is a feasible adaptive strategy to use PGPR to maintain the nutrient balance of plants in desert areas (Elser et al., 2003).

Plant C: N ratios are important indicators for exploring elemental allocation and adaptation strategies in plants (Zhang et al., 2020). An excessively high or low C: N ratio can impede the absorption of nutrients by plants, whereas a suitable C: N ratio can enhance the absorption and utilization of nutrients by plants. Previous studies showed that plants generally have a higher C: N ratio under drought stress (Jiang et al., 2021). Our study demonstrated that the C: N ratio of roots, stems and leaves of R. soongorica seedlings under salt stress was significantly lower than that of the control, but inoculated with strains P6, N20, and, N21, the C: N ratio of roots, stems and leaves significantly increased. C: N:P chemometrics of Trifolium repens inoculated with AMF have been studied, and consistent results have been observed (Dong et al., 2023). A higher C: N ratio indicated higher nitrogen use efficiency, which made the seedlings grow normally under salt stress.

The biomass of roots stems, and leaves is closely related to the C and N content of R. soongorica seedlings. Understanding the correlation between the C and N element content in plant leaves and biomass is crucial (Song et al., 2024). Studies have shown that the N in leaf content can influence the final allocation of plant biomass(Ding et al., 2011; Xu and Jiang, 2015); The improvement of biomass in mycorrhizal plants may also be due to the enhanced N uptake(Habibzadeh et al., 2013). This study found that root biomass was significantly positively correlated with leaf C content, stem biomass was significantly positively correlated with root stem leaf stem C content, significantly negatively correlated with root stem leaf stem N content, leaf biomass was significantly positively correlated with root stem leaf C content, and significantly negatively correlated with root stem leaf N content. The results indicated a significant correlation between the content of C and N in the roots, stems, and leaves of R. soongorica seedlings and their biomass allocation. Investigating this relationship was crucial for comprehending plant nutrient utilization strategies and the elemental balance within the plant.