Method for promoting rice growth by using composite microbial agent and humic acid

Abstract

This invention discloses a method for promoting rice growth using a compound microbial agent and humic acid. Humic acid and the compound microbial agent are added sequentially to rice-grown soil. The amount of humic acid added is 0.5-2% of the soil mass, preferably 1% of the soil mass. The solid-liquid ratio of humic acid to the compound microbial agent (g:ml) is 0.5-1.1:15-30, preferably 1:20. The compound microbial agent is obtained by mixing any two or more monofunctional bacteria in equal volume ratios. The monofunctional bacteria include *Bacillus amyloliquefaciens* FH1, *Bacillus shortwave diversicolor* NH1, *Glucosidobacterium tumefaciens* QZR14, *Bacillus cereus* S112, and *Pantothecinus flocculationensis*. 、 The compound microbial agent of this invention, along with humic acid, can effectively promote rice growth, optimize soil microbial community and function, and provide a scientific basis for the research and application of new compound microbial fertilizers, thus contributing to the development of sustainable agriculture.

Description

Technical Field

[0001] This invention relates to a method for promoting rice growth, specifically a method for promoting rice growth using compound microbial agents and humic acid, belonging to the field of agricultural production propagation technology. Background Technology

[0002] Rice, as one of the most important food crops globally (especially in Asia), plays a vital role in maintaining food security. However, traditional agricultural practices have long relied on the application of chemical fertilizers and pesticides. While this has increased rice yields to some extent, it has also led to a series of problems, including soil degradation, environmental pollution, and ecological imbalance. Therefore, researching and developing new agricultural fertilizers that can improve soil health while increasing rice yields is of significant practical importance.

[0003] Microbial inoculants can promote crop growth through various mechanisms, including: (1) enhancing soil fertility; (2) producing plant hormones to directly regulate plant growth and development; (3) inhibiting the growth and reproduction of harmful microorganisms; and (4) assisting plants in nutrient absorption. Compound microbial inoculants, which typically contain multiple species of microorganisms (such as nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and bacteria with ACC deaminase activity), exhibit a synergistic effect in promoting growth through multiple mechanisms. Furthermore, the microorganisms within the compound microbial inoculant can exchange metabolites (such as amino acids and carbohydrates) and signals. The improved adaptability and reduced metabolic burden of each microbial member in the compound microbial inoculant allow for a more stable and sustained growth-promoting function.

[0004] Humic acid (HA) is a class of natural organic macromolecular compounds found in soil, sediment and organic matter. Currently, the application and growth-promoting mechanisms of humic acid in agriculture mainly include the following aspects: (1) improving nutrient utilization; (2) improving soil structure; (3) regulating plant physiological activities and enhancing plant stress resistance; (4) regulating soil microbiome.

[0005] The synergistic application of microbial inoculants with humic acid is an important research direction in agriculture, environmental science, and soil health. In recent years, with the increasing demand for sustainable agricultural development and ecological environmental protection, researchers have gradually discovered that the combined use of microbial inoculants and humic acid can bring about significant benefits, such as improving soil fertility, reducing environmental stress, and enhancing crop resistance.

[0006] Although previous studies have explored the combined application of microbial inoculants with humic acid and biochar, research on the combined application of compound microbial inoculants with humic acid and biochar remains scarce. Most studies focus on the functional application of single microbial strains or the role of humic acid as a standalone soil conditioner in combination with inorganic fertilizers. The synergistic effects of multiple microorganisms in compound microbial inoculants and their combined effects with humic acid have not yet been fully explored and verified.

[0007] Therefore, conducting systematic research on the combination of compound microbial agents and humic acid may fill a gap in this field, not only reducing dependence on chemical fertilizers and pesticides but also improving soil quality through their ecological effects. This research aims to provide new theoretical basis and practical guidance for achieving sustainable agricultural production and the application of eco-friendly multi-microbial compound fertilizers. Summary of the Invention

[0008] The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a method for promoting rice growth using compound microbial agents and humic acid. The invention utilizes four functional single bacteria with nitrogen-fixing and phosphorus-solubilizing abilities, ACC deaminase production abilities, potassium-solubilizing abilities, and siderophore production abilities, two nitrogen-fixing cyanobacteria, and three functional single bacteria to construct compound microbial agents. These agents are then combined with humic acid. The effects of these combined applications on rice growth and soil microbial community structure are investigated using rice pot experiments and high-throughput sequencing technology.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for promoting rice growth using a compound microbial agent and humic acid. Humic acid and the compound microbial agent are added sequentially to soil in which rice is grown. The amount of humic acid added is 0.5-2% of the soil mass, and the solid-liquid ratio (g:ml) of humic acid and the compound microbial agent is 0.5-1.1:15-30.

[0010] In the above technical solution, the amount of humic acid added is preferably 1% of the mass of the soil.

[0011] In the above technical solution, the solid-liquid ratio (g:ml) of the humic acid and the compound bacterial agent is preferably 1:20.

[0012] In the above technical solution, the compound microbial agent is obtained by compounding any two or more monofunctional bacteria in equal volume ratios; the monofunctional bacteria include Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens FH1 (abbreviated as F), shortwave monocytogenes ( Brevundimonas sp. NH1 (abbreviated as N), Staphylococcus aureus ( Gluconacetobacter sp. )qzr14 (abbreviated as Q), Paleobacterium ( Ochrobactrum sp. S112 (abbreviated as S), Pantothecin clumps ( Pantoea agglomerans (abbreviated as B), *Microsporum sucroseum* ( Kosakonia sacchari (abbreviated as H), Pseudomonas linyingense ( Pseudomonas linyingensis (abbreviated as G), Trichoderma variegata ( Trichormus variabilis (abbreviated as K), Nostoc commune ( Nostoc sp. (abbreviated as J).

[0013] In the above technical solution, the monofunctional bacteria has an OD600 value of 0.2; OD600 refers to the absorbance value of the monofunctional bacterial solution at 600 nm, and the bacterial solution concentration is evaluated by OD600.

[0014] In the above technical solution, the compound bacterial agent is preferably composed of Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus starch liquefier FH1 (abbreviated as F), shortwave monocytogenes ( Brevundimonas sp. NH1 (abbreviated as N), Staphylococcus aureus ( Gluconacetobacter sp. )qzr14 (abbreviated as Q), Paleobacterium ( Ochrobactrum sp. The compound microbial agent is formed by mixing S112 (abbreviated as S) in equal volume ratios. This compound microbial agent is abbreviated as compound microbial agent FNQS. The aforementioned Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens FH1 (abbreviated as F), accession number CGMCC NO.17050; Shortwave Monoclonal ( Brevundimonas sp. NH1 (abbreviated as N), accession number CGMCC NO. 27793; Staphylococcus aureus ( Gluconacetobacter sp. QZR14 (abbreviated as Q), accession number CGMCC NO.10983; *Bacillus pallida* ( Ochrobactrum sp. S112 (abbreviated as S), collection number CGMCC NO.27794.

[0015] In the above technical solution, the compound microbial agent is preferably composed of Pantotheca acuminata (…). Pantoea agglomerans (abbreviated as B), *Microsporum sucroseum* ( Kosakonia sacchari (abbreviated as H), Pseudomonas linyingense ( Pseudomonas Linying The compound microbial agent is formed by mixing (abbreviated as G) in equal volume proportions. This compound microbial agent is abbreviated as compound microbial agent BHG. The aforementioned Pantoea agglomerans (abbreviated as B), accession number CGMCC 1.4006; Kosakonia sacchari (abbreviated as H), accession number CGMCC 1.12102; and Pseudomonas linyingensis (abbreviated as G), accession number CGMCC 1.10701.

[0016] In the above technical solution, the compound bacterial agent is preferably composed of Trichophyton variegatum (… Trichormus variabilis (abbreviated as K) and Nostoc commune ( Nostoc sp. The compound microbial agent (abbreviated as J) is formed by mixing in equal volume proportions. This compound microbial agent is abbreviated as compound microbial agent KJ.

[0017] The aforementioned polymorphic tufted algae ( Trichormus variabilis (abbreviated as K), accession number FACHB-164; Nostoc ( Nostoc sp. (abbreviated as J), accession number FACHB-87.

[0018] Compared with existing technologies, it has the following characteristics: (1) This invention uses Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens FH1 (abbreviated as F), shortwave monocytogenes ( Brevundimonas sp. NH1 (abbreviated as N), Staphylococcus aureus ( Gluconacetobacter sp. )qzr14 (abbreviated as Q), Paleobacterium ( Ochrobactrum sp. S112 (abbreviated as S), Pantothecin clumps ( Pantoea agglomerans (abbreviated as B), *Microsporum sucroseum* ( Kosakonia sacchari (abbreviated as H), Pseudomonas linyingense ( Pseudomonas Linying (abbreviated as G), Trichoderma variegata ( Trichormus variabilis (abbreviated as K), Nostoc commune ( We know sp. Three compound microbial agents (abbreviated as J) were combined with humic acid in a pot experiment. The results showed that all three compound microbial agents could significantly increase the plant height, root length and dry weight of rice. Humic acid significantly promoted the growth of rice roots. The synergistic application of compound microbial agents and humic acid further promoted the growth of rice.

[0019] (2) All three compound microbial agents and humic acid can reduce the α-diversity of rhizosphere bacteria and fungi to varying degrees. When the compound microbial agents and humic acid are used in combination, humic acid has a stronger effect on the diversity of rhizosphere soil microorganisms. When compounded with humic acid, the community structure of rice rhizosphere soil microorganisms treated by the three different compound microbial agents is significantly affected, and the regulatory response of different compound microbial agents to humic acid is different. Humic acid can enrich the microbial community related to nutrient cycling and some disease-resistant fungal groups to increase rice biomass.

[0020] (3) Compound microbial agent S1 (FNQS) enriched bacteria that facilitate nitrogen utilization, compound microbial agent S2 (BHG) enriched bacteria related to phosphorus and potassium solubilization, and compound microbial agent S3 (KJ) enhanced the abundance of bacteria related to plant stress resistance. The synergistic application of humic acid and compound microbial agents showed a stronger promoting effect. The synergistic application of the two enriched more bacteria and functions that were significantly positively correlated with rice biomass, which could further promote rice growth.

[0021] In conclusion, the combined application of compound microbial inoculants and humic acid can effectively promote rice growth and optimize soil microbial communities and functions. This invention provides a scientific basis for the research and application of novel compound microbial fertilizers and contributes to the development of sustainable agriculture. Attached Figure Description

[0022] Figure 1 To compare and verify the effects of different treatment groups on rice plant height in Example 1; Figure 2 To compare and verify the effects of different treatment groups on rice root length in Example 1; Figure 3 To compare and verify the effects of different treatment groups on the wet weight of rice in Example 1; Figure 4 To compare and verify the effects of different treatment groups on the dry weight of rice in Example 1. Detailed Implementation

[0023] The following describes in detail the specific embodiments of the technical solution of the present invention, but the present invention is not limited to the following description: The materials and processes involved in the following embodiments of the present invention are shown below: 1. Strains and materials: 1.1. Strains: The strain used in this invention is Bacillus amyloliquefaciens (BAM). Bacillus starch liquefier FH1 (abbreviated as F), shortwave monocytogenes ( Brevundimonas sp. NH1 (abbreviated as N), Staphylococcus aureus ( Gluconacetobacter sp. )qzr14 (abbreviated as Q), Paleobacterium ( Ochrobactrum sp. S112 (abbreviated as S), Pantothecin clumps ( Pantoea agglomerans (abbreviated as B), *Microsporum sucroseum* ( Kosakonia sacchari (abbreviated as H), Pseudomonas linyingense ( Pseudomonas linyingensis (abbreviated as G), Trichoderma variegata ( Trichormus variabilis (abbreviated as K), Nostoc commune ( Nostoc sp. (abbreviated as J), the source information of each bacterium is as follows: ① Bacillus amyloliquefaciens ( Bacillus amyloliquefaciensFH1, a microbial culture, accession number: CGMCCNO.17050, depositor: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (Wang Jingjing), depositary institution: China General Microbiological Culture Collection Center, deposit date: December 29, 2018; ② Shortwave monoclonal bacteria ( Brevundimonas sp. NH1, a culture strain, accession number: CGMCC NO. 27793, depositor: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (Wang Jingjing), depositary institution: China General Microbiological Culture Collection Center, deposit date: July 4, 2023; ③ Staphylococcus aureus ( Gluconacetobacter sp. )qzr14, deposited strain, accession number: CGMCCNO.10983, depositor: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (Wang Huan), depositary institution: China General Microbiological Culture Collection Center, deposit date: June 16, 2015; ④ Paleobacterium ( Ochrobactrum sp. S112, a microbial culture, accession number: CGMCC NO.27794, depositor: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (Wang Jingjing), depositary institution: China General Microbiological Culture Collection Center, deposit date: July 4, 2023; ⑤ Pantoea agglomerans in clusters, available for sale in the market, accession number: CGMCC 1.4006, available for sale in the market; ⑥ Kosakonia sacchari, available in the market, accession number: CGMCC 1.12102; ⑦ *Pseudomonas linyingensis*, available in the market, accession number: CGMCC1.10701; ⑧ Variegated Trichophyton ( Trichormus variabilis (), available for sale on the market, collection number: FACHB-164; ⑨ Nostoc commune ( Nostoc sp. (This item is available for sale on the market; collection number: FACHB-87.)

[0024] Among the aforementioned bacteria, F has a strong ability to produce siderophores; N has the ability to produce ACC deaminase; Q has the ability to solubilize phosphorus; S has the ability to solubilize potassium; K and J have the ability to fix nitrogen; and B, H, and G have the ability to solubilize phosphorus.

[0025] 1.2 Rice materials: The rice seed variety is Nei 5 You 8015, purchased from Zhejiang Changshan Lixin Seed Industry Co., Ltd.

[0026] Humic acid: Purchased from Shandong Cangyuan Biotechnology Co., Ltd.

[0027] Biochar: Purchased from Henan Lize Environmental Protection Technology Co., Ltd., it is made from corn stalks through anaerobic pyrolysis at 500℃.

[0028] Soil: The soil for the pot experiment was sourced from the Da'an Alkali Land Ecological Experimental Station in Jilin Province. After natural air drying, it was filtered through a 5 mm sieve. The soil physicochemical properties were as follows: total nitrogen 1.30 g / kg, total phosphorus 0.32 g / kg, total potassium 25.18 g / kg, organic matter 14.75 g / kg, organic carbon 8.56 g / kg, available nitrogen 88.87 mg / kg, available potassium 377.29 mg / kg, available phosphorus 33.15 mg / kg, electrical conductivity 1283.50 μS / cm, and pH 8.44.

[0029] 1.3 LB liquid culture medium: Weigh 10.0 g peptone, 5.0 g yeast extract and 10.0 g sodium chloride solid powder and dissolve them in 1 L of ultrapure water. Stir with a glass rod to dissolve and adjust the pH to 7.2±0.2. Then sterilize in an autoclave at 121℃ for 20 min and store at room temperature for later use.

[0030] LB solid medium: Based on the above LB liquid medium, add 18.0 g of agar powder, sterilize in a high-temperature autoclave at 121℃ for 20 min, and place in a constant temperature oven for later use.

[0031] PDA liquid culture medium: Weigh 5.0 g peptone, 2.0 g yeast extract, 20.0 g glucose, 1.0 g dipotassium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, and 0.5 g magnesium sulfate, dissolve in 1.0 L ultrapure water, stir with a glass rod to dissolve, sterilize in an autoclave at 115℃ for 30 min, and store at room temperature for later use.

[0032] PDA solid medium: Based on the above PDA liquid medium, add 15.0 g of agar powder, sterilize in a high-temperature autoclave at 115℃ for 30 min, and place in a constant temperature oven for later use.

[0033] TSB liquid culture medium: Weigh 17.0 g of tryptone, 5.0 g of sodium chloride, 3.0 g of soybean peptone, 2.5 g of glucose, and 2.5 g of dipotassium hydrogen phosphate, dissolve them in 1.0 L of ultrapure water, stir with a glass rod to dissolve, and adjust the pH to 7.3±0.2. Then sterilize in an autoclave at 115℃ for 30 min and store at room temperature for later use.

[0034] TSA solid medium: Based on the above TSB liquid medium, add 15.0 g of agar powder, sterilize in a high-temperature autoclave at 115℃ for 30 min, and place in a constant temperature oven for later use.

[0035] FP liquid culture medium: Weigh 10.0 g of wheat bran, 30.0 g of glucose, 5.0 g of ammonium sulfate, and 0.5 g of manganese sulfate, dissolve them in 1.0 L of ultrapure water, sterilize in an autoclave at 115℃ for 30 min, and store at room temperature for later use.

[0036] Trace metal A5 solution: Weigh 2.86 g boric acid, 1.81 g manganese chloride tetrahydrate, 0.222 g zinc sulfate heptahydrate, 0.39 g sodium permanganate dihydrate, 0.079 g copper sulfate pentahydrate, and 0.05 g cobalt nitrate hexahydrate, dissolve them in 1.0 L of ultrapure water, stir well, and set aside at room temperature for later use.

[0037] BG-11 liquid culture medium: Weigh out 0.3 g sodium nitrate, 0.04 g dipotassium hydrogen phosphate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride dihydrate, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g disodium EDTA, 0.02 g sodium carbonate, and 1.0 mL Trace metal A5 solution. Dissolve in 1.0 L ultrapure water, stir well, adjust pH to 7.1, sterilize in an autoclave at 115℃ for 30 min, and store at room temperature for later use.

[0038] BG-11 solid medium: Based on the above BG-11 liquid medium, add 10.0 g of agar powder, sterilize in a high-temperature autoclave at 115℃ for 30 min, and place in a constant temperature oven for later use.

[0039] Inorganic phosphorus culture medium: Weigh out 0.3 g magnesium sulfate heptahydrate, 0.3 g potassium chloride, 0.3 g sodium chloride, 0.03 g manganese sulfate, 5.0 g calcium phosphate, 0.5 g ammonium sulfate, 0.03 g ferrous sulfate heptahydrate, and 20.0 g agar, dissolve them in 980 mL deionized water, and adjust the pH to 7.2 to prepare component one; weigh out 10 g glucose and dissolve it in 20 mL deionized water to prepare component two. Then, sterilize components one and two in a high-temperature autoclave at 115 ℃ for 30 min, cool to about 60 ℃, and mix them thoroughly in a clean bench.

[0040] ACC stock solution: Weigh 0.1 g of ACC and dissolve it in 2.0 mL of ultrapure water. After filtration and sterilization using a 0.22 µm filter membrane, dispense the solution into sterile containers.

[0041] DF liquid culture medium: Weigh 2.0 g glucose, 2.0 g sodium gluconate, 6.0 g disodium hydrogen phosphate, 4.0 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, and 2.0 g citric acid, and dissolve them in 1.0 L deionized water, adjusting the pH to 7.2; Separately weigh 0.1 g boric acid, 0.112 g manganese sulfate monohydrate, 1.246 g zinc sulfate heptahydrate, 0.7822 g copper sulfate pentahydrate, and 0.1 g molybdenum trioxide, and dissolve them in 100 mL deionized water, then filter and sterilize using a 0.22 µm filter membrane to prepare component one; Finally, weigh 0.1 g ferrous sulfate heptahydrate and dissolve it in 10 mL of deionized sterile water to prepare component two.

[0042] ADF solid medium: Add 15.0 g of agar to the above DF liquid medium, sterilize, and then add ACC stock solution, controlling its concentration at 3.0-5.0 mmol / L.

[0043] Coomassie Brilliant Blue G250 solution: Dilute the 5 x Bradford Protein Assay Reagent solution from the kit 5 times with deionized water and use immediately.

[0044] Bovine serum albumin solution: Weigh 0.01 g of bovine serum albumin and dissolve it completely in 100 mL of deionized water. Store in a refrigerator at 4°C.

[0045] 2,4-Dinitrophenylhydrazine solution: Weigh 0.2 g of 2,4-dinitrophenylhydrazine and dissolve it in 100 mL of 2 mol / L HCl solution. Place the solution in an ultrasonic shaker and shake thoroughly until completely dissolved. After preparation, transfer the solution to a brown bottle, protect it from light, and store it in a refrigerator at 4 ℃.

[0046] 2. Process flow: 2.1 Activation of microbial strains 2.1.1 Activation of F, N, S, Q, B, H, and G bacterial strains: F, N, S, Q, B, H, and G bacterial strains stored in glycerol tubes (water to glycerol ratio 1:1) at -80℃ were removed and slowly thawed at 4℃. The strains were then inoculated onto TSB or LB liquid medium, and the inoculated flasks were placed on a shaker at 180 rpm and incubated at 30℃ for 48 h. After centrifugation at 6000 rpm for 8 min using a refrigerated centrifuge, the supernatant was discarded, and the cells were washed three times with 0.9% NaCl solution, retaining the bacterial cells. A small amount of sterile deionized water was added to resuspend the bacterial cells. The resuspended cells were then streaked on LB or TSB solid plates to check for contamination, and the cell count was performed under a microscope. 100-200 μL of the resuspended cells were spread onto solid medium and incubated under the same conditions until single colonies appeared on the plates, at which point the plates were stored at 4℃ for later use.

[0047] Strains were picked from single colonies on solid culture media and inoculated into 100 mL of autoclaved LB and TSB liquid media, respectively. The inoculated LB and TSB media were then incubated at 30 ℃ and 180 rpm in a shaking incubator for 2-3 days. After incubation, the cultures were centrifuged at 7500 rpm for 6 minutes using a refrigerated centrifuge. The supernatant was discarded, and the bacterial cells were washed twice with sterile deionized water to remove residual culture medium components. The bacterial cells were then resuspended in sterile deionized water, and the bacterial concentration was assessed by measuring the absorbance at 600 nm (OD600). To ensure similar initial concentrations of each strain in the compound bacterial agent, the OD600 values ​​of each tested strain were adjusted to 0.2 before use.

[0048] 2.1.2 Activation of K and J strains: The procedure is basically the same as 2.1.1, except that: ① K and J are inoculated into BG-11 liquid medium, and the inoculated conical flasks are placed on a shaker under light at 28 ℃ for 7 days with continuous aeration. ② Strains are picked from single colonies on solid medium and inoculated into BG-11 liquid medium. The BG-11 liquid medium is placed on a shaker under light at 28 ℃ for 7 days with continuous aeration until the bacteria reach the logarithmic growth phase. Bacterial suspensions with an OD600 value of 0.2 are then ready for use.

[0049] 2.2 Constructing a rice potted plant: 2.2.1 Soil Preparation: After the soil has been left to dry in a cool, shaded place, repeatedly pound it into powder with a hammer. Remove surface weeds and leaves, then filter the soil through a 5 mm sieve to remove large clumps and plant debris. Finally, mix the soil thoroughly. Place approximately 200 g of soil into each flowerpot (8 cm in diameter). The soil must be sterilized.

[0050] 2.2.2 Rice Seed Pretreatment: Select whole and plump rice seeds and soak them in room temperature water for 12 hours. Then, disinfect them in a 5% sodium hypochlorite solution for 10 minutes. Discard the sodium hypochlorite solution and soak them in a 75% ethanol solution for 5 minutes. Discard the ethanol solution and rinse the disinfected rice seeds repeatedly with sterile water. Spread the sterile water from the last rinse onto a plate. If no colonies grow on the plate, the disinfection is thorough; otherwise, the seeds need to be disinfected again. Place the thoroughly disinfected rice seeds in a cloth and seal it in a tray. Add an appropriate amount of sterile water to cover the cloth. Cover the mouth of the beaker with plastic wrap and place it in a dark environment. Change the water every other day. After 3 days, a large number of rice seeds will show signs of sprouting.

[0051] 2.2.3 Sowing and Watering: After the rice seeds show signs of germination, use sterile tweezers to sow 15 seeds of similar germination levels into each flowerpot and cover them with a thin layer of soil. Ensure the seeds are evenly distributed in the soil during sowing. After sowing, add 100 mL of tap water to the small dish at the bottom of the tray, and then place it in an artificial climate incubator. Maintain the temperature in the climate incubator at 28℃, the humidity at 75%, and the day / night cycle at 16 / 8 hours. Thin the seedlings on the 7th day after sowing. Based on the number of seedling roots, remove a certain number of seedlings from each tray to ensure that the number of seedling roots and their growth are consistent across all trays.

[0052] 2.2.4. Conduct pot experiment: Two days after sowing, add humic acid and compound bacterial agent to the soil where rice is planted. Every two days after sowing, add 40 mL of tap water along the small dish under the seed tray. At the same time, change the position of the pots in a directional manner (the artificial climate incubator has three layers. When changing, the position is circulated from top to bottom. The horizontal and vertical orientation of the pots in the same layer need to be changed) to reduce the growth differences caused by spatial heterogeneity.

[0053] 3. Detection indicators: Rice Sampling: Rice samples were collected 30 days after growth. The soil and rice seedlings were removed from the pots, and the soil attached to the roots was rinsed off with water. The seedlings were placed in 50 mL centrifuge tubes filled with tap water to maintain their viability. For measurement, the seedling and root lengths of individual rice plants were measured using a ruler placed on paper. The measured rice (including roots) was placed in envelopes, labeled with the corresponding pot number, and the wet weight of the rice was determined. The roots were then trimmed, labeled, and aliquoted into 5 mL centrifuge tubes, stored at 4 °C for subsequent bacterial screening. The rice envelopes with the roots removed were dried in a 60 °C oven to constant weight, and the dry weight of the above-ground parts was measured. The soil that fell off was considered the rhizosphere soil sample and aliquoted into 5 mL centrifuge tubes. 20 g of the sample was collected and stored at 4 °C for subsequent bacterial screening.

[0054] The method of the present invention is illustrated below with specific embodiments: Example 1:

[0055] A method for promoting rice growth using compound microbial agents and humic acid (labeled HAS1) involves adding humic acid and compound microbial agents sequentially to the soil planted with rice two days after sowing, with 2 g of humic acid and 40 mL of compound microbial agent FNQS added to each pot of soil.

[0056] In this embodiment, the humic acid is thoroughly ground, filtered through a 200-mesh sieve, and dried in a 55°C constant temperature oven for one week until constant weight, then sterilized and ready for use.

[0057] In this embodiment, the compound bacterial agent FNQS is formed by mixing Bacillus amyloliquefaciens FH1, Bacillus shortwave monocytogenes NH1, Gluconobacterium glutamicum QZR14, and Bacillus cereus S112 in equal volume ratios.

[0058] After 30 days of rice growth, the results are as follows: Figure 1-4 As shown, the rice plant height is greater than 35cm, the rice root length is greater than 10cm, the wet weight of the rice is greater than 3g, and the dry weight of the rice is about 0.6g. Example 2:

[0059] A method for promoting rice growth using a compound microbial agent and humic acid (labeled HAS2) is basically the same as in Example 1, except that the compound microbial agent is BHG. The compound microbial agent is formed by mixing *Pantotheca acuminata*, *Microsporum sucroseum*, and *Pseudomonas linyingensis* in equal volume ratios.

[0060] After 30 days of rice growth, the results are as follows: Figure 1-4 As shown, the rice plant is about 35cm tall, the rice root is more than 10cm long, the wet weight of the rice is about 3g, and the dry weight of the rice is about 0.6g. Example 3:

[0061] A method for promoting rice growth using a compound microbial agent and humic acid (labeled HAS3) is basically the same as in Example 1, except that the compound microbial agent is KJ. The compound microbial agent is formed by mixing *Trichophyton variegatum* and *Nostoc* in equal volume ratios.

[0062] After 30 days of rice growth, the results are as follows: Figure 1-4 As shown, the rice plant height is about 35cm, the root length is greater than 11cm, the wet weight of the rice is greater than 3g, and the dry weight of the rice is greater than 0.6g.

[0063] Comparative verification example 1:

[0064] The following experiment was conducted according to the method in Example 1. Two days after sowing, humic acid and / or compound microbial agent were added sequentially to the soil planted with rice. The different treatment groups are shown below: (1) Control group (CK): Soil was used only, without the addition of humic acid or biochar.

[0065] (2) Compound microbial agent FNQS treatment group (S1): 40 mL of compound microbial agent FNQS was added to each pot of soil.

[0066] (3) Compound microbial agent BHG treatment group (S2): 40 mL of compound microbial agent BHG was added to each pot of soil.

[0067] (4) Compound microbial agent KJ treatment group (S3): 40 mL of compound microbial agent KJ was added to each pot of soil.

[0068] (4) Humic acid treatment group (HA): 2 g of humic acid was added to each pot of soil.

[0069] (5) Humic acid + compound microbial agent FNQS treatment group (HAS1): 2 g of humic acid and 40 mL of compound microbial agent FNQS were added to each pot of soil (i.e. Example 1).

[0070] (6) Humic acid + compound microbial agent BHG treatment group (HAS2): 2 g of humic acid and 40 mL of compound microbial agent BHG were added to each pot of soil (i.e. Example 2).

[0071] (7) Humic acid + compound microbial agent KJ treatment group (HAS3): 2g of humic acid and 40 mL of compound microbial agent KJ were added to each pot of soil (i.e. Example 3).

[0072] After 30 days of rice growth, the results are as follows: Figure 1-4 As shown, compared with the control group (CK), the rice plant height in the HA treatment group was increased. Different compound microbial agents (S1, S2, S3) significantly increased the rice plant height and performed better than the HA treatment group. The combination of compound microbial agents and humic acid (HAS1, HAS2, HAS3) further enhanced rice growth, indicating that the synergistic effect of compound microbial agents and humic acid may help improve the nutrient absorption and growth vitality of rice.

[0073] Compared to the control (CK), different compound microbial agents (S1, S2, S3) all promoted root growth to varying degrees. The roots in the HA treatment group were longer and stronger, indicating that humic acid helps root growth. The HAS1, HAS2, and HAS3 treatment groups showed stronger root development, with a significant increase in both root mass and root length, suggesting that the synergistic effect of humic acid and microorganisms may significantly promote rice root growth and improve nutrient absorption capacity.

[0074] In conclusion, the synergistic treatment groups of compound microbial agents and humic acid (HAS1, HAS2, HAS3) showed the best performance. They not only increased the height of rice plants but also promoted root development, which may help enhance the stress resistance and nutrient absorption capacity of rice.

[0075] The above examples are merely illustrative of the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the essence of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for promoting rice growth using a compound microbial agent and humic acid, characterized in that, Add humic acid and compound microbial agent sequentially to the soil where rice is grown. The amount of humic acid added is 0.5-2% of the soil mass, and the solid-liquid ratio of humic acid and compound microbial agent (g:ml) is 0.5-1.1:15-30.

2. The method according to claim 1, characterized in that, The amount of humic acid added is 1% of the soil mass; the solid-liquid ratio of humic acid and compound microbial agent is 1:20 (g:ml).

3. The method according to claim 1, characterized in that, The compound microbial agent is obtained by combining any two or more monofunctional bacteria in equal volume ratios; the monofunctional bacteria include Bacillus amyloliquefaciens. Bacillus amyloliquefaciens FH1, abbreviated as F, is a shortwave monocytogenes. Brevundimonas sp. NH1, abbreviated as N, is a type of glucosamine. Gluconacetobactersp. qzr14, abbreviated as Q, is a type of bacillus. Ochrobactrum sp. S112, abbreviated as S, is a type of Pantotheca aggregata. Pantoea agglomerans, Abbreviation B, Microsporum sucrose Kosakonia sacchari Abbreviated as H, *Pseudomonas linyingensis* Pseudomonas linyingensis Abbreviated as G, *Trichoderma variegata* Trichormus variabilis Abbreviated as K, Nostoc commune Nostoc sp. Abbreviated as J.

4. The method according to claim 3, characterized in that, The monofunctional bacteria have an OD600 value of 0.

2. OD600 refers to the absorbance of the bacterial solution at 600 nm, and the bacterial concentration is evaluated by OD600.

5. The method according to claim 3, characterized in that, The aforementioned compound microbial agent is formed by mixing Bacillus amyloliquefaciens FH1, Shortwave Monoclonalella NH1, Gluconobacterium qzr14, and Aureobacterium lancifolium S112 in equal volume proportions. This compound microbial agent is referred to as compound microbial agent FNQS.

6. The method according to claim 5, characterized in that, The Bacillus amyloliquefaciens FH1, with accession number CGMCC NO.17050; Bacillus shortwave monocytogenes NH1, with accession number CGMCC NO. 27793; Gluconobacterium qzr14, with accession number CGMCC NO.10983; and Bacillus pallida S112, with accession number CGMCC NO.27794, are described.

7. The method according to claim 3, characterized in that, The aforementioned compound microbial agent is formed by mixing Pantotheca agglomerata, Microsporum sucrose, and Pseudomonas linyingensis in equal volume proportions. This compound microbial agent is referred to as compound microbial agent BHG.

8. The method according to claim 7, characterized in that, The aforementioned Pantotheca agglomerata has the accession number CGMCC 1.4006; Microsporum sucrose has the accession number CGMCC 1.12102; and Pseudomonas linyingensis has the accession number CGMCC 1.10701.

9. The method according to claim 3, characterized in that, The aforementioned compound microbial agent is formed by mixing Trichophyton variegata and Nostoc commune in equal volume ratios. This compound microbial agent is abbreviated as compound microbial agent KJ.

10. The method according to claim 9, characterized in that, The aforementioned *Trichoderma polymorpha*, accession number FACHB-164; and *Nostoc*, accession number FACHB-87.

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