Microbial agent for saline-alkali soil improvement and preparation method and application thereof

Abstract

The application discloses a kind of microbial inoculants for saline-alkali soil improvement and preparation method and application thereof, it is related to microbial inoculants technical field, the microbial inoculants for saline-alkali soil improvement includes by saline-alkali soil fern rhizosphere soil extraction soil microorganism enrichment liquid, EM bacteria fermentation liquor, trichoderma harzianum activation solution and fern powder mixed solution;The soil microorganism enrichment liquid and EM bacteria fermentation liquor contain nitrogen-fixing bacteria, phosphorus-solubilizing bacteria and plant rhizosphere growth-promoting bacteria;The OD600 value of microbial inoculants is 0.5-1.0, and the effective viable bacterial number in each gram of microbial inoculants is 4.0×10^8 CFU / g-8.0×10^8 CFU / g;Endemic microorganism enrichment liquid, EM bacteria fermentation liquor, trichoderma harzianum activation solution and fern powder mixed solution are compounded and fermented.EM bacteria provide lactic acid bacteria, yeast, photosynthetic bacteria and other various beneficial bacteria, trichoderma harzianum has the ability to inhibit soil-borne pathogenic bacteria, and endemic bacteria provide efficient nitrogen fixation, phosphorus solubilization and growth promotion functions.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a microbial inoculant for improving saline-alkali land, its preparation method, and its application. Background Technology

[0002] Saline-alkali land is a significant obstacle to agricultural production and ecological environment improvement. The area of ​​saline-alkali land is expanding globally, and it is widely distributed in my country, especially in the Northwest, North China, and coastal areas, where soil salinization is severe, leading to stunted crop growth, reduced yields, and even land abandonment. Traditional methods for improving saline-alkali land mainly include physical methods (such as topsoil import and deep plowing), water conservancy methods (such as irrigation and drainage for salt leaching), and chemical methods (such as applying gypsum and aluminum sulfate). These methods are often costly, involve large-scale engineering projects, are prone to secondary pollution, and their improvement effects are difficult to sustain.

[0003] In recent years, microbial inoculants have gradually become a research hotspot for the biological improvement of saline-alkali land due to their environmentally friendly, efficient, and sustainable characteristics. Microbial inoculants improve the rhizosphere microenvironment and promote crop growth through mechanisms such as nitrogen fixation, phosphorus and potassium solubilization, secretion of plant hormones, and inhibition of pathogens. However, most commercially available microbial inoculants are single-strain or simple compound formulations, lacking adaptability to specific saline-alkali land ecological conditions, and exhibiting problems such as poor colonization ability, weak synergistic effects of functional bacteria, and unstable improvement effects. Furthermore, the development of specialized microbial inoculants for characteristic crops of saline-alkali land (such as *Fern Root*) is still lacking.

[0004] Therefore, developing a microbial agent for saline-alkali land improvement, its preparation method, and its application are of great significance for saline-alkali land improvement and increasing the yield and quality of specialty crops. Summary of the Invention

[0005] In view of this, the present invention addresses the deficiencies of existing technologies, and its main objective is to provide a microbial inoculant for saline-alkali land improvement, its preparation method, and its application. The present invention involves a compound fermentation of a mixture of indigenous microbial enrichment solution, EM (Effective Microorganisms) fermentation broth, Trichoderma harzianum activation solution, and fern fiber powder. EM provides various beneficial bacteria such as lactic acid bacteria, yeast, and photosynthetic bacteria; Trichoderma harzianum has the ability to inhibit soil-borne pathogens; and indigenous bacteria provide efficient nitrogen fixation, phosphorus solubilization, and growth promotion functions. The synergistic effect of these multiple functional bacteria can simultaneously achieve multiple objectives, including salinity reduction, pH adjustment, nutrient activation, disease suppression, and root development promotion.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A microbial agent for improving saline-alkali land, comprising a soil microbial enrichment solution extracted from the rhizosphere soil of *Pteris vittata* in saline-alkali land, an EM (Effective Microorganisms) fermentation broth, a *Trichoderma harzianum* activation broth, and a mixture of *Pteris vittata* powder; the soil microbial enrichment solution and the EM fermentation broth contain nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant rhizosphere growth-promoting bacteria; the OD600 value of the microbial agent is 0.5-1.0, and the effective viable bacteria count per gram of microbial agent is 4.0×10^8 CFU / g-8.0×10^8 CFU / g.

[0007] As a preferred embodiment: the fern root powder mixture is prepared by mixing fern root powder and water at a mass ratio of 1:10 and then letting it stand at 25-30℃ for 5 days; the EM fermentation liquid is prepared by sealing and fermenting brown sugar, EM bacteria and water at 25-30℃ for 7-10 days; the Trichoderma harzianum activation liquid is prepared by mixing Trichoderma harzianum and warm water at 30℃ at a mass ratio of 1:10 and then activating it at 28-30℃ for 2-3 hours.

[0008] A method for preparing a microbial inoculant for improving saline-alkali land includes the following steps: S1. Soil microbial extraction: Fresh soil from the rhizosphere of *Fernonia chinensis* in saline-alkali land was mixed with sterile water and shaken. After standing, the supernatant was centrifuged to obtain the soil rhizosphere microbial mother liquor. S2. Component preparation: Prepare fern root powder mixture, EM fermentation broth and Trichoderma harzianum activation broth separately; S3. Compound fermentation: Mix the fern root powder mixture, EM fermentation liquid and Trichoderma harzianum activation liquid in proportion, and let it stand at 25-30℃ for 12-15 hours to obtain the microbial agent.

[0009] As a preferred embodiment: during soil microbial extraction in step S1, the mass ratio of soil to sterile water is 1:9, the shaking conditions are 28-30℃, shaking at 180r / min for 50 minutes, and centrifugation conditions are 5000r / min for 10 minutes.

[0010] As a preferred embodiment: in the compound fermentation step S3, the mixing ratio of each component by volume is: fern root powder mixture: EM fermentation broth: Trichoderma harzianum mixture = (2-5): (6-9): (0.5-2).

[0011] As a preferred embodiment: in the compound fermentation step S3, the mixing ratio of each component by volume is: fern root powder mixture: EM fermentation broth: Trichoderma harzianum mixture = 3.75 : 7.5 : 1.

[0012] The application of the aforementioned microbial agent in the improvement of saline-alkali land.

[0013] As a preferred embodiment: the microbial agent is used to improve the physicochemical properties of saline-alkali soil and / or promote crop growth; the improvement of the physicochemical properties of saline-alkali soil includes any one or more of the following: reducing soil pH, reducing soil electrical conductivity, reducing soil total salt content, improving the stability of soil water-stable aggregates, increasing soil organic matter content, increasing soil nitrogen and phosphorus nutrient content, and increasing soil enzyme activity.

[0014] As a preferred embodiment, the application rate of the microbial agent in saline-alkali land is 100-150 kg / hm². 2 .

[0015] As a preferred embodiment, the application rate of the microbial agent in saline-alkali land is 120 kg / hm². 2 .

[0016] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution: First, it exhibits good local adaptability and strong colonization ability: This invention extracts an enriched solution of indigenous microorganisms from the rhizosphere soil of *Pteris vittata* in saline-alkali land. This solution contains nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant rhizosphere growth-promoting bacteria (PGPR) that have adapted to the local saline-alkali environment. These indigenous strains possess natural ecological adaptability and can rapidly colonize and reproduce after being applied to the soil, resulting in a lasting improvement effect.

[0017] Secondly, the synergistic effect of multiple microorganisms provides comprehensive functionality: This invention involves the compound fermentation of a mixture of indigenous microbial enrichment solution, EM fermentation broth, Trichoderma harzianum activation solution, and fern root powder. EM provides various beneficial bacteria such as lactic acid bacteria, yeast, and photosynthetic bacteria; Trichoderma harzianum has the ability to inhibit soil-borne pathogens; and indigenous bacteria provide efficient nitrogen fixation, phosphorus solubilization, and growth promotion functions. The synergistic effect of multiple functional bacteria can simultaneously achieve multiple goals, including salt reduction, pH adjustment, nutrient activation, disease inhibition, and root development promotion.

[0018] Third, the fern root powder mixture provides nutrients and promotes the proliferation of bacterial strains: fern root powder is rich in nutrients such as starch, sugars, and amino acids. As a natural carbon and nitrogen source, it can significantly promote the growth and metabolic activity of various microorganisms in the compound bacterial agent and increase the effective live bacteria count of the bacterial agent.

[0019] Fourth, it significantly improves the physical and chemical properties of saline-alkali soil: After applying the microbial agent of this invention, the soil pH value is significantly reduced, and the electrical conductivity (EC) and total salt content are significantly reduced; the stability of soil water-stable aggregates is improved, and the proportion of large aggregates increases; the content of soil organic matter, available phosphorus, total phosphorus and other nutrients is increased; and the activity of soil enzymes such as alkaline phosphatase is enhanced.

[0020] Fifth, it promotes crop growth and improves yield and quality: Application results on *Pteris vittata* show that the microbial agent of this invention can significantly increase plant height, root length, and above-ground biomass, reduce disease incidence, and significantly increase yield. Simultaneously, it improves the density and appearance of *Pteris vittata* bulbs, increasing their commercial value.

[0021] Sixth, the raw materials used in this invention are widely available and inexpensive, the preparation process does not require complex equipment, and the fermentation conditions are mild (room temperature and pressure), making it suitable for grassroots agricultural production units to propagate and use on their own, and it has good prospects for promotion and application.

[0022] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the effect of different application rates of microbial agents of the present invention on the pH of rhizosphere and non-rhizosphere soils of *Fernonia spp.* in saline-alkali land. Figure 2 This is a schematic diagram illustrating the effect of different application rates of microbial inoculants on the EC (excessive osmosis) of rhizosphere and non-rhizosphere soils in saline-alkali land. Figure 3 This is a schematic diagram illustrating the effect of different application rates of microbial inoculants on the TDS of rhizosphere and non-rhizosphere soils of *Pteris vittata* in saline-alkali land. Figure 4 This is a schematic diagram illustrating the effect of different application rates of microbial agents of the present invention on the organic matter content of rhizosphere and non-rhizosphere soils of *Fernonia chinensis* in saline-alkali land. Figure 5 This is a schematic diagram illustrating the effect of different application rates of microbial inoculants on the total nitrogen content in the rhizosphere and non-rhizosphere soils of *Fernonia acutissima* in saline-alkali land. Figure 6 This is a schematic diagram illustrating the effect of different microbial inoculant application rates on the total phosphorus content in the rhizosphere and non-rhizosphere soils of *Fernonia acutissima* in saline-alkali land. Figure 7 This is a schematic diagram illustrating the effect of different application rates of microbial inoculants on the available phosphorus content in the rhizosphere and non-rhizosphere soils of *Fernonia acutissima* in saline-alkali land. Figure 8 This is a schematic diagram illustrating the effect of different application rates of microbial inoculants on CBH activity in the rhizosphere and non-rhizosphere soils of *Pteris vittata* in saline-alkali land. Figure 9 This is a schematic diagram illustrating the effect of different application rates of microbial agents of the present invention on LAP activity in the rhizosphere and non-rhizosphere soils of *Pteris vittata* in saline-alkali land. Figure 10 This is a schematic diagram illustrating the effect of different application rates of microbial agents of the present invention on NAG activity in the rhizosphere and non-rhizosphere soils of *Pteris vittata* in saline-alkali land. Detailed Implementation

[0024] The present invention is as follows Figure 1 As shown in Figure 10, a microbial agent for improving saline-alkali land comprises a soil microbial enrichment solution extracted from the rhizosphere soil of *Pteris vittata* in saline-alkali land, an EM fermentation broth, a *Trichoderma harzianum* activation broth, and a mixture of *Pteris vittata* powder. The soil microbial enrichment solution and the EM fermentation broth contain nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant rhizosphere growth-promoting bacteria. The OD600 value of the microbial agent is 0.5-1.0, and the effective viable bacteria count per gram of microbial agent is 4.0×10^8 CFU / g-8.0×10^8 CFU / g.

[0025] The fern root powder mixture is prepared by mixing fern root powder and water at a mass ratio of 1:10 and then letting it stand at 25-30℃ for 5 days; the EM fermentation liquid is prepared by fermenting brown sugar, EM bacteria and water in a sealed container at 25-30℃ for 7-10 days; the Trichoderma harzianum activation liquid is prepared by mixing Trichoderma harzianum and warm water at 30℃ at a mass ratio of 1:10 and then activating it at 28-30℃ for 2-3 hours.

[0026] A method for preparing a microbial inoculant for improving saline-alkali land includes the following steps: S1. Soil microbial extraction: Fresh soil from the rhizosphere of *Fernonia chinensis* in saline-alkali land was mixed with sterile water and shaken. After standing, the supernatant was centrifuged to obtain the soil rhizosphere microbial mother liquor. S2. Component preparation: Prepare fern root powder mixture, EM fermentation broth and Trichoderma harzianum activation broth separately; S3. Compound fermentation: Mix the fern root powder mixture, EM fermentation broth and Trichoderma harzianum activation broth in a certain proportion, and let it stand at 25-30℃ for 12-15 hours to obtain the microbial agent.

[0027] In step S1, when extracting soil microorganisms, the mass ratio of soil to sterile water is 1:9, the shaking conditions are 28-30℃, shaking at 180r / min for 50 minutes, and centrifugation conditions are 5000r / min for 10 minutes.

[0028] In step S3 of the compound fermentation process, the mixing ratio of each component by volume is: fern root powder mixture: EM fermentation broth: Trichoderma harzianum mixture = (2-5): (6-9): (0.5-2).

[0029] In step S3 of the compound fermentation process, the mixing ratio of each component by volume is: fern root powder mixture: EM fermentation broth: Trichoderma harzianum mixture = 3.75 : 7.5 : 1.

[0030] An application of this microbial agent in the improvement of saline-alkali land.

[0031] This microbial agent is used to improve the physicochemical properties of saline-alkali soil and / or promote crop growth; the improvement of the physicochemical properties of saline-alkali soil includes any one or more of the following: reducing soil pH, reducing soil electrical conductivity, reducing soil total salt content, improving the stability of soil water-stable aggregates, increasing soil organic matter content, increasing soil nitrogen and phosphorus nutrient content, and increasing soil enzyme activity.

[0032] The application rate of this microbial agent in saline-alkali land is 100-150 kg / hm². 2 .

[0033] The application rate of this microbial agent in saline-alkali land is 120 kg / hm². 2 .

[0034] Example: Application of a microbial agent in the improvement of saline-alkali land Effects of different application rates of microbial inoculants on soil physical properties Effects on soil pH, electrical conductivity and total salt content Soil pH, electrical conductivity (EC), and total salt content (TDS) are the core indicators for evaluating the improvement effect of saline-alkali land. As shown in Table 1, the application of microbial inoculants had a significant impact on the salinity and alkali indicators of both rhizosphere and non-rhizosphere soils of *Pteris vittata*.

[0035] Soil pH values ​​in the rhizosphere ranged from 7.69 to 8.03 across all treatments. The T2 treatment (80 kg / hm²) had the lowest pH value at 7.69, significantly lower than treatments T0 and T3. P <0.05), a decrease of 2.9% compared to T0 (7.92); T3 treatment (120 kg / hm 2 The pH value rose to 8.03, significantly higher than other treatments. P <0.05), an increase of 1.4% compared to T0; the differences between T1, T4, and T5 treatments and T0 were not significant ( P >0.05), and remained at 7.94, 7.93, and 7.90 respectively. This indicates that the regulation of rhizosphere pH by microbial inoculants is not a one-way decrease; the low to medium dose (80 kg / hm²) has the best alkalinity-reducing effect, while the medium dose (120 kg / hm²) shows a rebound, and the overall change is limited.

[0036] In non-rhizosphere soil, the T0 treatment had the highest pH value (8.37), significantly higher than the T1-T4 treatments. P <0.05; T5 treatment was the second best (8.33), with no significant difference from T0 ( P >0.05), but significantly higher than T1-T4 ( P<0.05), with no significant differences among treatments T1-T4, and pH values ​​maintained between 8.15 and 8.22. The response range of non-rhizosphere pH was smaller than that of the rhizosphere, indicating that the regulatory effect of the microbial agent on soil pH is mainly concentrated in the rhizosphere microdomain.

[0037] Soil electrical conductivity (EC) and rhizosphere electrical conductivity showed a fluctuating trend of first increasing, then decreasing, and then increasing again with increasing inoculant application rate. The T4 treatment (160 kg / hm²) had the lowest EC value (832 uS / cm), significantly lower than other treatments. P <0.05, a decrease of 23.3% compared to T0 (1085 uS / cm). The EC values ​​of T2 and T5 treatments were 1262 and 1313 uS / cm, respectively, both significantly higher than T0 ( P <0.05, T1 and T3 treatments (1078 and 1089 uS / cm) were not significantly different from T0 ( P >0.05). This result indicates that only medium to high doses of inoculant (160 kg / hm²) can effectively reduce rhizosphere salt content, while low or excessive doses may lead to salt accumulation.

[0038] In non-rhizosphere soil, the differences in EC values ​​among treatments were even more pronounced. Treatment T0 had the highest EC at 4907 uS / cm, significantly higher than all other inoculant treatments (P<0.05). Treatment T2 (80 kg / hm²) had the lowest EC at 985 uS / cm, a 79.9% decrease compared to T0, significantly lower than all other treatments (P<0.05). As the inoculant dosage continued to increase, non-rhizosphere EC gradually recovered: T3 was 1680 uS / cm, T4 was 2357 uS / cm, and T5 rebounded to 4077 uS / cm, with significant differences between these three and T0 (P<0.05). Treatment T1 had an EC of 1211 uS / cm, a decrease of 75.3%.

[0039] Total salt content (TDS) showed a trend in rhizosphere TDS that was highly consistent with EC. Treatment T4 had the lowest TDS (0.58 g / kg), significantly lower than treatments T0, T2, and T5 (P<0.05), and a 23.7% decrease compared to T0 (0.76 g / kg). Treatments T2 and T5 showed TDS increases to 0.89 and 0.91 g / kg, respectively, significantly higher than T0 and T4 (P<0.05). Treatments T1 and T3 (0.75 and 0.75 g / kg) showed no significant difference from T0 (P>0.05). Non-rhizosphere TDS also exhibited a pattern of highest T0 and lowest T2. The TDS in the T0 treatment reached 3.42 g / kg, while that in the T2 treatment decreased to 0.69 g / kg, a reduction of 79.8%. Although the TDS in the T1 (0.83 g / kg), T3 (1.16 g / kg), T4 (1.64 g / kg), and T5 (2.84 g / kg) treatments were all significantly lower than that in the T0 treatment, they gradually rebounded with increasing application rates, with T5 approaching the T0 level. This trend further illustrates that 40–80 kg / hm² of microbial inoculant can significantly reduce non-rhizosphere background salinity by improving soil permeability, but excessive application (195 kg / hm²) leads to salinity rebound.

[0040] Table 1: Soil pH, EC and TDS under different treatments

[0041] Impact on soil aggregate stability: Soil aggregates are the basic units of soil structure, and their particle size distribution and stability are key indicators for evaluating soil physical quality. This study used a wet sieving method to classify rhizosphere soil aggregates into four levels: large aggregates (2 mm), medium aggregates (1-2 mm), small aggregates (0.25-1 mm), and microaggregates (<0.25 mm), and further classified them using a wet sieving method. 0.25 Aggregate stability was comprehensively evaluated by (total content of 0.25mm water-stable aggregates), mean weight diameter (MWD), and geometric mean diameter (GMD).

[0042] The content of large aggregates (2 mm) showed a trend of first increasing and then decreasing with increasing application rate of microbial inoculant. The content was lowest in treatment T0 (1.22), while treatments T4 and T5 reached 3.43 and 3.12, respectively, significantly higher than that in treatment T0. P <0.05), representing increases of 181.1% and 155.7%, respectively. The T1-T3 treatments (2.39-2.67) did not show a significant difference from T0. PThe concentration of microbial inoculants (>0.05) was not significantly different from that of T4 and T5. This indicates that medium to high concentrations of microbial inoculants (160–195 kg / hm²) can significantly promote the formation of large aggregates (>2 mm). Among medium aggregates (1–2 mm), the concentration was highest in treatment T3 (2.52) and lowest in treatment T0 (1.44), but only some treatments showed significant differences, with the overall variation being smaller than that of large aggregates. Among small aggregates (0.25–1 mm), the concentration was also highest in treatment T4 (5.78) and lowest in treatment T0 (3.16), with a significant difference between T4 and T0. P <0.05%, the other treatments were in the middle, and the differences between the two were not significant. The content of small aggregates in treatment T4 increased by 82.9% compared with T0. Among microaggregates (<0.25mm), the effect of different microbial agent application rates on the content of microaggregates was not significant. P The value was >0.05, and fluctuated between 8.48% and 12.54%. This indicates that microbial agents mainly affect the formation and transformation of larger particle size aggregates, and have limited direct impact on micro-aggregates.

[0043] WR 0.25 (Total content of water-stable agglomerates >0.25 mm): This index comprehensively reflects the total amount of agglomerates of all particle sizes >0.25 mm. T3 and T4 treatments WR 0.25 The scores were 20.20 and 20.09 respectively, significantly higher than T0 (11.64) and T1 (13.90). P <0.05), which is 73.5% and 72.6% higher than T0, respectively; T2 and T5 treatments (16.25 and 16.30) are in the middle, with no significant difference from other groups. Overall, it shows a unimodal change characteristic of "low amount promotes, medium amount is optimal, and high amount slightly decreases".

[0044] Table 2: Content of aggregates at various levels in rhizosphere soil under different treatments (mean ± standard deviation, n=3)

[0045] Weight-mean diameter (MWD) and geometric mean diameter (GMD) are important parameters for evaluating the stability of soil aggregates. Table 3 shows that the amount of microbial agent applied has a significant impact on both. Weight-mean diameter (MWD): [This is similar to] WR... 0.25 The variation showed a highly consistent unimodal change, initially rising and then falling. The MWD of treatment T3 (120 kg / hm²) reached 0.170 mm, the highest among all treatments, significantly higher than T0 (0.076 mm), T4 (0.127 mm), and other treatments. P<0.05%, an increase of 124.2% compared to T0. Treatment T5 (0.136 mm) was the second highest, while treatment T0 had the lowest content. This result indicates that an application rate of 120 kg / hm² of microbial agent can maximize the water stability of soil aggregates and achieve the best structural improvement effect. Beyond this application rate, MWD significantly decreased. Geometric mean diameter (GMD): The trend differed from MWD. ​​Treatments T0, T1, and T5 all had the highest GMD values ​​(0.915–0.918 mm), with no significant difference among the three. Treatment T4's GMD decreased to 0.847 mm, significantly lower than all other treatments. P < 0.05), with treatments T2 and T3 (0.894 and 0.895 mm) falling in the middle. The abnormal decrease in GMD in treatment T4 indicates that while the 160 kg / hm² inoculant dosage promoted the formation of large aggregates, it may have altered the original distribution pattern of microaggregates, leading to an abnormal decrease in the geometric mean diameter.

[0046] Table 3: Stability index of rhizosphere soil aggregates under different treatments (mean ± standard deviation, n=3)

[0047] Comprehensive distribution of four-level aggregates, WR 0.25 Based on the changing patterns of MWD and GMD, the following conclusions can be drawn: the application of microbial agents mainly increases the total content of water-stable aggregates (WR) by promoting the formation of large aggregates (2 mm) and small aggregates (0.25–1 mm). 0.25 This improves the soil structure of saline-alkali land. All aggregate stability indices reached optimal or near-optimal levels at 120 kg / hm² (T3 treatment), which was determined to be the optimal application rate for structural improvement. Excessive application (above 160 kg / hm²) still promoted the formation of large aggregates, but the mean squared displacement (MWD) began to decrease, the gross medley density (GMD) significantly decreased, and the structural improvement effect declined.

[0048] Effects of different application rates of microbial inoculants on soil chemical properties Impact on soil organic matter content Table 4 shows that the application rate of the microbial agent had a relatively limited effect on the organic matter content of both rhizosphere and non-rhizosphere soils. In the rhizosphere soil, the T1 treatment had the highest organic matter content (8.78 g / kg), which was 9.5% higher than that of T0 (8.02 g / kg), and the difference between the two was significant. P <0.05, the T5 treatment had the lowest (7.65 g / kg), which was significantly different from the T1 treatment. P <0.05), but the difference from T0, T2 (7.94 g / kg), T3 (8.21 g / kg) and T4 (7.83 g / kg) was not significant. P>0.05). Rhizosphere organic matter showed a slight overall trend of "increasing at low levels and decreasing at high levels," with variations within ±10%. T3 was slightly higher than T0 by 2.4%, but the difference was not statistically significant. This indicates that the net accumulation effect of single-season application of microbial agents on the total organic matter pool of the rhizosphere soil is limited, and the organic matter carried by the microbial agents is almost in balance with the consumption by microbial decomposition.

[0049] In non-rhizosphere soils, the response of organic matter content was more complex. Treatment T1 had the lowest organic matter content (6.34 g / kg), and both T1 and T3 (6.51 g / kg) were significantly lower than T0 (7.18 g / kg) (P<0.05). This may be related to the activation of microbial activity by the inoculant and the acceleration of the decomposition of existing organic matter in the non-rhizosphere. Treatment T2 had the highest organic matter content (7.45 g / kg), but the difference from T0 was not significant; treatment T5 (7.15 g / kg) recovered to the T0 level.

[0050] A comparison between the rhizosphere and non-rhizosphere showed that the organic matter content in the rhizosphere was higher than that in the non-rhizosphere (6.34–7.45 g / kg) under all treatments (7.65–8.78 g / kg), reflecting the enrichment effect of rhizosphere deposition and root exudation on organic matter.

[0051] Table 4: Soil organic matter content at different locations (g / kg, mean ± standard deviation, n=3)

[0052] Impact on soil nitrogen and phosphorus nutrient content As shown in Table 5, the amount of microbial agent applied had a significant impact on nitrogen and phosphorus nutrients in both rhizosphere and non-rhizosphere soils of *Fernonia spp.*, and the direction of the impact varied depending on the element type and soil location.

[0053] In the rhizosphere soil, the total nitrogen content initially increased slightly with increasing inoculant dosage, then continuously decreased. The total nitrogen levels in treatments T1 and T2 were 0.514 and 0.512 g / kg, respectively, representing the highest levels, and were not significantly different from T0 (0.495 g / kg). P >0.05); significantly lower than T1 from T3 (0.478 g / kg). P <0.05%, the T5 treatment reduced to 0.452 g / kg, the lowest among all treatments, a significant decrease of 8.7% compared to T0. P <0.05). This indicates that low-level inoculant (40–80 kg / hm²) has no significant depletion of total soil nitrogen, but above 120 kg / hm², the inoculant may accelerate organic nitrogen mineralization, and coupled with increased absorption by *Fernella asiatica*, this leads to a net loss of total nitrogen. In non-rhizosphere soils, treatment T1 had the highest total nitrogen (0.486 g / kg), significantly higher than T0 (0.442 g / kg). P<0.05), with an increase of 10.0%. T3 and T0 were at the lowest levels, while T2, T4, T5 and T0 showed no significant difference from T0. Non-rhizosphere total nitrogen increased under low-dose inoculant treatment, possibly related to the input of nitrogenous organic matter from the inoculant itself.

[0054] In the rhizosphere soil, total phosphorus showed the most positive response to the amount of inoculant applied. The total phosphorus levels in treatments T3 and T4 were 3.07 and 3.09 g / kg, respectively, representing the highest levels and significantly higher than T0 (2.78 g / kg) and T2 (2.81 g / kg). P <0.05), T4 increased by 11.2% compared to T0. Treatments T1 and T5 (2.95 and 2.96 g / kg) were in the middle, showing significant differences from T0. P <0.05), but the difference from T3 and T4 was not significant. This indicates that the application rate of 120-160 kg / hm² of microbial agent can effectively activate fixed phosphorus in the soil and increase the total phosphorus content. In non-rhizosphere soil, the total phosphorus content was highest in treatment T0 (3.01 g / kg) and lowest in treatment T1 (2.72 g / kg), significantly lower than T0 (P<0.05). Treatment T4 (2.95 g / kg) had the highest content among the microbial agent treatments, with no significant difference from T0. The total phosphorus content in non-rhizosphere soil was mostly highest in the control, suggesting that the phosphorus activated by the microbial agent was mainly captured and utilized in the rhizosphere, and the accumulation effect was difficult to observe in the non-rhizosphere. Available phosphorus is the most responsive indicator among phosphorus nutrients.

[0055] In the rhizosphere soil, the available phosphorus content in treatment T4 reached a high of 65.13 mg / kg, the highest level, significantly higher than that in treatment T0 (35.23 mg / kg). P <0.05%, an increase of 84.9% compared to T0, nearly doubling; T5 was second (48.89 mg / kg), and T0 was the lowest. There was no significant difference in available phosphorus between the T1-T2 treatments and T0. Rhizosphere available phosphorus remained at a high level overall in the range of 120–195 kg / hm², clearly indicating the strong activation effect of phosphate-solubilizing bacteria on soil fixed phosphorus. In non-rhizosphere soils, the response of available phosphorus was significantly weaker than in the rhizosphere. The highest was in the T5 treatment (31.62 mg / kg), and the lowest was in the T2 treatment (22.72 mg / kg), with a significant difference between the two. P The phosphorus concentrations in the rhizosphere were <0.05, with T0, T1, T3, and T4 showing moderate differences from T5 and T2. This indicates that the inoculant primarily exerts its phosphorus-solubilizing effect in the rhizosphere, with little effect on phosphorus activation outside the rhizosphere. The available phosphorus in the rhizosphere T4 treatment was nearly 2.9 times that in the non-rhizosphere T0 treatment, fully demonstrating the rhizosphere enrichment characteristic of the inoculant's nutrient activation effect.

[0056] Table 5: Soil nitrogen and phosphorus content under different treatments (mean ± standard deviation, n=3)

[0057] Effects of Different Microbial Inoculant Application Rates on Soil Enzyme Activity: Soil enzyme activity is an important biological indicator reflecting the intensity of microbial metabolism and the rate of nutrient cycling. This experiment measured the activities of five key hydrolytic enzymes in rhizosphere and non-rhizosphere soils: β-glucosidase (BG, carbon cycle related), alkaline phosphatase (AP, phosphorus cycle related), cellobiase (CBH, carbon cycle related), leucine aminopeptidase (LAP, nitrogen cycle related), and acetylglucosidase (NAG, nitrogen / carbon cycle related). Rhizosphere soil enzyme activity: Table 6 shows that the response patterns of the five rhizosphere soil enzymes to the dosage of inoculant differed significantly. The activities of β-glucosidase (BG), cellobiase (CBH), and leucine aminopeptidase (LAP) all peaked in the T5 treatment (195 kg / hm²), significantly higher than those in the T0 treatment. P <0.05. BG activity was highest in T5 treatment (152.76 nmol·g). -1 ·h -1 ), compared to T0 (nmol·g -1 ·h -1 The concentration increased by 205.5%, significantly higher than that of T3 (86.22 nmol·g). -1 ·h -1 T4 (34.45 nmol·g) -1 ·h -1 ) and T0 ( P <0.05) T2 treatment (134.26 nmol·g -1 ·h -1 The activity was also at a relatively high level. BG activity decreased significantly to its lowest value (nmol·g⁻¹) after T4 treatment. -1 ·h -1 It presents a complex pattern of "low volume increase, medium volume fluctuation, and high volume surge".

[0058] CBH activity reached 492.16 and 473.63 nmol·g⁻¹ after treatments at T5 and T2, respectively. -1 ·h -1 Both belong to the highest level, compared to T0 (102.21 nmol·g -1 ·h -1 The levels of T3 and T4 increased by 381.5%, and were significantly higher than those of T3 (205.34 nmol·g). -1 ·h -1 ) and T4 (208.96 nmol·g -1 ·h -1 () P <0.05), T1 treatment (123.00 nmol·g) -1 ·h -1 The difference between T0 and T0 is not significant.

[0059] LAP activity was highest in the T5 treatment (225.23 nmol·g). -1 ·h -1 ), significantly higher than other treatments ( P <0.05, followed by the T2 treatment (210.93 nmol·g). -1 ·h -1 The lowest value was achieved with treatment T4 (153.23 nmol·g). -1 ·h -1 ), significantly lower than T5 and T2 ( P <0.05). LAP activity generally showed a trend of "stable in the early stage, fluctuating in the middle stage, and surging in the high stage".

[0060] The activity of these three carbon and nitrogen cycle-related enzymes was strongly stimulated under high doses (195 kg / hm²) of bacterial agent, indicating that the relevant functional microorganisms were still significantly activated up to the highest dose in this experiment.

[0061] Alkaline phosphatase (AP) activity peaked at 176.39 nmol·g⁻¹ under the T3 treatment (120 kg / hm²). -1 ·h -1 ), compared to T0 (120.67 nmol·g -1 ·h -1 The T4 treatment increased by 46.2% (168.09 nmol·g). -1 ·h -1 ) and T5 treatment (161.11 nmol·g -1 h -1 The levels of these three also remained at a high level, and were similar to T0 and T1 (131.56 nmol·g). -1 ·h -1 Significant differences () P <0.05, T2 treatment had the lowest (108.47 nmol·g) -1 h -1 ), significantly lower than T0 ( P <0.05). AP activity exhibited a pattern of "low-level fluctuations, medium-level surges, and high-level maintenance," clearly indicating that 120–160 kg / hm² is the optimal range for phosphorus invertase activity. This pattern is highly consistent with the trend of higher total phosphorus and available phosphorus content in the T3–T4 treatments.

[0062] The activity of acetylglucosidase (NAG) reached 163.04 nmol·g in both T1 and T2 treatments. -1 ·h -1 Compared to T0 (99.90 nmol·g -1 ·h -1 Significantly increased by 63.2% ( P<0.05); the activity dropped sharply to 45.47 nmol·g under T3 treatment. -1 ·h -1 It was only 45.5% of T0, the lowest level, and significantly different from all treatments. P <0.05); T5 treatment (139.54 nmol·g) -1 ·h -1 The levels of NAG significantly increased, but remained below the T1-T2 levels. NAG exhibited a unique pattern of "low-level promotion, medium-level inhibition, and high-level recovery"—the T3 treatment (120 kg / hm²) produced a strong selective inhibition of NAG activity, which was the most unique among the five enzymes. This is presumably related to the competitive exclusion of specific microbial groups (such as actinomycetes) at this dosage.

[0063] Table 6: Rhizosphere soil enzyme activities under different treatments (U / g dry soil, mean ± standard deviation, n=3)

[0064] Non-rhizosphere soil enzyme activity: As shown in Table 7, the overall non-rhizosphere soil enzyme activity was significantly lower than that in the rhizosphere ( P <0.05), and the response patterns to different inoculant dosages differed significantly from those in the rhizosphere: β-glucosidase BG activity showed an abnormally high value (370.71 nmol·g) in the T3 treatment. -1 ·h -1 ), significantly higher than all other treatments ( P <0.05, which is T0 (20.82 nmol·g -1 ·h -1 17.8 times that of T4; followed by T4 (213.62 nmol·g). -1 ·h -1 T5 again (166.20 nmol·g) -1 ·h -1 Non-rhizosphere BG activity generally increased and then decreased with increasing inoculant dosage, peaking at T3 rather than T5 in the rhizosphere. This indicates that moderate amounts of inoculant may form enrichment zones in localized non-rhizosphere areas, temporarily stimulating BG activity. Alkaline phosphatase (AP): showed a completely opposite trend to the rhizosphere treatment, exhibiting "inoculant inhibition." AP activity was highest at T0 treatment (154.02 nmol·g⁻¹). -1 ·h -1 ), significantly higher than T2 (74.27 nmol·g). -1 ·h -1 ) and T3 (72.79 nmol·g -1 ·h -1 () P <0.05), T5 treatment (120.48 nmol·g -1 ·h-1 The levels recovered somewhat, but remained below T0. The continued decrease in non-rhizosphere AP contrasted sharply with the significant increase in rhizosphere AP, further confirming that the promoting effect of the inoculant on phosphorus conversion was mainly concentrated in the rhizosphere. Cellobiose hydrolase (CBH): highest in T1 treatment (131.70 nmol·g⁻¹). -1 ·h -1 T0 was the lowest (54.93 nmol·g). -1 ·h -1 The T2-T5 values ​​were in the middle range, with little overall difference between treatments. Non-rhizosphere CBH activity was significantly lower than the highest rhizosphere value (492.16 nmol·g⁻¹). -1 ·h -1 This indicates that cellulose decomposition is mainly active in the rhizosphere. Leucine aminopeptidase (LAP): The highest levels were observed at T0, T1, T3, and T4 (130.36-148.38 nmol·g⁻¹). -1 ·h -1 The enzyme activity was significantly reduced in T2 and T5 treatments (95.30 and 82.28 nmol·g, respectively). -1 ·h -1 () P <0.05%. Non-rhizosphere LAP activity remained stable in most treatments, decreasing only at low to medium levels (T2) and high levels (T5). NAG (acetylglucosidase): Activity was highest at T2 treatment (166.58 nmol·g). -1 ·h -1 ), significantly higher than other treatments ( P <0.05, which is T0 (62.04 nmol·g -1 ·h -1 The response of non-rhizosphere NAG was 2.7 times that of rhizosphere NAG; the other treatments from T1 to T5 showed no significant difference from T0. The response of non-rhizosphere NAG was similar to that of rhizosphere NAG, peaking at T2, but no strong inhibition was observed at T3.

[0065] Table 7: Non-rhizosphere soil enzyme activity (U / g dry soil, mean ± standard deviation, n=3)

[0066] The key design focus of this invention is: First, it exhibits good local adaptability and strong colonization ability: This invention extracts an enriched solution of indigenous microorganisms from the rhizosphere soil of *Pteris vittata* in saline-alkali land. This solution contains nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant rhizosphere growth-promoting bacteria (PGPR) that have adapted to the local saline-alkali environment. These indigenous strains possess natural ecological adaptability and can rapidly colonize and reproduce after being applied to the soil, resulting in a lasting improvement effect.

[0067] Secondly, the synergistic effect of multiple microorganisms provides comprehensive functionality: This invention involves the compound fermentation of a mixture of indigenous microbial enrichment solution, EM fermentation broth, Trichoderma harzianum activation solution, and fern root powder. EM provides various beneficial bacteria such as lactic acid bacteria, yeast, and photosynthetic bacteria; Trichoderma harzianum has the ability to inhibit soil-borne pathogens; and indigenous bacteria provide efficient nitrogen fixation, phosphorus solubilization, and growth promotion functions. The synergistic effect of multiple functional bacteria can simultaneously achieve multiple goals, including salt reduction, pH adjustment, nutrient activation, disease inhibition, and root development promotion.

[0068] Third, the fern root powder mixture provides nutrients and promotes the proliferation of bacterial strains: fern root powder is rich in nutrients such as starch, sugars, and amino acids. As a natural carbon and nitrogen source, it can significantly promote the growth and metabolic activity of various microorganisms in the compound bacterial agent and increase the effective live bacteria count of the bacterial agent.

[0069] Fourth, it significantly improves the physical and chemical properties of saline-alkali soil: After applying the microbial agent of this invention, the soil pH value is significantly reduced, and the electrical conductivity (EC) and total salt content are significantly reduced; the stability of soil water-stable aggregates is improved, and the proportion of large aggregates increases; the content of soil organic matter, available phosphorus, total phosphorus and other nutrients is increased; and the activity of soil enzymes such as alkaline phosphatase is enhanced.

[0070] Fifth, it promotes crop growth and improves yield and quality: Application results on *Pteris vittata* show that the microbial agent of this invention can significantly increase plant height, root length, and above-ground biomass, reduce disease incidence, and significantly increase yield. Simultaneously, it improves the density and appearance of *Pteris vittata* bulbs, increasing their commercial value.

[0071] Sixth, the raw materials used in this invention are widely available and inexpensive, the preparation process does not require complex equipment, and the fermentation conditions are mild (room temperature and pressure), making it suitable for grassroots agricultural production units to propagate and use on their own, and it has good prospects for promotion and application.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A microbial inoculant for improving saline-alkali land, characterized in that: The microbial agent comprises a soil microbial enrichment solution, EM fermentation broth, Trichoderma harzianum activation broth, and a mixture of fern root powder extracted from the rhizosphere soil of *Pteris vittata* in saline-alkali land; the soil microbial enrichment solution and EM fermentation broth contain nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant rhizosphere growth-promoting bacteria; the OD600 value of the microbial agent is 0.5-1.0, and the effective viable bacteria count per gram of microbial agent is 4.0×10^8 CFU / g-8.0×10^8 CFU / g.

2. The microbial inoculant for improving saline-alkali land according to claim 1, characterized in that: The fern root powder mixture is prepared by mixing fern root powder and water at a mass ratio of 1:10 and then letting it stand at 25-30℃ for 5 days; the EM fermentation liquid is prepared by sealing and fermenting brown sugar, EM bacteria and water at 25-30℃ for 7-10 days; the Trichoderma harzianum activation liquid is prepared by mixing Trichoderma harzianum and warm water at 30℃ at a mass ratio of 1:10 and then activating it at 28-30℃ for 2-3 hours.

3. A method for preparing a microbial inoculant for improving saline-alkali land as described in any one of claims 1-2, characterized in that: Includes the following steps: S1. Soil microbial extraction: Fresh soil from the rhizosphere of *Fernonia chinensis* in saline-alkali land was mixed with sterile water and shaken. After standing, the supernatant was centrifuged to obtain the soil rhizosphere microbial mother liquor. S2. Component preparation: Prepare fern root powder mixture, EM fermentation broth and Trichoderma harzianum activation broth separately; S3. Compound fermentation: Mix the fern root powder mixture, EM fermentation liquid and Trichoderma harzianum activation liquid in proportion, and let it stand at 25-30℃ for 12-15 hours to obtain the microbial agent.

4. The preparation method according to claim 3, characterized in that: In step S1, when extracting soil microorganisms, the mass ratio of soil to sterile water is 1:9, the shaking conditions are 28-30℃, shaking at 180r / min for 50 minutes, and centrifugation conditions are 5000r / min for 10 minutes.

5. The preparation method according to claim 3, characterized in that: In step S3, the mixing ratio of each component by volume is: fern root powder mixture: EM fermentation liquid: Trichoderma harzianum mixture = (2-5): (6-9): (0.5-2).

6. The preparation method according to claim 5, characterized in that: In step S3, the mixing ratio of each component by volume is: fern root powder mixture: EM fermentation broth: Trichoderma harzianum mixture = 3.75 : 7.5 :

1.

7. The application of the microbial agent according to any one of claims 1-2 in the improvement of saline-alkali land.

8. The application according to claim 7, characterized in that: The microbial agent is used to improve the physicochemical properties of saline-alkali soil and / or promote crop growth; the improvement of the physicochemical properties of saline-alkali soil includes any one or more of the following: reducing soil pH, reducing soil electrical conductivity, reducing soil total salt content, improving the stability of soil water-stable aggregates, increasing soil organic matter content, increasing soil nitrogen and phosphorus nutrient content, and increasing soil enzyme activity.

9. The application according to claim 7, characterized in that: The application rate of the microbial agent in saline-alkali land is 100-150 kg / hm. 2 .

10. The application according to claim 9, characterized in that: The application rate of the microbial agent in saline-alkali land is 120 kg / hm². 2 .

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