Composite microbial inoculant for planting rice in saline-alkali soil

Abstract

The invention discloses a microbial complex microbial inoculant for saline-alkali soil rice planting, and belongs to the field of microorganisms. The microbial compound bacterial agent is prepared from bacillus subtilis and bacillus licheniformis, the bacillus subtilis is bacillus subtilis Z21, and the bacillus licheniformis is bacillus licheniformis Z8. The invention further discloses a preparation method of the microbial compound bacterial agent. The compound microbial inoculant has a synergistic effect in promoting rice population development, and Z21 and Z8 form a superimposed advantage in physiological regulation of enhancing stress resistance and promoting tillering. Long-term application is expected to break through soil hardening limitation and further improve saline-alkali soil improvement efficiency. After long-term application of the composite microbial inoculant, a rhizosphere microbial network structure is remodeled, so that more stable improvement of soil ecological functions is realized. According to the microbial compound inoculant, the functional advantages of Z8 and Z21 strains are integrated, the number of rice holes and stems is remarkably increased, the reduction of the conductivity of rhizosphere soil is influenced by the continuous reduction of the conductivity of a water body, and ion poison and osmotic stress are relieved. The composite microbial inoculant can be used for planting rice in saline-alkali soil.

Description

Technical Field

[0001] This application belongs to the field of microbiology, specifically relating to a microbial compound agent for rice cultivation in saline-alkali land. Background Technology

[0002] The Songnen Plain soda-saline-alkali land is recognized as one of the world's three major types of soda-saline-alkali land, covering an area of ​​approximately 3.73 million hectares. Practice has shown that promoting rice cultivation in saline-alkali land is one of the most effective methods for soil improvement, not only sustainably restoring damaged soil ecosystems but also yielding considerable food production. However, the high pH, ​​high salinity, and soil compaction of saline-alkali land significantly hinder rice yield, especially during rice growth, where seedling survival rate directly determines the foundation for plant colony establishment and yield formation.

[0003] Microbial inoculants, due to their environmentally friendly characteristics, offer a new solution for salt-tolerant rice cultivation, demonstrating unique advantages in saline-alkali land ecological restoration and agricultural utilization. As a green fertilizer, microbial inoculants can regulate rhizosphere microecology, promote nutrient absorption, and alleviate salt-alkali stress, showing great potential in improving the soil environment and increasing crop yield in saline-alkali land. Among numerous microbial strains, Bacillus has gained widespread recognition in my country. Recent studies indicate that Bacillus may be the dominant factor promoting plant growth and soil health in saline-alkali soils, and screening for Bacillus strains with efficient survival strategies is key to solving the problem of salt damage in agricultural production. However, current research on the regulation of rice growth and development in saline-alkali land by Bacillus remains relatively limited. Summary of the Invention

[0004] This invention provides a microbial compound inoculant for rice cultivation in saline-alkali land.

[0005] The microbial compound agent for rice cultivation in saline-alkali land is composed of Bacillus subtilis Z21 and Bacillus licheniformis Z8.

[0006] Furthermore, the ratio of viable Bacillus subtilis Z21 to Bacillus licheniformis Z8 was 1:1.

[0007] Furthermore, Bacillus subtilis Z21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36477 and deposit date November 3, 2025.

[0008] Furthermore, the identified Bacillus licheniformis is Bacillus licheniformis Z8, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36476 and accession date November 3, 2025.

[0009] Furthermore, the microbial compound inoculant consists of fermentation broth of Bacillus subtilis Z21 and fermentation broth of Bacillus licheniformis Z8.

[0010] Furthermore, the preparation method of Bacillus subtilis Z21 fermentation broth: LB medium was used to culture at 37℃ and 150rpm for 24h.

[0011] Furthermore, the preparation method of Bacillus licheniformis Z8 fermentation broth: cultured in LB medium at 37℃ and 150rpm for 24h.

[0012] The microorganism used in this invention for rice cultivation in saline-alkali land is Bacillus subtilis Z21, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36477, deposit date November 3, 2025, and address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0013] The microorganism used in this invention for rice cultivation in saline-alkali land is Bacillus licheniformis Z8, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36476, deposit date November 3, 2025, and address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0014] The beneficial effects of the microbial compound inoculant of this invention:

[0015] 1. In saline-alkali pot experiments, the microbial compound inoculant significantly improved seedling survival rate. In saline-alkali soil seedling tray experiments, Bacillus subtilis Z21 alone significantly increased proline content and decreased malondialdehyde content in rice seedlings, indicating that Bacillus subtilis Z21 can effectively alleviate oxidative damage to rice seedlings in saline-alkali environments, enhance plant stress resistance, and effectively alleviate the inhibitory effect of saline-alkali stress on early rice growth.

[0016] Meanwhile, although Bacillus licheniformis Z8 alone did not significantly improve the survival rate of rice after transplanting, it showed a clear growth advantage during the tillering stage. Bacillus licheniformis Z8 started tillering earlier, indicating that Bacillus licheniformis Z8 can promote the adaptation of rice seedlings to the saline-alkali environment more quickly and help rice enter the rapid tillering stage, laying the foundation for the accumulation of panicles in the later stage.

[0017] The microbial compound agent of this invention exhibits the best overall performance in terms of rice survival rate and tillering dynamics. It significantly improves seedling survival rate and accelerates tillering process. The number of tillers per hole is also significantly better than that of Bacillus subtilis Z21 or Bacillus licheniformis Z8 used alone. This indicates that the microbial compound agent of this invention has a synergistic effect in promoting rice population development. The synergistic effect of the microbial compound agent stems from the metabolic complementarity and ecological niche synergy among different functional strains. The physiological regulation of Bacillus subtilis Z21 in enhancing stress resistance and Bacillus licheniformis Z8 in promoting tillering forms a superimposed advantage.

[0018] 2. Improving the health of saline-alkali soil is crucial for achieving stable and high crop yields. Changes in pH and electrical conductivity in saline-alkali soil are two core elements in its improvement. The microbial compound inoculant described in this invention significantly reduced the changes in water electrical conductivity during rice cultivation, and these changes exhibited a significant progression over time. Similar patterns were also detected in the rice rhizosphere soil. The rhizosphere electrical conductivity decreased in the microbial compound inoculant treatment group, with reductions observed in both Bacillus subtilis Z21 and Bacillus licheniformis Z8. The microbial compound inoculant treatment group showed significantly higher levels of electrical conductivity than the single-bacterial treatment group, indicating that the synergistic effect of functional microbial communities more effectively regulated rhizosphere salinity dynamics. Changes in water pH also indicate a gradual improvement in the rhizosphere microenvironment. At 50 days after transplanting, the water pH of Bacillus subtilis Z21, Bacillus licheniformis Z8, and the microbial compound inoculant groups all showed a decrease compared to the control group (CK). Although the pH changes among the treatment groups were significant, the small magnitude of the changes meant the interaction between microbial treatment and time was not significant. No significant changes were observed in rhizosphere soil pH among the treatments, indicating that the regulatory effect of microbial inoculants on soil pH is a slow and long-term process. In saline-alkali soil improvement, the core principle is "salt comes with water, salt goes with water." Water is the only carrier of salt migration; therefore, regulating salt transport through water management is crucial. However, during water-salt transport, soil compaction restricts the penetration of salt ions from the water. Combined with the small fluctuations in water pH, this is a significant reason why the pH of the rice rhizosphere soil did not show significant fluctuations. Furthermore, the rate of decrease in water conductivity of Bacillus subtilis Z21 was slower than that of the other two groups during 30 to 50 days after transplanting, resulting in a relatively weaker effect on regulating soil conductivity in the later stages, consistent with the aforementioned problem of insignificant pH changes caused by soil compaction. In summary, microbial agents regulate the rhizosphere salinity environment by slowly influencing water pH and conductivity, creating a more suitable microenvironment for rice root growth. Microbial compound agents enhance this regulatory ability through multi-bacterial synergy, particularly in reducing conductivity. Although short-term pH changes are not significant, long-term application is expected to overcome the limitations of soil compaction and further improve the efficiency of saline-alkali land improvement.

[0019] 3. Soil viable bacteria count is a comprehensive indicator reflecting soil material cycling capacity, soil nutrient supply, and soil ecological health. Using *Bacillus licheniformis* Z8 alone resulted in a higher soil viable bacteria count, while using *Bacillus subtilis* Z21 alone showed no significant fluctuation. However, in terms of soil acid phosphatase levels, using *Bacillus subtilis* Z21 alone was more effective, and the microbial compound agent showed higher results in both soil viable bacteria count and soil acid phosphatase levels. When *Bacillus licheniformis* Z8 is used alone, it exhibits strong soil colonization ability and metabolic activity, effectively promoting the reproduction and activity of indigenous microbial communities. However, the introduction of *Bacillus subtilis* Z21 into the microbial compound agent may not have significantly increased the total viable bacteria count in the short term due to the interaction of different salt-alkali tolerance mechanisms, which may have prevented sufficient synergistic activation of the indigenous microbial community. However, from the perspective of the complementarity of functional microbial communities, after long-term application, microbial compound inoculants significantly reduce soil electrical conductivity by reshaping the rhizosphere microbial network structure, gradually enhancing the driving effect on indigenous microbial communities, thereby achieving a more stable improvement in soil ecological functions.

[0020] Bacillus subtilis Z21 maintained strong growth activity under conditions of 10% NaCl and pH 9.0, significantly improving the survival rate of rice in saline-alkali environments and significantly increasing the activity of acid phosphatase in paddy soil. Bacillus licheniformis Z8 maintained good growth ability under conditions of 6% NaCl and pH 9.0, effectively promoting rice tillering and significantly increasing the number of viable bacteria in the soil. Both strains significantly enhanced the osmotic regulation capacity and antioxidant defense system of rice seedlings in saline-alkali soils, improved leaf photosynthetic efficiency, and facilitated biomass accumulation. After rice transplanting, both Bacillus licheniformis Z8 and Bacillus subtilis Z21 effectively reduced pH and electrical conductivity in water over time and increased the number of effective panicles, thereby significantly increasing yield. The microbial compound inoculant significantly increases the number of rice seedlings by integrating the functional advantages of Bacillus licheniformis Z8 and Bacillus subtilis Z21 strains, and alleviates ion toxicity and osmotic stress by influencing the reduction of rhizosphere soil conductivity through the continuous decrease of water conductivity.

[0021] The synergistic effect of the optimized microbial compound inoculant combination of the present invention can be used for the cultivation of rice in saline-alkali land, thereby achieving a continuous increase in the yield of rice in saline-alkali land and providing a more valuable technical model for the growth of rice in saline-alkali land with the assistance of microorganisms. Attached Figure Description

[0022] Figure 1 Colony morphology for Z21 and Z8;

[0023] Figure 2 A phylogenetic tree of strains based on the 16S sequence;

[0024] Figure 3 The results are from the test of the growth-promoting performance of the strain;

[0025] Figure 4 Results for strains Z21 and Z8 at different salt contents;

[0026] Figure 5 Results for strains Z21 and Z8 at different pH values;

[0027] Figure 6 The plant height of rice seedlings after culturing strains Z21 and Z8;

[0028] Figure 7 Fresh rice seedlings cultured from strains Z21 and Z8;

[0029] Figure 8 The results show the determination of proline content in rice seedling leaves;

[0030] Figure 9 The results show the determination of malondialdehyde content in rice seedling leaves;

[0031] Figure 10 For the determination of chlorophyll content in rice seedling leaves;

[0032] Figure 11 To determine the carotenoid content in rice seedling leaves;

[0033] Figure 12 The results show the survival rate of transplanted rice seedlings;

[0034] Figure 13 To investigate the changes in the number of rice seedlings per planting hole after transplanting;

[0035] Figure 14 Statistics on pH changes in water bodies after rice transplanting;

[0036] Figure 15 Statistics on the changes in water conductivity after rice transplanting;

[0037] Figure 16 Correlation analysis of effective panicle number and yield in rice;

[0038] Figure 17 Correlation analysis of rice dry weight and yield;

[0039] Figure 18 The results of pH testing of the rhizosphere soil of rice in a pot experiment;

[0040] Figure 19 The results of electrical conductivity measurements in the rhizosphere soil of rice in a pot experiment;

[0041] Figure 20 The results are from the determination of viable bacteria count in the soil.

[0042] Figure 21 The results are from the determination of soil acid phosphatase. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0045] Example 1: Preparation of the compound microbial agent of the present invention

[0046] The microbial compound agent described in this embodiment is composed of Bacillus subtilis Z21 and Bacillus licheniformis Z8; the ratio of viable Bacillus subtilis to Bacillus licheniformis is 1:1.

[0047] The Bacillus subtilis Z21 and Bacillus licheniformis Z8 were obtained in July 2025 from saline-alkali soil (experimental field of Da'an Alkali Land Ecological Experimental Station, Chinese Academy of Sciences) using a gradient dilution method.

[0048] Preparation method of Bacillus subtilis Z21 fermentation culture medium: cultured in LB medium at 37℃ and 150rpm for 24h.

[0049] Preparation method of Bacillus licheniformis Z8 fermentation culture medium: cultured in LB medium at 37℃ and 150rpm for 24h.

[0050] Example 2: Comparative Experiment

[0051] Four experimental groups were set up: Group Z21 used Bacillus subtilis Z21 alone (CGMCC No. 36477), Group Z8 used Bacillus licheniformis Z8 alone (CGMCC No. 36476), the compound microbial agent group used the microbial compound microbial agent of the present invention as described in Example 1, and CK was the blank control group.

[0052] 1. Materials

[0053] The rice variety used was Longdao 185, which came from the Institute of Cultivation and Farming of the Heilongjiang Academy of Agricultural Sciences.

[0054] The soil used was soda saline-alkali soil, which came from the experimental field of the Da'an Alkali Land Ecological Experimental Station of the Chinese Academy of Sciences. The soil pH was 8.6, the organic matter content was 11.3 g / kg, the available nitrogen was 29.02 mg / kg, the available phosphorus was 11.2 mg / kg, the available potassium was 192.58 mg / kg, and the electrical conductivity was 426.5 μS / cm.

[0055] Test media: LB liquid medium was used for strain culture; CAS medium was used for siderophore production detection; Monkina medium with lecithin as the sole phosphorus source was used for organic phosphorus detection; starch-dissolving and protein-dissolving media were used for strain performance testing.

[0056] 2. Experiment

[0057] 2.1 Identification of growth-promoting characteristics of Bacillus subtilis Z21 (strain Z21) and Bacillus licheniformis Z8 (strain Z8)

[0058] Strains Z21 and Z8 were inoculated onto functional culture media capable of producing siderophores, dissolving organophosphates, dissolving starch, and dissolving proteins, respectively, and their colorimetric reactions or lysing zones on each functional culture medium were observed.

[0059] 2.2 Stress resistance test of Bacillus subtilis Z21 (strain Z21) and Bacillus licheniformis Z8 (strain Z8)

[0060] The stress resistance of the strain was assessed by a gradient stress experiment. LB liquid medium with NaCl contents of 2%, 4%, 6%, 8%, 10%, and 12% and pH gradients of 7.0, 8.0, 9.0, and 10.0 was prepared, and 1% liquid seed culture was inoculated into the medium. The culture was incubated at 37℃ with shaking, and the OD of the bacterial culture was measured every 4 hours. 600 The value was used to assess the growth ability of the strain under different salt and alkali stresses.

[0061] 2.3 Seedbed Experiment

[0062] This embodiment uses a standard blanket-type seedbed system to conduct a rice seedbed cultivation experiment. There are three treatment groups: a blank control group (CK), and groups Z21 and Z8. One rice seed is precisely sown in each seedbed hole. After the rice seeds germinate, a bacterial suspension cultured to the mid-logarithmic growth stage is extracted, diluted 100 times, and sprayed onto the seedbed soil of the corresponding treatment group. The blank control group is sprayed with an equal amount of water as a negative control. Twenty days later, growth indicators such as seedling height, fresh weight, proline, malondialdehyde, chlorophyll, and carotenoid content are measured.

[0063] 2.4 Pot Experiment

[0064] Eight kilograms of saline-alkali soil were placed in plastic buckets, and 3 g of compound fertilizer (NPK≥30%) was applied to each bucket as base fertilizer. The experiment was divided into four groups: a blank control group, a Z21 microbial agent treatment group, a Z8 microbial agent treatment group, and a compound microbial agent treatment group (Z21+Z8). For the microbial agent treatment groups, 3 mL of microbial solution was added to the water before transplanting, with three replicates per group. Normally cultured rice seedlings were transplanted into plastic buckets after 30 days of growth. The growth of the rice seedlings and changes in the pH and conductivity of the water were monitored every 10 days until the booting stage (50 days). At the rice maturity stage (90 days), plant physiological indicators (plant height, dry weight, effective panicles, etc.), soil physicochemical indicators (pH, EC), and biological activity indicators (effective viable bacteria count, acid phosphatase) were measured.

[0065] 2.4 Data Processing

[0066] Experimental data were statistically analyzed and plotted using Graphpad Prism 5.0 and SPASS 28.0 software.

[0067] 3. Results

[0068] 3.1 Screening and Identification of Strains

[0069] Strains Z21 and Z8 were screened from saline-alkali soil using a gradient dilution method. Figure 1 These are colony morphology diagrams for Z21 and Z8. Figure 1 -A shows the colony morphology of strain Z21. Figure 1 -B is a colony morphology diagram of strain Z8; from Figure 1 The Z21 colony is grayish-white in color, with a rough, opaque surface, wrinkles, a dry texture, and generally irregular edges. Figure 1 -A); Z8 colonies are round, grayish-white in color, with a raised center, and a smooth and bright surface. Figure 1 -B). A target fragment of approximately 1500 bp was amplified using specific 16S rDNA primers. Sequence alignment using BLAST software showed that strain Z21 shared 100% sequence homology with *Bacillus subtilis*, and strain Z8 shared 99.85% sequence homology with *Bacillus licheniformis*. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11. Figure 2 The phylogenetic tree of the strains based on the 16S sequence is shown in the figure. Strain Z21 belongs to the same branch as Bacillus subtilis, and strain Z8 belongs to the same branch as Bacillus licheniformis, with a confidence level of 100%. Based on the 16S rDNA sequence identification results, strain Z21 belongs to Bacillus subtilis, and strain Z8 belongs to Bacillus licheniformis.

[0070] 3.2 Identification of growth-promoting characteristics of different strains

[0071] Strains Z21 and Z8 were inoculated onto functional culture media capable of producing siderophores, dissolving organophosphates, dissolving starch, and dissolving proteins, respectively, and their colorimetric reactions or lysing zones on each functional culture medium were observed. Figure 3 The results of the strain's growth-promoting performance test are from... Figure 3 It can be seen that strain Z21 forms a distinct blue transparent zone on CAS medium. Figure 3 -B indicates that it has a strong ability to produce siderophores; a clear phosphatase ring is produced on lecithin-containing medium ( Figure 3 -A indicates that it has the ability to dissolve organic phosphorus; strain Z8 shows hydrolysis zones on starch medium ( Figure 3 -D), confirming its ability to break down starch; a clear zone also appeared on casein medium ( Figure 3 -C) indicates that it possesses protease activity. Table 1 shows the results of the growth-promoting performance of the strains. As can be seen from Table 1, strain Z21 has the functions of producing iron carriers and solubilizing phosphorus, which helps to enhance the absorption and utilization of nutrients by plants, especially in saline-alkali soils where it can effectively alleviate iron deficiency and phosphorus fixation problems; while strain Z8 shows the ability to degrade starch and decompose protein, which may promote seedling growth by improving rhizosphere organic matter metabolism. The two strains each have unique growth-promoting mechanisms.

[0072] Table 1 Growth-promoting properties of the strains

[0073]

[0074] 3.3 Growth curves at different salt and alkali concentrations

[0075] Strains Z21 and Z8 were inoculated into LB media with different salt concentrations and pH values, and their growth was observed. The results are as follows: Figure 4 As shown ( Figure 4 (Results of inoculating strains Z21 and Z8 with different salt concentrations) Strain Z21 grew well in media with NaCl concentrations of 2%, 4%, 6%, 8%, and 10%, but strain Z21 could not grow normally in media with a NaCl concentration of 12%. Figure 5 The results of inoculating strains Z21 and Z8 with LB medium at different pH values ​​showed that strain Z21 could grow stably at pH 7.0 and 8.0, but its growth activity was significantly inhibited at pH 9.0 and it could not grow at pH 10.0. These results indicate that strain Z21 still exhibits strong growth activity at a NaCl content of 10% and a pH of 9.0, making it suitable for application in saline-alkali soil environments.

[0076] Strain Z8 grew well at NaCl concentrations of 2%, 4%, and 6%. At 8% NaCl concentration, strain Z8 showed a certain growth trend during the lag phase, but growth was significantly limited during the logarithmic and stationary phases. Strain Z8 grew stably at pH 7.0, 8.0, and 9.0, but could not survive at pH 10.0. These results indicate that strain Z8 still possesses good growth ability at a NaCl concentration of 6% and a pH of 9.0, and no pH limit was detected.

[0077] 3.4 Seedbed Experiment

[0078] 3.4.1 Plant physiological indicators

[0079] The fermentation broths of strains Z21 and Z8 were diluted 100-fold and inoculated into newly emerged rice seedlings in saline-alkali soil substrate. Plant growth was recorded after 30 days. Results are as follows: Figure 6 and Figure 7 As shown ( Figure 6 The plant height of rice seedlings after culturing strains Z21 and Z8. Figure 7(Fresh rice seedlings cultured from strains Z21 and Z8) The plant height and fresh weight of rice seedlings inoculated with strain Z21 were significantly higher than those of the control group (p < 0.05 and p < 0.001), with an average increase of 13.98% in plant height and 66.23% in fresh weight. The plant height and fresh weight of seedlings inoculated with strain Z8 were also significantly higher than those of the control group (p < 0.001 and p < 0.001), with increases of 27.44% and 70.13%, respectively. These results indicate that both strains can significantly promote the growth of rice seedlings in saline-alkali soil.

[0080] 3.4.2 Plant biochemical indicators

[0081] Figure 8 The results show the determination of proline content in rice seedling leaves. Figure 9 The results of the determination of malondialdehyde content in rice seedling leaves were obtained by... Figure 8 and Figure 9 The determination of proline and malondialdehyde (MDA) content in rice seedling leaves revealed that inoculation with strains Z21 and Z8 significantly increased the accumulation of proline, an osmotic regulator. The proline content in the Z21 group was significantly higher than that in the control group (p < 0.05), while the proline content in the Z8 group was 17.96% higher than that in the control group. The content of MDA, a membrane damage substance, was significantly reduced, with the MDA content in the Z21 and Z8 groups decreasing by 54.55% and 33.13% respectively compared to the control group (p < 0.01 and p < 0.05). These results indicate that both strains can enhance the osmotic regulation capacity of rice seedlings and alleviate membrane lipid peroxidation damage.

[0082] Figure 10 The results show the determination of chlorophyll content in rice seedling leaves. Figure 11 The results of the determination of carotenoid content in rice seedling leaves were obtained by analyzing... Figure 10 and Figure 11 The determination of chlorophyll and carotenoid content in rice seedling leaves revealed that after inoculation with strains Z21 and Z8, the chlorophyll content of the plants was significantly higher than that of the CK group (p < 0.001 and p < 0.01), and the carotenoid content of the Z21 group was significantly higher than that of the CK group (p < 0.01). The carotenoid content of the Z8 group was also significantly higher than that of the CK group, but the difference was not statistically significant. These results indicate that both strains can effectively promote the synthesis of photosynthetic pigments and enhance the photosynthetic capacity of rice seedlings.

[0083] 3.5 Potted Plant Experiment

[0084] 3.5.1 Survival rate of transplanted rice seedlings

[0085] Figure 12The results for rice transplanting survival rates showed that the survival rates of rice seedlings inoculated with strains Z21, Z8, and the microbial compound agent of this invention in saline-alkali soil were 87.78%, 58.05%, and 96.97%, respectively, significantly higher than the control group's 32.17%. Among them, the Z21 group and the compound agent group performed best, significantly higher than the CK group (p < 0.05 and p < 0.01), indicating that it has a strong promoting effect on seedling root adaptability and stress recovery.

[0086] Figure 13 To investigate the changes in the number of rice seedlings in each planting hole after transplanting, statistical analysis showed that there was no significant difference in the initial number of seedlings among the treatment groups, with the compound microbial agent group having a slightly lower number than the other three groups. The results of the two-way ANOVA showed that 10 days after transplanting, seedlings in all treatment groups died. The number of stems per hole decreased significantly in the CK and Z8 groups, while the number of stems per hole decreased slowly in the Z21 group and the compound microbial agent group. The difference between the Z21 group and the CK group was statistically significant (p < 0.05). 20 days after transplanting, seedlings in the Z21 and CK groups still died, but the number of stems per hole in the Z21 group was still higher than that in the CK group. Rice in the Z8 group and the compound microbial agent group began to tiller, and the number of stems per hole in the compound microbial agent group was significantly higher than that in the CK group (p < 0.001). From 30 to 50 days after transplanting, the number of stems per hole in all treatment groups continued to increase, and the number of stems per hole in the microbial treatment groups was significantly higher than that in the CK group (p < 0.05, p < 0.05 and p < 0.001; p < 0.05, p < 0.01 and p < 0.001; p < 0.05, p < 0.05 and p < 0.001). A two-way ANOVA on the effects of different treatments on the number of rice stalks over time showed (Table 2: Two-way ANOVA on the effects of different treatments on the number of rice stalks over time) that both the different microbial inoculant treatments and time had significant effects on the number of stalks (p < 0.0001 and p < 0.0001), and the interaction between the two was also significant (p < 0.05), indicating that the promoting effect of the inoculant treatment on the number of stalks increased over time. The compound inoculant group showed the strongest growth-promoting effect throughout the entire growth period, indicating that it has a more stable colonization and synergistic regulatory ability under saline-alkali stress.

[0087] Table 2. Two-way ANOVA analysis of the effect of different treatments on the number of rice stems per hole over time.

[0088]

[0089] 3.5.2 Periodic variations in pH and conductivity + repeated measures ANOVA

[0090] Statistical analysis of pH changes in water bodies after rice transplanting showed that there was no significant difference in initial pH among the treatment groups. However, as the transplanting time progressed, the pH of the water body exhibited an unstable trend, and after 30 days, the pH of the water body in the microbial agent treatment group gradually diverged from that of the control group (CK group). Combined with two-way ANOVA results, it was found that the pH of the Z21 group was significantly lower than that of the CK group at 40 and 50 days after transplanting (p < 0.05 and p < 0.01, respectively). Two-way ANOVA on the effects of different treatments on the pH changes of rice pot water over time showed (Table 2) that the effects of different microbial agent treatment groups and time on the number of stems per hole were both significant (p < 0.0001 and p < 0.0001, respectively), but the interaction between the two was not significant (p > 0.05).

[0091] Figure 14 Statistics on pH changes in water bodies after rice transplanting. Figure 15 To statistically analyze the changes in water conductivity after rice transplanting, by... Figure 14 and Figure 15 Statistical analysis showed that there was no significant difference in the initial conductivity among the treatment groups. As the transplanting time progressed, the overall conductivity of the water body showed a trend of first increasing and then decreasing. Combined with the results of two-way ANOVA, it was found that at 10 days after transplanting, the conductivity of each treatment group was not significantly different from the CK group; at 20 days after transplanting, the conductivity of groups Z21 and Z8 was significantly lower than that of the CK group (p < 0.001 and p < 0.05, respectively); at 30 days after transplanting, the conductivity of group Z8 and the compound microbial agent group was significantly lower than that of the CK group (p < 0.001 and p < 0.001, respectively); and at 40 and 50 days after transplanting, the conductivity of each microbial agent treatment group was significantly lower than that of the CK group (p < 0.001 and p < 0.05; p < 0.001 and p < 0.05; p < 0.001 and p < 0.05). A two-way ANOVA on the effects of different treatments on the changes in water conductivity of potted rice plants over time showed (Table 3: Two-way ANOVA on the effects of different treatments on pH and conductivity of potted water over time). The effects of different microbial inoculant treatments and time on water conductivity were all significant (p < 0.0001 and p < 0.0001), and the interaction between the two was also significant (p < 0.01). This indicates that the effect of inoculant treatment on water conductivity reduction increases over time, suggesting that the continuous effect of microbial inoculants not only changes the chemical environment of the water but also has a time-dependent effect.

[0092] Table 3. Two-way ANOVA analysis of the effects of different treatments on pH and conductivity of potted water over time.

[0093]

[0094] 3.5.3 Pot Experiment

[0095] Table 4 shows the effects of different treatments on the components of rice growth and yield. As can be seen from Table 4, the improvement in different indicators varied among the microbial agent treatment groups. Among them, the Z8 group and the compound microbial agent group showed outstanding performance in plant dry matter accumulation, with their dry weight significantly higher than the control group (p < 0.01 and p < 0.05). The dry weight of the Z21 group was also significantly higher than that of the CK group, with an increase of 123.54%, but the difference was not statistically significant. In terms of the number of effective panicles, the Z8 group was significantly higher than the CK group (p < 0.05). Although the Z21 group and the compound microbial agent group were higher than the CK group, the differences were not statistically significant, with average increases of 65.19% and 73.79%, respectively. Regarding the total number of grains, both the Z8 group and the compound microbial agent group were significantly higher than the CK group (p < 0.05 and p < 0.05), while the Z21 group increased by 102.02%, but the difference was not statistically significant. In terms of yield per pot, all microbial agent treatment groups were significantly higher than the CK group (p < 0.05, p < 0.05, and p < 0.05), resulting in a significant yield increase. In addition, all microbial agent treatment groups showed an increasing trend in plant height, grains per panicle, panicle length, seed setting rate, and thousand-grain weight, but the differences were not statistically significant, indicating that the impact of microbial agents on rice yield is mainly reflected in key factors such as dry weight, effective panicle number, and total grain number.

[0096] Table 4. Effects of different treatments on the components of rice growth and yield.

[0097]

[0098] 3.5.4 Relationship between output and constituent factors

[0099] Figure 16 Correlation analysis of effective panicle number and yield in rice. Figure 17 For the correlation analysis of rice dry weight and yield, based on Figure 16 and Figure 17 The analysis results showed that the coefficients of determination (R²) between the number of effective panicles and dry weight and yield were 0.7473 and 0.7370, respectively, and the p-values ​​for the significance test of the regression relationship were 0.0003 and 0.0004 (P<0.001), respectively, both reaching the highly significant level, indicating that there is a highly significant positive correlation between the number of effective panicles and dry weight and yield of rice.

[0100] 3.5.5 pH and electrical conductivity of rhizosphere soil in pot experiments

[0101] Figure 18 The results show the pH values ​​of the rhizosphere soil in a pot experiment of rice. Figure 19The results of the pot experiment on the electrical conductivity of rice rhizosphere soil showed that there was no significant change in pH among the treatment groups. The electrical conductivity of the microbial agent treatment was lower than that of the control group, and the electrical conductivity of the compound microbial agent group was significantly different from that of the CK group (p < 0.05). The electrical conductivity of the Z21 and Z8 groups was reduced by 2.35% and 12.76% compared with the CK group, respectively. The microbial agent treatment effectively reduced soil salinity, improved the rhizosphere environment, and provided favorable conditions for rice growth.

[0102] 3.5.6 Effects of different treatments on soil viable bacteria count and enzyme activity

[0103] Figure 20 The results of soil viable bacteria count determination showed that the number of bacteria in group Z8 was significantly increased compared with that in group CK (p < 0.001), and the number of bacteria in the compound microbial agent group was also significantly increased compared with that in group CK, but the difference was not statistically significant, with an increase of 382.87%. The number of bacteria in group Z21 was not significantly different from that in group CK. Figure 21 The results of soil acid phosphatase determination show that the levels of microbial inoculant treatment were significantly higher than those of the control (p<0.01, p<0.05, and p<0.05), with group Z21 showing better performance than group Z8 and the compound inoculant group.

[0104] 4. Results

[0105] 4.1 Effects of the microbial compound inoculant of the present invention on rice yield

[0106] Rice is the preferred crop for saline-alkali land management, but salinity, as a core environmental stressor restricting crop growth and yield, severely affects rice growth, development, and yield. Microbial inoculants, as a green fertilizer that has emerged in recent years, have shown great potential in improving the soil environment and increasing crop yield in saline-alkali land by regulating rhizosphere microecology, promoting nutrient absorption, and alleviating saline-alkali stress. During the growth of rice in saline-alkali land, the survival rate of rice seedlings directly determines the foundation for population establishment and yield formation. In pot experiments, the Z21 group and the compound inoculant group significantly improved seedling survival rates. In saline-alkali soil seedling tray experiments, the Z21 group significantly increased the proline content and decreased the malondialdehyde content in rice seedlings, indicating that strain Z21 can effectively alleviate oxidative damage to rice seedlings in a saline-alkali environment, enhance plant resistance, and effectively alleviate the inhibitory effect of saline-alkali stress on early rice growth. Meanwhile, although the survival rate of rice seedlings in group Z8 did not increase significantly after transplanting, it showed a clear growth advantage during the tillering stage. The tillering initiation time of group Z8 and the compound microbial agent group was earlier than that of group CK, indicating that strain Z8 can promote the adaptation of rice seedlings to the saline-alkali environment more quickly and help rice enter the rapid tillering stage, laying the foundation for the accumulation of panicles in the later stage. The compound microbial agent group showed the best overall performance in terms of rice survival rate and tillering dynamics, which not only significantly improved the seedling survival rate but also accelerated the tillering process. The number of panicles was also significantly better than that of groups Z21 and Z8, indicating that the microbial compound microbial agent of this invention has a synergistic effect in promoting rice population development. The synergistic effect of the microbial compound microbial agent of this invention stems from the metabolic complementarity and ecological niche synergy between different functional strains. The enhanced stress resistance of strain Z21 and the physiological regulation of tillering by strain Z8 form a superimposed advantage. In a two-way ANOVA of the change in the number of rice panicles over time, the interaction between treatment and time had a significant impact on the number of panicles (p=0.0199), indicating that microbial agents have a significant impact on the dynamic changes in rice tillering over time. Furthermore, in the correlation analysis, there was a highly significant positive correlation between the number of effective panicles and yield (p=0.0003), and the number of effective panicles in the microbial agent treatment group was significantly higher than that in the control group (CK group). This indicates that microbial agents significantly improve yield by enhancing rice seedling survival rate and promoting tillering (p<0.05). These results show that strain Z21 can ensure uniform and robust seedling growth by strengthening stress resistance in the early stages, while strain Z8 can activate the growth potential of rice in the early tillering stage. The dominant roles of the two strains differ significantly at different growth stages. The microbial compound agent integrates a dual mechanism, achieving a sustained increase in rice yield in saline-alkali land through the synergistic effect of optimized agent combinations, providing a more valuable technical model for the promotion of microbial-assisted rice growth in saline-alkali land.

[0107] 4.2 The impact of the microbial compound inoculant of the present invention on soil health

[0108] Improving the health of saline-alkali soil is crucial for achieving stable and high crop yields. Changes in pH and electrical conductivity in saline-alkali soil are two core elements in its improvement. This embodiment demonstrates that the application of the microbial compound agent of this invention significantly reduced the changes in water electrical conductivity during rice cultivation, and the changes in electrical conductivity showed a significant progression over time. Similar patterns were also detected in the rhizosphere soil of rice. The rhizosphere electrical conductivity of the microbial compound agent of this invention decreased by 18.17%, with reductions of 2.35% and 12.76% in groups Z21 and Z8, respectively. The compound agent treatment group showed significantly higher levels of activity than the single-strain treatment group, indicating that the synergistic effect of functional microbial communities more effectively regulated the dynamics of rhizosphere salinity. Changes in water pH also indicate a gradual improvement in the rhizosphere microenvironment. In this example, at 50 days after transplanting, the water pH in groups Z21, Z8, and the compound microbial agent group decreased by 5.61%, 2.52%, and 3.43% respectively compared to the CK group. Although the changes in water pH among the treatment groups showed significant differences, the interaction between microbial treatment and time was not significant due to the small range of change. Furthermore, there were no significant changes in rhizosphere soil pH among the treatments, indicating that the regulatory effect of microbial agents on soil pH is a slow and long-term process. In saline-alkali soil improvement, the core principle of saline-alkali soil management is "salt comes with water and salt goes with water." Water is the only carrier for salt migration; therefore, regulating salt transport through water management is key to treatment. However, during water-salt transport, soil compaction restricts the penetration of salt ions from the water, and the small fluctuations in water pH are important reasons why the pH of the rice rhizosphere soil did not show significant fluctuations. Furthermore, the rate of decrease in water conductivity in group Z21 was slower than that of the other two groups during 30 to 50 days after transplanting, resulting in a relatively weaker effect on regulating soil conductivity in the later stages, consistent with the aforementioned issue of insignificant pH changes caused by soil compaction. In summary, microbial agents regulate the rhizosphere salinity environment by slowly influencing water pH and conductivity, creating a more suitable microenvironment for rice root growth. Compound microbial agents enhance this regulatory capacity through multi-microbial synergy, particularly excelling in reducing conductivity. Although short-term pH changes are not significant, long-term application can overcome the limitations of soil compaction and further improve the efficiency of saline-alkali land improvement.

[0109] Soil viable bacteria count is a comprehensive indicator reflecting soil material cycling capacity, soil nutrient supply, and soil ecological health. In this embodiment, the soil viable bacteria count in group Z8 was significantly higher than that in group CK (p < 0.001), while group Z21 showed no significant change compared to group CK. Although the compound microbial agent group was significantly higher than group CK, the difference was not statistically significant. Regarding soil acid phosphatase results, all microbial treatment groups were significantly better than group CK, with group Z21 being superior to the other two groups, representing different salt-alkali tolerance mechanisms between strains Z21 and Z8. These results indicate that strain Z8 has strong soil colonization ability and metabolic activity, effectively promoting the reproduction and activity enhancement of indigenous microbial communities. However, the introduction of the microbial compound microbial agent into strain Z21 failed to fully synergistically activate the indigenous microbial community in the short term due to the interaction of different salt-alkali tolerance mechanisms, resulting in no significant increase in the total viable bacteria count. However, from the perspective of functional microbial community complementarity, the microbial compound microbial agent of this invention, after long-term application, significantly reduces soil electrical conductivity by reshaping the rhizosphere microbial network structure, gradually enhancing the driving effect on the indigenous microbial community, thereby achieving a more stable improvement in soil ecological function.

[0110] 4.3 Strain Z21 maintained strong growth activity under conditions of 10% NaCl and pH 9.0, significantly improving the survival rate of rice in saline-alkali environments and significantly increasing the activity of acid phosphatase in rice soil. Strain Z8 maintained good growth ability under conditions of 6% NaCl and pH 9.0, effectively promoting rice tillering and significantly increasing the number of viable bacteria in the soil. Both strains significantly enhanced the osmotic regulation capacity and antioxidant defense system of rice seedlings in saline-alkali soil, improved leaf photosynthetic efficiency, and accumulated biomass. After rice transplanting, both strains Z8 and Z21 effectively reduced the pH and conductivity of water over time and increased the number of effective panicles, thereby significantly increasing yield. This invention's microbial compound agent, by integrating the functional advantages of strains Z8 and Z21, significantly increases the number of rice seedlings in planting holes and, through the continuous decrease in water conductivity, affects the reduction of rhizosphere soil conductivity, alleviating ion toxicity and osmotic stress.

Claims

1. A microbial complex microbial agent for saline-alkali paddy rice planting, characterized in that The microbial compound inoculant consists of Bacillus subtilis Z21 and Bacillus licheniformis Z8.

2. The microbial complexing agent for saline-alkali paddy planting according to claim 1, characterized in that The ratio of viable Bacillus subtilis Z21 to Bacillus licheniformis Z8 was 1:

1. 3.The microbial compound microbial inoculant for saline-alkali paddy field planting according to claim 1, characterized in that Bacillus subtilis Z21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36477 and accession date November 3, 2025.

4. The microbial compound inoculant for rice cultivation in saline-alkali land according to claim 1, characterized in that... The Bacillus licheniformis strain Z8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36476 and accession date November 3, 2025.

5. The microbial compound inoculant for rice cultivation in saline-alkali land according to claim 1, characterized in that, The microbial compound inoculant consists of fermentation broth of Bacillus subtilis Z21 and Bacillus licheniformis Z8.

6. The microbial compound inoculant for rice cultivation in saline-alkali land according to claim 5, characterized in that, Preparation method of Bacillus subtilis Z21 fermentation broth: cultured in LB medium at 37℃ and 150rpm for 24h.

7. The microbial compound inoculant for rice cultivation in saline-alkali land according to claim 5, characterized in that, Preparation method of Bacillus licheniformis Z8 fermentation broth: cultured in LB medium at 37℃ and 150rpm for 24h.

8. Microorganisms for rice cultivation in saline-alkali land, characterized in that... The microorganism is Bacillus subtilis Z21, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36477 and deposit date November 3, 2025.

9. Microorganisms for rice cultivation in saline-alkali land, characterized in that... The microorganism is Bacillus licheniformis Z8, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36476 and deposit date November 3, 2025.

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