Complex microbial inoculant and method for rapidly curing cultivated land through collaborative deep ploughing of complex microbial inoculant and humic acid

Abstract

The invention belongs to the technical field of soil improvement, and particularly relates to a complex microbial inoculant and a rapid ripening method for cultivated land deeply ploughed through cooperation of the complex microbial inoculant and humic acid. In the method, the used complex microbial inoculants comprise bacillus velezensis, stenotrophomonas maltophilia C-GD and bacillus subtilis. The complex microbial inoculants and humic acid are mixed to form a modifier mixture, and deep ploughing is carried out cooperatively, so that quick curing of cultivated land is realized. The method is suitable for rapid improvement of soil quality of degraded soil, newly cultivated land or facility agriculture.

Description

Technical Field

[0001] This invention belongs to the field of soil improvement technology, specifically a method for rapid soil maturation through deep tillage using a compound microbial agent and humic acid in synergistic effects. Background Technology

[0002] Currently, global arable land degradation is leading to declining productivity. Certain technological means are needed to improve soil fertility and tillage performance. Traditional arable land improvement relies heavily on natural weathering and organic fertilizers for slow improvement, a process that takes 3-5 years and requires continuous fertilizer input to maintain yields (annual fertilizer application > 400 kg / hectare). This method inevitably leads to soil structural deterioration: such as increased compaction area, impeded water and nutrient infiltration in the plow pan, decreased biological activity, reduced microbial diversity by 40%-60%, decreased enzyme activity by 50%, exacerbated environmental pollution, nitrogen fertilizer utilization rate of less than 35%, and leaching losses causing excessive nitrate levels in groundwater.

[0003] Currently, rapid farmland improvement technologies mainly focus on soil structure construction, barrier layer reduction, and organic matter enhancement. Soil structure optimization: Addressing the common problems of thin topsoil and obstructive barrier layers in newly reclaimed farmland, key technological breakthroughs have been achieved in areas such as rapid site spatial survey and detection, barrier layer reduction, and ecological protection enhancement. For example, engineering methods are used to construct a high-quality topsoil layer, improving soil aeration and root penetration.

[0004] Rapid fertilization technology: Using carbon source materials (such as mineral or biomass sources) as the core, it creates artificial humus-based products to accelerate the accumulation of soil organic matter. Simultaneously, it integrates technologies such as straw-based soil conditioners, biochar-based fertilizers, and humic acid soil conditioners to achieve rapid replenishment of soil nutrients and structural improvement.

[0005] Existing technologies include research on the application of microbial agents or bio-organic fertilizers for farmland improvement. However, existing agents mostly focus on single functions (such as nitrogen fixation or disease resistance) and do not systematically integrate multiple effects such as decomposition, nitrogen fixation, and growth promotion. Furthermore, these agents are easily disinfected by ultraviolet radiation in shallow soil (<20 cm) and have difficulty reaching deep root systems. Therefore, further research and development of this technology is needed to enable rapid farmland maturation. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a method for rapid soil maturation based on the synergistic deep tillage of a compound microbial agent and humic acid. This method involves preparing a compound (microbial) agent and combining it with humic acid for deep tillage and mixed application to achieve rapid soil maturation. This method is suitable for the rapid improvement of soil quality in degraded soils, newly reclaimed farmland, or facility agriculture. To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain of maltophilic oligotrophomonas ( 嗜麦芽窄食单胞菌 C-GD was deposited on October 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.36448. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] This maltophilic oligotrophomonas ( 嗜麦芽窄食单胞菌 C-GD has the property of converting atmospheric nitrogen into ammoniacal nitrogen that can be absorbed by plants. During screening, the nitrogenase activity of this bacterium was tested to be 45.90 (ng / g / 24h). Therefore, it can be used to improve soil nitrogen cycling, thereby increasing soil nitrogen use efficiency and promoting nitrogen absorption by crops.

[0008] This application also protects the above-mentioned Stenotrophomonas maltophilia ( 嗜麦芽窄食单胞菌 Application of C-GD and humic acid in rapid land maturation through synergistic deep tillage.

[0009] This application also protects a compound microbial agent comprising Bacillus belye (B. belye). 芽孢杆菌属 贝莱斯芽孢杆菌 The above-mentioned maltophilic oligotrophomonas ( 嗜麦芽窄食单胞菌 C-GD and Bacillus subtilis ( 枯草芽孢杆菌 ).

[0010] As a preferred embodiment of this application, in the compound bacterial agent, Bacillus belye ( 贝莱斯芽孢杆菌 ), Stenotrophomonas maltophilia ( 嗜麦芽窄食单胞菌 C-GD and Bacillus subtilis ( 枯草芽孢杆菌 All exist in the form of bacterial suspension, bacterial powder, bacterial liquid, or other forms containing effective strains; in compound bacterial agents, Bacillus belyssus ( 贝莱斯芽孢杆菌 The viable count of bacteria is ≥1×10⁻⁶. 6 CFU / g; Stenotrophomonas maltophilia ( 嗜麦芽窄食单胞菌 C-GD viable count ≥ 1 × 10⁻⁶ 6 CFU / g; Bacillus subtilis ( 枯草芽孢杆菌 The viable count is ≥1×10⁻⁶. 6 CFU / g.

[0011] This application also protects the application of the above-described compound microbial agent in the rapid maturation of farmland through synergistic deep tillage with humic acid.

[0012] Furthermore, in the aforementioned application, the specific application method is as follows: the compound microbial agent is mixed with humic acid to form a soil conditioner mixture, and then the soil conditioner mixture is evenly spread on the soil surface, and then it is mixed with the soil by tilling, so as to achieve rapid soil maturation.

[0013] In a preferred embodiment of this application, the mass ratio of the compound microbial agent to humic acid is 1:5 to 1:15.

[0014] In a preferred embodiment of this application, the compound microbial agent is applied at a rate of 1-2 kg / mu; the humic acid is applied at a rate of 10-20 kg / mu; the tillage is carried out using deep tillage machinery, with 1-3 tillage passes; and the tillage depth is 25-30 cm.

[0015] This application also protects a method for rapid soil maturation based on the above-mentioned compound microbial agent and its synergistic deep tillage with humic acid, specifically including the following steps: (1) Mix the compound microbial agent with humic acid to form a mixture of improvers; (2) Use deep tillage machinery to plow the soil in one go to break up the plow pan and spread the soil amendment mixture from step (1) evenly on the soil surface; (3) The land is tilled a second time to ensure that the soil conditioner is fully mixed with the soil.

[0016] In a preferred embodiment of this application, the mass ratio of compound microbial agent to humic acid in the method is 1:5-1:15; the application rate of compound microbial agent is 1-2 kg / mu; the application rate of humic acid is 10-20 kg / mu; the tillage depth is 25-30 cm; the soil pH is 6.0-8.0; and the soil relative humidity is 60%-70%.

[0017] Furthermore, the humic acid content is ≥50%.

[0018] In this invention, the combined use of humic acid and functional microbial agents not only improves saline-alkali soil and increases organic matter, but also activates the soil microbial network, resulting in a faster release of fertilizer efficacy. This study found that when humic acid is applied alone, its effect on soil aggregate structure is limited to a depth of 15-20 cm. However, when combined with the compound microbial agent described in this application, conventional deep plowing (25-30 cm) can break up the plow pan, significantly improving soil fertility, microbial activity, and crop yield within 3-4 months, thereby reducing reliance on chemical fertilizers.

[0019] The method of rapid land maturation through deep tillage using compound microbial agents and humic acid does not affect the normal use of arable land and can be carried out before crop sowing or after crop harvesting.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The three microbial agents work synergistically to form a stable microbial community and jointly improve soil biological activity.

[0021] (2) Through multiple mechanisms such as microbial decomposition of organic matter, microbial nitrogen fixation and microbial disease resistance, soil fertility and crop yield can be comprehensively improved.

[0022] (3) Reduce the use of chemical fertilizers and pesticides, lower agricultural production costs, and promote sustainable agricultural development.

[0023] (4) It significantly improves soil fertility, microbial activity and crop yield within 3-4 months, achieves rapid land maturation and reduces dependence on chemical fertilizers. Attached Figure Description

[0024] Figure 1 Stenotrophomonas maltophilia ( 嗜麦芽窄食单胞菌 Phylogenetic tree of C-GD; Figure Standard curve of indoleacetic acid for microorganisms (Bacillus subtilis and Bacillus belye); ​ Standard curves for ACC deaminase in microorganisms (Bacillus subtilis and Bacillus belye); ​ This image shows the positive result of Bacillus belyssus on day 3 during siderogenetic assay. ​ This image shows the positive results of Bacillus belychnophorus on day 8 in the phosphorus solubility test. ​ Bacillus subtilis ( ​ (Figure showing the results of antibacterial experiments on rice blast disease 189, 193 and 199 respectively) ​ Bacillus repens ( ​ ), Stenotrophomonas maltophilia ( ​ C-GD and Bacillus subtilis ( ​ The antagonistic experiment results are shown in the figure. ​ Box plots of bacterial alpha diversity were obtained for Example 4 and Comparative Examples 1-3, respectively. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] In the description of this invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0029] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "connection" should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral connection; of course, it can also be a mechanical connection or an electrical connection; furthermore, it can refer to a direct connection, an indirect connection through an intermediate medium, or a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0030] In this invention, some conventional operating equipment, devices and components have been omitted or only briefly described.

[0031] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0032] In this application, all percentages not explicitly stated are mass percentages, i.e., wt%.

[0033] In this invention, Bacillus belye ( ​ The strain in question is an existing strain that was deposited on May 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.34429. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It has been disclosed in the patent application number 2025110533227, therefore no deposit certificate is provided.

[0034] Bacillus subtilis ( ​ Purchased from the China Agricultural Microbial Culture Collection Center, with the serial number: ACCC10655.

[0035] The main indicators of farmland improvement are: Topsoil thickness: ≥ 25 cm; Organic matter content: ≥ 20 g / kg (2.0%); Alkaline nitrogen uptake: >90 mg / kg; Available phosphorus: >20 mg / kg; Available potassium: >120 mg / kg, pH: 6.0 - 7.5.

[0036] Example 1: Stenotrophomonas maltophilia ( ​ Isolation, purification, and preservation of C-GD: On August 17, 2024, soil samples were collected from a farmland in Longquanyi District, Chengdu City, Sichuan Province. The samples were collected from the rhizosphere of the crops at a depth of approximately 2-5 cm. The collected samples were stored in sterile plastic bags and transported back to the laboratory in a foam box filled with ice packs.

[0037] 1. Soil pretreatment and enrichment: Take 5g of fresh rhizosphere soil, add 45mL of sterile physiological saline, shake for 20min, and prepare 10 -1 Soil suspension. Then, it was serially diluted 10-fold until 10... -6 Take 10 -4 10 -5 10 -6 Three gradient dilutions, 100 μL each, were spread on Assumption nitrogen-free solid plates, with three replicates for each gradient.

[0038] 2. Cultivation and purification: Invert the plate in a 30℃ constant temperature incubator and incubate in the dark for 5-7 days. Select single colonies with vigorous growth and different morphologies from the surface of the plate and perform three-zone streak purification on a new Assoube plate until a pure culture strain is obtained.

[0039] 3. Initial screening and preservation: The purified strain was inoculated into Assumption nitrogen-free liquid medium and cultured with shaking at 30℃ and 180 rpm for 3 days. The turbidity of the culture medium was observed to preliminarily determine its growth ability under nitrogen-free conditions. The well-growing strains were simultaneously transferred to LB slant (short-term preservation at 4℃) and LB liquid containing 20% ​​glycerol (long-term preservation at -80℃).

[0040] 4. Strain Identification: The isolated strains were labeled CA, CC, and C-GD (GD3), respectively. 16S rRNA gene sequencing was used to identify the isolated strains. Total DNA was extracted using a bacterial genomic DNA kit (GenElute™, Merck, Germany) according to the manufacturer's instructions. The sequences obtained from sequencing were entered into the NCBI database's BLAST database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for searching and obtaining alignment results for similar sequences. The three strains were identified as *Serratia liquefaction*, *Enterobacter bungandr*, and *Stenotrophomonas maltophilia*. The nitrogen fixation efficiency of the three strains was determined using the acetylene reduction method (existing technology, not described in detail), yielding nitrogenase activities of 41.6 (ng / g / 24h), 42.35 (ng / g / 24h), and 45.90 (ng / g / 24h), respectively. Therefore, *Stenotrophomonas maltophilia*, which had the highest nitrogenase activity, was selected. ​ (C-GD) was deposited as a nitrogen-fixing bacterium on October 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.36448. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0041] Example 2: Functional assays of Bacillus subtilis (ACCC10655) and Bacillus belyssus: The indoleacetic acid (IAA) content and ACC deaminase (ACCD) activity in Bacillus subtilis were determined using a kit. Specific results are shown in Table 1. ​ and ​ ,in ​ This is the standard curve for indoleacetic acid; ​ This is the standard curve for ACC deaminase.

[0042] Based on the microbial indoleacetic acid standard curve ( ​ The calculated IAA content of Bacillus subtilis was 56.48 μg / L, based on the microbial ACC deaminase standard curve ( ​ The calculated ACC activity in Bacillus subtilis was 174.437 μg / L, indicating that it has good growth-promoting ability.

[0043] Table 1:

[0044] Bacillus subtilis was used to conduct plate confrontation inhibition experiments against rice blast disease strains 189, 193, and 199. Rice blast disease strains that had grown for 3 days were selected. Holes were punched at the edges of the colonies using a 5mm diameter punch. The resulting mycelial cakes were placed in the center of the solid culture medium for both the control and experimental groups. In the experimental groups, one loopful of Bacillus subtilis was inoculated on each side of the rice blast disease mycelial cake using an inoculation loop. Five days later, when the mycelial cakes in the control group had covered two-thirds of the plate, the inhibition rate was measured. Specific results are shown below. ​ The inhibition rates were measured to be 70.6%, 64.0%, and 65.5%, respectively. This indicates that Bacillus subtilis (ACCC10655) can effectively inhibit the pathogen of rice blast.

[0045] The indoleacetic acid (IAA) content and ACC deaminase (ACCD) activity of Bacillus belyssus were determined using a kit. The specific results are shown in Table 2. Based on the microbial indoleacetic acid standard curve (…),… ​ The IAA content of Bacillus belyssus was calculated to be 57.039 μg / L, based on the microbial ACC deaminase standard curve ( ​ The calculated ACC activity of Bacillus belyssus was 187.465 μg / L, indicating that it has good growth-promoting ability.

[0046] Table 2:

[0047] Siderogenesis detection of Bacillus belyssus (CAS medium, blue-green): Prepare CAS solid medium. After activating the Bacillus belye, inoculate it onto CAS plates (approximately 5 mm in diameter). Invert the inoculated plates and incubate them in the dark at a suitable temperature (32℃) for 1-3 days. See below for specific results. ​ , ​ This image shows a positive result of Bacillus belyssus on day 3 during siderophore production testing. As can be seen from the image, an orange-yellow to orange-red halo appeared around the colony after three days. This is because the siderophores secreted by this strain interact with CAS-Fe... 3+ Fe in the complex 3+ The stronger binding force displaced the CAS dye (orange) from the complex, causing the local color to change from blue-green to orange-red, proving that the strain has the ability to produce siderophores.

[0048] Phosphate-solubilizing ability of Bacillus belyssus (NBRIP medium, white): Prepare NBRIP solid medium. After activating the *Bacillus belyssus* to be tested, inoculate it onto NBRIP plates (approximately 5 mm in diameter). Invert the inoculated plates and incubate in the dark at a suitable temperature (32℃) for 5-10 days. See below for specific results.​ , ​ This image shows a positive result of Bacillus belyssus on day 8 in a phosphate-solubilizing assay. As can be seen from the image, a clear zone appeared around the colony after eight days. This clear zone is formed by the dissolution of insoluble phosphates below and around the colony by organic acids and other substances secreted by the strain. This demonstrates the strain's phosphate-solubilizing ability.

[0049] The above tests prove that Bacillus belye has the ability to promote growth.

[0050] Example 3: Qualitative tests (using existing technology) were performed on the phosphorus-solubilizing and siderophore-producing abilities of the three bacterial strains. The results showed that after 3 days of culture, orange-yellow to orange-red halos appeared around the colonies of all three strains. After 8 days, clear halos appeared around the colonies of *Bacillus subtilis* and *Bacillus maltophilia*. The 48-hour growth curves of the three strains were measured. *Bacillus subtilis* and *Bacillus maltophilia* showed a "J"-shaped growth pattern from 8 to 40 hours, while *Oligotroph ...

[0051] Strain antagonism experiment Antagonistic effect of three bacterial strains was tested using the streak plating method. The results showed that the three strains could coexist. The three strains were sequentially inoculated onto solid LB medium, and straight lines were drawn along the edge of the medium, with the ends of the three lines intersecting. After 3 days of incubation, the growth status at the interface between the strains was observed to determine whether antagonistic activity existed. The results showed that no antagonistic zone or inhibition zone was formed at the interface, indicating that the three strains could coexist.

[0052] Example 4: A method for rapid soil maturation through synergistic deep tillage using a compound microbial agent and humic acid includes the following steps: (1) Preparation of compound microbial agent.

[0053] Bacillus berberis ( ​ Bacillus subtilis ( ​ ) and the maltophilic oligotrophosome preserved in Example 1 ( ​ (C-GD) were inoculated into LB liquid medium and cultured with shaking at 30℃ and 180 rpm until the late logarithmic growth stage (approximately 48 hours) to obtain bacterial suspensions of three strains, among which Bacillus belye ( ​ The number of viable bacteria in the bacterial solution is ≥1×10⁻⁶ 8 CFU / g; Bacillus subtilis ( ​ The number of viable bacteria in the bacterial solution is ≥1×10⁻⁶ 8 CFU / g; Stenotrophomonas maltophilia ( ​ (C-GD) bacterial culture with a viable count ≥1×10 7CFU / g. Three bacterial solutions were mixed in an equal volume ratio (1:1:1) to prepare a compound (microbial) inoculum (total viable count ≥1×10⁻⁶). 8 CFU / g).

[0054] (2) The compound (microbial) inoculant and humic acid are prepared in a certain proportion and stirred evenly. The resulting mixture is also called the improver mixture. The mass percentage of humic acid in the humic acid is ≥50%.

[0055] The humic acid was purchased from Huaneng Company. Before use, the humic acid and deionized water were mixed in a mass ratio of 1:10 to form a humic acid solution.

[0056] (4) Use deep tillage machinery to plow to a depth of 25-30 cm to break up the plow pan; spread the soil amendment mixture evenly on the soil surface, and then plow a second time (plowing depth of 15 cm) to fully mix the amendment with the soil. The compound microbial agent dosage is 1.0 kg / mu, and the humic acid dosage is 10 kg / mu. After the second plowing, the amendment mixture is evenly distributed in the soil tillage layer (0-30 cm).

[0057] During the first and second tillage, the soil pH value is 6.0-8.0; the soil relative humidity is 60%-70%.

[0058] Comparative Example 1: The same method as in Example 4 was used for rapid soil conditioning, the only difference being the addition of only a compound (microbial) inoculant, without the addition of humic acid; all other steps were the same. The application rate of the compound (microbial) inoculant was 1.0 kg / mu (total viable count ≥ 5 × 10⁻⁶). 8 CFU / g) Comparative Example 2: The same method as in Example 4 was used for rapid soil conditioning, except that the soil conditioner mixture was used instead of deep tillage machinery to plow to a depth of 25-30 cm to break up the plow pan and to perform a second plowing (to a depth of 15 cm) to fully mix the soil conditioner with the soil. Instead, the traditional tillage method was used.

[0059] Comparative Example 3: The same method as in Example 4 was used for rapid soil maturation, except that no substances were added, i.e. no compound (microbial) inoculants and humic acid were added at the same time.

[0060] experiment: A field experiment will be conducted in Xihe in May 2025, with soil samples collected before the experiment.

[0061] The soil physicochemical properties were measured before the experiment as follows:

[0062] The experiment began with soil treatments performed according to the methods described in Example 4 and Comparative Examples 1-3, followed by rice planting. Soil samples were collected in September 2025 when the rice matured.

[0063] Soil physicochemical properties were measured on the land after cultivation in Example 4 and Comparative Examples 1-3.

[0064] Measurement methods: Soil pH was accurately determined using glass electrode technology; soil organic matter content was quantitatively analyzed using potassium dichromate oxidation combined with external heating; total nitrogen was determined using a pretreatment step of sulfuric acid and accelerator digestion, followed by Kjeldahl nitrogen determination; total phosphorus was determined by first treating with NaOH alkali fusion, followed by quantitative analysis using molybdenum-antimony spectrophotometry; total potassium content was determined by NaOH fusion flame spectroscopy; soil alkaline-available nitrogen was determined based on the principle of alkaline diffusion; available phosphorus was determined by first extracting with ammonium fluoride-hydrochloric acid solution and sodium bicarbonate solution, followed by determination using molybdenum-antimony colorimetry. Each soil sample was measured at least three times, and the average value was calculated.

[0065] The measured items and results are shown in Table 1: Table 1

[0066] The comparison and comprehensive analysis show that Example 4 (deep tillage + compound microbial agent + humic acid) has the best soil improvement effect: its pH value was significantly reduced to 6.13±0.01, effectively alleviating salinization; the organic matter content (14.29±0.03 g / kg) and total phosphorus (0.77±0.00 g / kg) increased significantly, and the available potassium (195.67±0.58 mg / kg) increased by 104%, far exceeding other groups. The synergistic effect of humic acid and microbial agent not only optimized the soil physicochemical properties, but also greatly improved nutrient availability through a dual mechanism of chelation and biological activation, while deep tillage of 30 cm ensured uniform mixing of the soil conditioner with the deeper soil layers. Although the activation effect of microbial agent on nitrogen and phosphorus is limited in the short term, Example 4 shows significant advantages in both rapid maturation (120 days) and continuous nutrient supply, making it the optimal solution that balances efficiency and ecological sustainability.

[0067] Experimental Example 2 Soil microbial alpha diversity was measured using conventional techniques on the land after tillage in Example 4 and Comparative Examples 1-3, respectively.

[0068] Assay Methods: Genomic DNA was extracted from the samples using the SDS method. An appropriate amount of extracted DNA was placed in a centrifuge tube and diluted to 1 ng / μl with sterile water. Using the diluted genomic DNA as a template, the bacterial V5-V7 hypervariable region of the 16S rRNA gene was amplified. Libraries were constructed using a library construction kit, and the constructed libraries were quantified by Qubit and Q-PCR. Qualified libraries were sequenced using NovaSeq 6000 at PE 250. To analyze the microbial community diversity within the samples, the Alpha Diversity indices (observed_otus, shannon, simpson, chao1) (n=3) for different samples were calculated using QIIME2 software.

[0069] The measured items and results are as follows ​ As shown: Depend on ​ Data analysis revealed that, based on microbial diversity analysis, Example 4 (deep tillage + compound microbial agent + humic acid) exhibited the best soil ecological improvement effect: its observed_species value reached a maximum of 5361, significantly higher than other groups, and the Chao1 index showed that this group had the highest potential for species richness. Simultaneously, the Shannon and Simpson indices indicated that Example 4 maintained optimal levels of microbial community diversity and evenness. This result confirms that Example 4, through the "microbial agent-humic acid-deep tillage" strategy, not only rapidly increased the number of beneficial bacteria but also constructed a complex, balanced, and highly resistant soil micro-ecosystem.

[0070] In summary, this application provides a method for rapid land ripening based on the synergistic deep tillage of compound microbial agents and humic acid, including a method for preparing the compound microbial agent, a method and proportion for mixing and applying the microbial agent and humic acid, and a deep tillage method. This rapid land ripening method is simple, feasible, and highly efficient.

[0071] Those skilled in the art should understand that the methods described in this invention are not limited to the embodiments described in the specific implementation details. The above detailed description is merely for illustrative purposes and is not intended to limit the invention. Other implementation methods derived by those skilled in the art based on the technical solutions of this invention also fall within the scope of this invention's technical innovation. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A strain of Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia C-GD was deposited on October 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.36448. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. The Stenotrophomonas maltophilia (S. maltophilia) of claim 1, wherein the S. maltophilia is a strain of S. maltophilia deposited with the Korean Research Institute of Bioscience and Biotechnology (KRIBB) on May 31, 2012 under Accession No. KCTC 13207HP. Stenotrophomonas maltophilia ) Application of C-GD and humic acid in rapid maturation of plowed land in synergistic deep plowing.

3. A complex microbial agent, characterized in that: The complex microbial agent comprises Bacillus velezensis (Bacillus velezensis) Bacillus velezensis Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) as claimed in claim 1, Stenotrophomonas maltophilia C-GD and Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ​ 4. The complex bacterial agent according to claim 1, characterized by: The complex microbial agent contains Bacillus velezensis (Bacillus velezensis) Bacillus velezensis , Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia , C-GD and Bacillus subtilis (Bacillus subtilis) Bacillus subtilis . In the complex microbial agent, the viable count of Bacillus velezensis (Bacillus velezensis) Bacillus velezensis is ≥1×10 6 CFU / g; the viable count of Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia C-GD is ≥1×10 6 CFU / g; and the viable count of Bacillus subtilis (Bacillus subtilis) Bacillus subtilis is ≥1×10 6 CFU / g.

5. The application of the complex microbial agent in claim 3 or 4 in the rapid maturation of ploughed land in cooperation with humic acid.

6. The use according to claim 5, characterized in that: Once ploughing is carried out to a depth of 25-30 cm using a deep ploughing machine to break the plough pan; the mixture of the amendment is uniformly applied to the surface of the soil, and then secondary ploughing (ploughing depth of 15 cm) is carried out to fully mix the amendment with the soil, and finally rapid maturation of the ploughed land is achieved.

7. The use according to claim 5, wherein: The mass ratio of the complex microbial agent to humic acid is 1:5-1:

15.

8. The use according to claim 5, characterized in that: The application amount of the complex microbial agent is 1-2 kg / mu; the application amount of humic acid is 10-20 kg / mu; the form of ploughing is deep ploughing by a deep ploughing machine; the number of times of ploughing is 1-3 times; and the depth of ploughing is 25-30 cm.

9. A method for rapid soil maturation of ploughing based on the synergistic ploughing of the complex microbial agent according to claim 3 or 4 and humic acid, characterized by The method comprises the following steps: (1) mixing the complex microbial agent with humic acid to form a mixture of the amendment; (2) breaking the plough pan by once ploughing using a deep ploughing machine; (3) uniformly applying the mixture of the amendment in step (1) to the surface of the land, (4) secondary ploughing of the land to fully mix the amendment with the soil.

10. The method of claim 9, wherein: The mass ratio of the complex microbial agent to humic acid is 1:5-1:15; the application amount of the complex microbial agent is 1-2 kg / mu; the application amount of humic acid is 10-20 kg / mu; the depth of ploughing is 25-30 cm; the pH value of the soil is 6.0-8.0; and the relative humidity of the soil is 60%-70%.

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