Bio-organic fertilizer containing plant growth-promoting rhizobacteria, and preparation method and application thereof

Abstract

The application discloses a bio-organic fertilizer containing rhizosphere growth-promoting bacteria and a preparation method and application thereof, and belongs to the technical field of agricultural microbial fertilizers. Bacillus velezensis The bio-organic fertilizer contains an organic fertilizer carrier, a spore inducer, a spore protective agent, an amino acid hydrolysate and bacillus velezensis HP26 bacteria, wherein the mass of the spore inducer is 0.1-0.2% of the dry weight of the organic fertilizer carrier, the mass of the bacillus velezensis HP26 bacteria is 5-10% of the dry weight of the organic fertilizer carrier, the mass of the spore protective agent is 5-10% of the dry weight of the organic fertilizer carrier, and the mass of the amino acid hydrolysate is 15-20% of the dry weight of the organic fertilizer carrier. The bio-organic fertilizer containing the rhizosphere growth-promoting bacteria can be used in combination with reduced chemical fertilizers to improve soil fertility, significantly increase the content of soil organic matter, adjust the pH value of soil and relieve the problem of soil acidification caused by long-term use of chemical fertilizers.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial fertilizer technology, specifically relating to a bio-organic fertilizer containing rhizosphere growth-promoting bacteria, its preparation method, and its application. Background Technology

[0002] The long-term excessive application of chemical fertilizers has led to a series of ecological problems, including soil degradation, a continuous decline in nutrient utilization, and a decrease in rhizosphere microbial diversity. Nitrogen and phosphorus nutrients enter the environment through ammonia volatilization, runoff, leaching, and infiltration, causing not only huge economic losses but also ecological security problems such as eutrophication of water bodies. Developing environmentally friendly bio-fertilizers and constructing a technical system that synergistically achieves the reduction of chemical fertilizer use and the improvement of crop yield and quality has become a core direction of agricultural green development research.

[0003] Bio-organic fertilizer is a special type of organic fertilizer containing specific functional microbial strains. Its core components include organic matter and functional microorganisms. The synergistic effect of these two components gives bio-organic fertilizer significant advantages in improving soil microecology, increasing fertilizer utilization, and enhancing crop resistance. Among numerous functional microbial groups, Bacillus spp., due to its strong resistance, rapid reproduction, and ability to secrete various growth-promoting substances, has become the most extensively researched and widely used functional microbial group in the field of bio-organic fertilizer. Among them, Bacillus belesiensis (… Bacillus velezensis Due to its excellent growth-promoting, disease-resistant, and environmental adaptability, it has received widespread attention in recent years.

[0004] However, existing bio-organic fertilizer technologies still have significant shortcomings. First, research on process parameters from superior strains to highly active products is not systematic. The survival rate and shelf-life stability of functional bacteria in organic fertilizer carriers are core bottlenecks restricting product commercialization. In existing technologies, the problems of poor survival and stability of functional bacteria in organic fertilizer carriers are common, with the number of effective viable bacteria in some products dropping below the national standard after 3-6 months of storage. Second, the appropriate substitution threshold for bio-organic fertilizers and reduced chemical fertilizer application lacks systematic verification, and precise application technical parameters are lacking for field application. Third, existing bio-organic fertilizer products mostly focus on a single growth-promoting function, lacking a systematic evaluation of the synergistic regulatory effects of the soil-microbe-crop system.

[0005] Therefore, developing bio-organic fertilizer products that combine high survival rate, long shelf life, and stable field efficacy, and establishing a precise application technology system for reducing fertilizer use and increasing efficiency, is of great practical significance for promoting the large-scale application of bio-organic fertilizers and achieving the goal of reducing fertilizer use and increasing efficiency. Summary of the Invention

[0006] The first objective of this invention is to provide a bio-organic fertilizer containing rhizosphere growth-promoting bacteria, which has good shelf-life stability and can reduce the amount of chemical fertilizer applied and promote wheat growth when used in combination with reduced-volume fertilizer.

[0007] The second objective of this invention is to provide a method for preparing a bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

[0008] The third objective of this invention is to provide an application of a bio-organic fertilizer containing rhizosphere growth-promoting bacteria in replacing 15-45% by weight of compound fertilizer.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a bio-organic fertilizer containing rhizosphere growth-promoting bacteria, including an organic fertilizer carrier, a spore inducer, a spore protectant, an amino acid hydrolysate, and a Bacillus vesiculosus HP26 bacterial solution. Based on the dry weight of the organic fertilizer carrier, the mass of the spore inducer is 0.1-0.2% of the dry weight of the organic fertilizer carrier, the mass of the Bacillus vesiculosus HP26 bacterial solution is 5-10% of the dry weight of the organic fertilizer carrier, the mass of the spore protectant is 5-10% of the dry weight of the organic fertilizer carrier, and the mass of the amino acid hydrolysate is 15-20% of the dry weight of the organic fertilizer carrier.

[0010] Furthermore, the *Bacillus belye* HP26 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35936, and the effective viable count of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria is ≥2×10⁻⁶. 8 CFU / g, moisture content ≤20%.

[0011] Furthermore, the method for preparing the Bacillus belyss HP26 bacterial culture includes: activating Bacillus belyss HP26 plates, transferring them to NB liquid medium, and culturing them with shaking at 30–37 ℃ and 180–200 r / min for 12 h to prepare a seed culture; then transferring the seed culture at a volume ratio of 0.5–1% and culturing it at 30–37 ℃ and 180–200 r / min for 12–24 h to obtain the inoculum; the plate activation conditions are constant temperature activation at 30–37 ℃ for 12–24 h.

[0012] Furthermore, the effective viable count of the *Bacillus vesiculosus* HP26 bacterial suspension is 1.0 × 10⁻⁶. 8 ~5.0×10 9 CFU / mL.

[0013] Furthermore, the organic fertilizer carrier includes well-rotted pig manure; the moisture content of the organic fertilizer carrier is 25-30%.

[0014] Furthermore, the spore inducer is a manganese sulfate solution, and the mass fraction of the manganese sulfate solution is 5-10%.

[0015] Furthermore, the spore protectant includes trehalose, glycerol and fulvic acid, wherein the mass ratio of trehalose to glycerol is (0.8-1.2):1, and the mass ratio of trehalose to fulvic acid is 1:(2.5-3.5).

[0016] Furthermore, the nitrogen content of the amino acid hydrolysate is 13–18 g / L, and the pH is 1.8–2.0.

[0017] Secondly, the present invention provides a method for preparing a bio-organic fertilizer containing rhizosphere growth-promoting bacteria as described above, comprising the following steps: adding a formula amount of organic fertilizer carrier to a formula amount of amino acid hydrolysate, adjusting the pH to 5.5-6.5, then adding a formula amount of Bacillus vesiculosus HP26 bacterial solution, a spore inducer and a spore protectant, and drying after fermentation to obtain a bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

[0018] Furthermore, the fermentation includes static fermentation at 30–35 °C for 5–7 days, with the pile turned over once a day.

[0019] Thirdly, the present invention provides an application of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria as described above in replacing 15-45% by weight of compound fertilizer, wherein the application rate of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria is 300-500 kg / mu.

[0020] Furthermore, the bio-organic fertilizer containing rhizosphere growth-promoting bacteria is mixed with 50% by weight of compound fertilizer and applied as a base fertilizer before wheat sowing, while the remaining 50% by weight of compound fertilizer is applied as a top dressing during the wheat jointing stage.

[0021] The beneficial effects of this invention are: The bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention uses Bacillus vesiculosus HP26 (CGMCC No. 35936) as the core functional bacterial source. This strain has a significant growth-promoting effect on wheat and can improve the above-ground fresh weight, underground fresh weight, above-ground dry weight, underground dry weight and other phenotypic indicators of wheat seedlings.

[0022] The method for preparing bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention ensures the survival rate and stability of Bacillus berberis HP26 in the organic fertilizer carrier by optimizing the water content of the organic fertilizer carrier and the amount of amino acid hydrolysate added. The bio-organic fertilizer containing rhizosphere growth-promoting bacteria prepared by this method has an effective viable bacteria count ≥2×10⁻⁶. 8The CFU / g count of effective live bacteria remains stable within 6 months of storage at room temperature, demonstrating excellent shelf-life stability and meeting the national standard for bio-organic fertilizer (NY 884-2012).

[0023] The bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention not only provides high-density live bacteria, but also provides continuous nutritional support for the colonization and function of functional bacteria in the rhizosphere through the synergistic effect of amino acid hydrolysate and organic fertilizer carrier, breaking through the limitation of single function of existing bio-organic fertilizer products.

[0024] This invention provides a precise fertilizer-reducing and yield-enhancing application technique for wheat: The conventional recommendation is to apply 300-400 kg / mu of bio-organic fertilizer combined with a 15-30% reduction in ternary compound fertilizer. For scenarios with high fertilizer reduction requirements, 400-500 kg / mu of bio-organic fertilizer combined with a 45% reduction in ternary compound fertilizer can be used. This technique can reduce fertilizer usage while simultaneously increasing wheat yield by more than 10%.

[0025] The bio-organic fertilizer containing rhizosphere growth-promoting bacteria of this invention, when applied in combination with reduced-volume fertilizer, can improve soil fertility. The application of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria can significantly increase soil organic matter content, regulate soil pH, and alleviate soil acidification caused by long-term application of chemical fertilizers. When the application rate of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria reaches 400-500 kg / mu, even under a fertilization mode with a 45% reduction in chemical fertilizer application, the contents of total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, and available potassium in the soil can still maintain a stable high level.

[0026] When the bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention is applied in combination with the reduced-volume fertilizer, it can activate soil enzyme activity. The application of bio-organic fertilizer containing rhizosphere growth-promoting bacteria can significantly increase the activity of soil β-glucosidase, sucrase, urease, catalase, neutral phosphatase and nitrate reductase, and comprehensively improve the soil's ability to cycle and transform carbon, nitrogen and phosphorus nutrients and its microecological antioxidant function.

[0027] The bio-organic fertilizer containing rhizosphere growth-promoting bacteria of this invention, when applied in combination with reduced-volume fertilizer, can increase wheat yield. When 300-400 kg / mu of bio-organic fertilizer containing rhizosphere growth-promoting bacteria is applied in combination with a 15-30% reduction in chemical fertilizer, wheat yield can reach 500-600 kg / mu, which is more than 15% higher than conventional fertilization treatment. When the reduction in chemical fertilizer application is increased to 45%, the application of 400-500 kg / mu of bio-organic fertilizer containing rhizosphere growth-promoting bacteria can still maintain a stable yield level.

[0028] The preparation process of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention is simple, cost-controllable, and environmentally friendly. It is suitable for the production practice of reducing chemical fertilizer use and increasing efficiency in wheat-producing areas, and has economic, ecological and social benefits. Attached Figure Description

[0029] Figure 1 The graphs show the effects of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and reduced application of chemical fertilizer on soil pH, organic matter, total nitrogen, and total phosphorus in Example 2. In the graphs, A is the effect on soil pH, B is the effect on soil organic matter, C is the effect on soil total nitrogen, and D is the effect on soil total phosphorus. Figure 2 The graphs show the effects of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and reduced application of chemical fertilizer on total potassium, alkaline nitrogen, available phosphorus, and available potassium in the soil in Example 2. In the graphs, A is the effect on total potassium in the soil, B is the effect on alkaline nitrogen in the soil, C is the effect on available phosphorus in the soil, and D is the effect on available potassium in the soil. Figure 3 The diagram shows the effects of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and reduced application of chemical fertilizer on S-β-GC, S-SC, S-UE and S-CAT in soil in Example 2. In this diagram, A is the effect on S-β-GC in soil, B is the effect on S-SC in soil, C is the effect on S-UE in soil, and D is the effect on S-CAT in soil. Figure 4 The diagram shows the effects of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and reduced application of chemical fertilizer on S-NP and S-NR in soil in Example 2. In this diagram, A represents the effect on S-NP in soil, and B represents the effect on S-NR in soil. Figure 5 The graphs show the effects of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and reduced application of chemical fertilizer on the agronomic traits and yield of wheat in the first year, as described in Example 2. In the graphs, A represents the effect on wheat plant height in the first year, B represents the effect on wheat thousand-grain weight in the first year, C represents the effect on the number of wheat spikelets in the first year, D represents the effect on the number of grains per spike in the first year, E represents the effect on the number of wheat spikes in the first year, and F represents the effect on wheat yield in the first year. Figure 6 The graphs show the effects of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and reduced application of chemical fertilizer on the agronomic traits and yield of wheat in the second year, as described in Example 2. In the graphs, A represents the effect on wheat plant height in the second year, B represents the effect on wheat thousand-grain weight in the second year, C represents the effect on the number of wheat spikelets in the second year, D represents the effect on the number of grains per ear in the second year, E represents the effect on the number of wheat ears in the second year, and F represents the effect on wheat yield in the second year. Figure 7 The graphs show the changes in effective viable bacteria during the fermentation process of bio-organic fertilizers containing rhizosphere growth-promoting bacteria in Experiment Examples 1 and 2, where A represents Experiment Example 1 and B represents Experiment Example 2. Figure 8 This is a graph showing the change in the number of viable bacteria in the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Experiment Example 3 during its shelf life of 1 to 6 months. Detailed Implementation

[0030] In the following examples, the Bacillus belye HP26 used was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35936.

[0031] In the following examples, the NB medium used included the following components: 10 g peptone, 3 g beef extract powder, 5 g sodium chloride, and 1 L distilled water. The pH of the resulting NB medium was 7.2–7.4.

[0032] In the following examples, the untreated, well-rotted pig manure used had a moisture content of 20.56%, an organic matter content of 27.83%, a total nitrogen content of 1.83%, a P2O5 content of 2.76%, and a K2O content of 1.29%.

[0033] The amino acid hydrolysate used in the following examples was purchased from Lianye Biotechnology Co., Ltd.

[0034] The following will provide further details with reference to embodiments of the present invention.

[0035] Example 1 The bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Example 1 includes the following raw materials: The organic fertilizer carrier has a dry weight of 1000 g, 200 g of amino acid hydrolysate, 1 g of spore inducer, 100 g of spore protectant, and 100 g of Bacillus vesiculosus HP26 bacterial solution.

[0036] The spore inducer was a 10% manganese sulfate solution; the spore protectant was a mixture of 20 g trehalose, 20 g glycerol and 60 g humic acid; the organic fertilizer carrier was well-rotted pig manure; and the amino acid hydrolysate had a nitrogen content of 13 g / L and a pH of 1.8.

[0037] The preparation method of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Example 1 includes the following steps: After activating Bacillus belycei HP26 on a plate at 30 ℃ for 12 h, a single colony of Bacillus belycei HP26 was picked and inoculated into 100 mL of NB liquid medium. After culturing at 30 ℃ and 180 r / min for 12 h, a seed culture of Bacillus belycei HP26 was obtained. One mL of this seed culture was then inoculated into 100 mL of NB liquid medium and cultured at 30 ℃ and 180 r / min for 12 h to obtain a bacterial suspension of Bacillus belycei HP26. The viable count of the obtained Bacillus belycei HP26 bacterial suspension was 1.0 × 10⁻⁶. 9 CFU / mL.

[0038] After drying and crushing the formulated amount of well-rotted pig manure, pass it through a 20-mesh sieve and adjust the moisture content to 25%. Then, add the formulated amount of amino acid hydrolysate and adjust the pH to 6.5. Next, add the formulated amount of Bacillus vesiculus HP26 bacterial solution, spore inducer, and spore protectant. Ferment at 35 ℃ for 7 days, turning the pile once a day. Measure the number of viable Bacillus vesiculus HP26 in the bio-organic fertilizer daily. After fermentation, ventilate and dry until the moisture content is 18.7%, thus obtaining bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

[0039] The bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 1 was prepared using the same method as the bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 1. The bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 1 had an effective viable bacteria count of 4.10 × 10⁻⁶. 8 The product has a CFU / g content, a water content of 18.6%, an organic matter content of ≥40%, and a pH of 6.5.

[0040] Example 2 The bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Example 2 includes the following raw materials: The organic fertilizer carrier has a dry weight of 1000 g, 150 g of amino acid hydrolysate, 2 g of spore inducer, 50 g of spore protectant, and 50 g of Bacillus vesiculosus HP26 bacterial solution.

[0041] The spore inducer was a 5% manganese sulfate solution; the spore protectant was a mixture of 8 g trehalose, 10 g glycerol and 32 g humic acid; the organic fertilizer carrier was well-rotted pig manure; and the amino acid hydrolysate had a nitrogen content of 18 g / L and a pH of 2.0.

[0042] The preparation method of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Example 2 includes the following steps: After activating Bacillus belycei HP26 on a plate at 30 ℃ for 12 h, a single colony of Bacillus belycei HP26 was picked and inoculated into 100 mL of NB liquid medium. After culturing at 30 ℃ and 180 r / min for 12 h, a seed culture of Bacillus belycei HP26 was obtained. One mL of this seed culture was then inoculated into 100 mL of NB liquid medium and cultured at 30 ℃ and 180 r / min for 12 h to obtain a bacterial suspension of Bacillus belycei HP26. The viable count of the obtained Bacillus belycei HP26 bacterial suspension was 1.0 × 10⁻⁶. 9 CFU / mL.

[0043] After drying and crushing the formulated amount of well-rotted pig manure, pass it through a 20-mesh sieve and adjust the moisture content to 30%. Then, add the formulated amount of amino acid hydrolysate and adjust the pH to 5.5. Next, add the formulated amount of Bacillus vesiculus HP26 bacterial solution, spore inducer, and spore protectant. Ferment at 30 ℃ for 5 days, turning the pile once a day. Measure the number of viable Bacillus vesiculus HP26 in the bio-organic fertilizer daily. After fermentation, ventilate and dry until the moisture content is 18.7%, thus obtaining bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

[0044] The bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 2 was prepared using the same method as the bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 2. The bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 2 had an effective viable bacteria count of 3.25 × 10⁻⁶. 8 The product has a CFU / g content, a water content of 18.2%, an organic matter content of ≥40%, and a pH of 6.7.

[0045] Example 3 Application of bio-organic fertilizer containing rhizosphere growth-promoting bacteria in replacing part of the application of chemical fertilizers and achieving fertilizer reduction and efficiency improvement. Wheat was selected as the crop, and a two-factor completely randomized block design was adopted: 6 gradients of bio-organic fertilizer (0, 100, 200, 300, 400, 500 kg / mu) and 4 gradients of reduced chemical fertilizer application (0%, 15%, 30%, 45%), for a total of 24 treatments with 3 replicates. The planting area of ​​each treatment group was 1.5 × 1.5 m². 2 .

[0046] The conventional fertilizer is Stanley ternary compound fertilizer, with a standard application rate of 50 kg / mu; the bio-organic fertilizer containing rhizosphere growth-promoting bacteria was prepared using the preparation method of Example 1.

[0047] Table 1 shows the application rates of bio-organic fertilizer containing rhizosphere growth-promoting bacteria and ternary compound fertilizer in groups T01 to T24, as well as the reduction ratio of chemical fertilizer.

[0048] Table 1. Fertilizer composition and fertilizer reduction ratio for groups T01 to T24

[0049] Fertilization method: Before wheat sowing, apply a mixture of all bio-organic fertilizer containing rhizosphere growth-promoting bacteria and 50% by weight of NPK compound fertilizer evenly, then plow it into the soil. The remaining 50% by weight of NPK compound fertilizer is applied as topdressing during the wheat jointing stage. Rhizosphere soil samples are collected during the wheat grain-filling stage, and soil physicochemical properties are measured. The effects of applying bio-organic fertilizer containing rhizosphere growth-promoting bacteria in combination with reduced-volume fertilizer on the regulation of the physicochemical properties of wheat rhizosphere soil are compared and analyzed.

[0050] The results are as follows Figures 1-4As shown, with the increase in the application rate of bio-organic fertilizer containing rhizosphere-promoting bacteria, the soil pH gradually increases, and the contents of organic matter, total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, and available potassium continue to rise. When the application rate of bio-organic fertilizer containing rhizosphere-promoting bacteria reaches 400-500 kg / mu, reducing the application rate of ternary compound fertilizer by 0-45% has no significant negative impact on various physicochemical indicators of the soil. This indicates that the bio-organic fertilizer containing rhizosphere-promoting bacteria of this invention can effectively alleviate soil acidification, stably maintain soil fertility levels, and increase the application of bio-organic fertilizer containing rhizosphere-promoting bacteria can comprehensively activate the activity of enzymes related to carbon, nitrogen, and phosphorus cycles and antioxidant enzymes in the soil. When 400–500 kg / mu of bio-organic fertilizer containing rhizosphere growth-promoting bacteria is applied in combination with ternary compound fertilizer with a reduction of 0–30% by weight, the activities of β-glucosidase (S-β-GC), sucrase (S-SC), urease (S-UE), catalase (S-CAT), neutral phosphatase (S-NP), and nitrate reductase (S-NR) in the soil are maintained at a high level, resulting in excellent nutrient conversion function.

[0051] Two consecutive years of field trials were conducted, and yield components such as wheat plant height, number of spikes per unit area, number of spikelets per spike, number of grains per spike, and thousand-grain weight, as well as grain yield, were measured. The agronomic traits and yields of wheat in the first and second years are shown below. Figure 5 and 6 As shown, increased application of bio-organic fertilizer containing rhizosphere growth-promoting bacteria is the dominant positive factor for yield improvement, while reduced application of a single high-proportion ternary compound fertilizer inhibits wheat growth and development and has an adverse effect on yield formation. The response trends of each component factor are consistent: plant height increases significantly with increasing application of bio-organic fertilizer containing rhizosphere growth-promoting bacteria, and gradually decreases with increasing reduction in the proportion of ternary compound fertilizer. When the application rate of bio-organic fertilizer containing rhizosphere growth-promoting bacteria reaches 300 kg / mu or more, the difference in plant height between different fertilizer reduction ratios significantly narrows. The number of spikes per unit area, the number of spikelets per spike, and the number of grains per spike all steadily increase with increased application of bio-organic fertilizer, and show a decreasing trend with increasing fertilizer reduction ratio. A high application rate of 400–500 kg / mu of bio-organic fertilizer can effectively compensate for the insufficient number of spikes and limited spike development caused by reduced ternary compound fertilizer, stabilizing spike traits under a 15–30% fertilizer reduction ratio gradient. The thousand-grain weight shows a relatively small response range to fertilization patterns, and high application rates of bio-organic fertilizer containing rhizosphere growth-promoting bacteria can maintain grain plumpness.

[0052] Grain yield results showed that, under the conventional yield-increasing model, the combined application of 300–400 kg / mu of bio-organic fertilizer and 15–30% reduced-weight compound fertilizer resulted in a wheat yield increase of over 15% compared to the conventional fertilization treatment, exhibiting the best overall yield performance and representing the recommended application scheme in the field. Under the high-ratio fertilizer reduction model, the combined application of 400–500 kg / mu of bio-organic fertilizer and 45% reduced-weight compound fertilizer maintained a stable yield level, making it suitable for production scenarios with strict fertilizer reduction requirements.

[0053] Comparative Examples 1-2 The preparation methods of the bio-organic fertilizers containing rhizosphere growth-promoting bacteria in Comparative Examples 1 and 2 are basically the same as those in Example 1. The difference between the preparation methods of the bio-organic fertilizers containing rhizosphere growth-promoting bacteria in Comparative Examples 1 and 2 and those in Example 1 is as follows: No spore inducer was added in Comparative Example 1; No spore protectant was added in Comparative Example 2.

[0054] In Comparative Example 1, the number of effective viable bacteria in the bio-organic fertilizer containing rhizosphere growth-promoting bacteria was 2.57 × 10⁻⁶. 8 The bio-organic fertilizer from Comparative Example 1 had a CFU / g concentration, a moisture content of 18.5%, an organic matter content ≥40%, and a pH of 6.8. After being stored at room temperature for 6 months, the effective viable bacteria count of the fertilizer decreased to 1.25 × 10⁻⁶. 8 The CFU / g is below the national standard threshold, making long-term storage impossible. Compared to the preparation method of bio-organic fertilizer containing rhizosphere-promoting bacteria in Example 1, the preparation method of Comparative Example 1 lacks a spore inducer, resulting in a significant reduction in the spore formation rate of Bacillus belyss HP26. The bacteria mostly exist in vegetative form, exhibiting weak resistance and significant bacterial loss during fermentation. Consequently, the number of effective viable bacteria in the resulting bio-organic fertilizer containing rhizosphere-promoting bacteria is significantly lower than that in Example 1, and its shelf stability is extremely poor.

[0055] In Comparative Example 2, the number of effective viable bacteria in the bio-organic fertilizer containing rhizosphere growth-promoting bacteria was 3.16 × 10⁻⁶. 8 The bio-organic fertilizer from Comparative Example 2 had a CFU / g concentration, a moisture content of 18.4%, an organic matter content ≥40%, and a pH of 6.5. After being stored at room temperature for 6 months, the effective viable bacteria count of the fertilizer decreased to 1.83 × 10⁻⁶. 8 The CFU / g concentration indicates moderate stability. Compared to the preparation method of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Example 1, the preparation method of Comparative Example 1 lacks a spore protectant composed of trehalose, glycerol, and fulvic acid. High temperatures and dehydration stress during fermentation and drying cause significant spore inactivation. Furthermore, the microorganisms are susceptible to continuous decay due to environmental temperature and humidity fluctuations during storage, resulting in poor stability and failing to meet the requirements for commercial application.

[0056] Experimental Example 1 The effect of organic fertilizer carrier moisture content on the effective viable bacteria count of bio-organic fertilizer containing rhizosphere growth-promoting bacteria A single colony of *Bacillus belyssus* HP26 was picked and inoculated into 100 mL of NB liquid medium. After incubation at 30 ℃ for 12 h, a *Bacillus belyssus* HP26 seed culture was obtained. 1 mL of this seed culture was then inoculated into 100 mL of NB liquid medium and incubated at 30 ℃ for 12 h to obtain a *Bacillus belyssus* HP26 bacterial suspension. The viable count of the obtained *Bacillus belyssus* HP26 bacterial suspension was 1 × 10⁻⁶. 9 CFU / mL.

[0057] 1000 g of well-rotted pig manure was dried, crushed, and passed through a 20-mesh sieve. The moisture content was adjusted to 0%, 5%, 10%, 15%, 20%, 25%, and 30%, respectively. The manure was fermented at 35 ℃ for 7 days, with the pile turned over once a day. The number of viable Bacillus vesiculosus HP26 bacteria in the bio-organic fertilizer was measured daily. After fermentation, the manure was ventilated and dried until the moisture content was 18.7%, thus obtaining bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

[0058] The effective viable bacteria count change curve of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Experiment Example 1 during the fermentation process from 1 to 7 days is shown in the figure below. Figure 7 As shown in Figure A, the moisture content of the organic fertilizer carrier significantly affects the survival and reproduction of Bacillus belyssus HP26. When the moisture content of the organic fertilizer carrier is 15–20%, Bacillus belyssus HP26 grows slowly and has a low number of viable bacteria; when the moisture content of the organic fertilizer carrier is 25%, the number of Bacillus belyssus HP26 maintains a stable increase over 7 days, and the number of viable bacteria remains at 2.23 × 10⁻⁶. 8 CFU / g or higher; when the moisture content of the organic fertilizer carrier is ≥30%, the number of Bacillus belye HP26 increases rapidly in the early stage, but the rate of appearance slows down significantly in the later stage. Therefore, in the bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention, the optimal moisture content of the organic fertilizer carrier is 25%.

[0059] Experimental Example 2 The effect of amino acid hydrolysate dosage on the number of viable bacteria in bio-organic fertilizer containing rhizosphere growth-promoting bacteria. A single colony of *Bacillus belyssus* HP26 was picked and inoculated into 100 mL of NB liquid medium. After incubation at 30 ℃ for 12 h, a *Bacillus belyssus* HP26 seed culture was obtained. 1 mL of this seed culture was then inoculated into 100 mL of NB liquid medium and incubated at 30 ℃ for 12 h to obtain a *Bacillus belyssus* HP26 bacterial suspension. The viable count of the obtained *Bacillus belyssus* HP26 bacterial suspension was 1 × 10⁻⁶. 9 CFU / mL.

[0060] 1000 g of well-rotted pig manure was dried, crushed, and passed through a 20-mesh sieve. Then, 0 g, 50 g, 100 g, 150 g, 200 g, and 250 g of amino acid hydrolysate were added, respectively, so that the added mass of amino acid hydrolysate was 0%, 5%, 10%, 15%, 20%, and 25% of the dry weight of the well-rotted pig manure. Next, 100 mL of the obtained Bacillus vesicularis HP26 bacterial solution was added, and fermentation was carried out at 35 ℃ for 7 days. The pile was turned once a day, and the number of effective viable Bacillus vesicularis HP26 in the bio-organic fertilizer was measured daily. After fermentation, the fertilizer was ventilated and dried until the moisture content was 18.7%, thus obtaining bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

[0061] The effective viable bacteria count change curve of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Experiment Example 2 during the fermentation process from 1 to 7 days is shown in the figure below. Figure 7 As shown in Figure B, amino acid hydrolysate, as a nutrient additive, can significantly promote the survival and reproduction of Bacillus belyssus HP26. When the added mass of amino acid hydrolysate is 0-10% of the dry weight of the organic fertilizer carrier, nutrients are insufficient, the number of Bacillus belyssus HP26 increases slowly, and the effective viable count is less than 1.0 × 10⁻⁶. 8 CFU / g; When the added mass of amino acid hydrolysate is 20% of the dry weight of the organic fertilizer carrier, the effective viable count of Bacillus vesiculosus HP26 remains at a high level for 7 days, reaching 3.69 × 10⁻⁶. 8 CFU / g or higher; when the added mass of amino acid hydrolysate is not less than 25% of the dry weight of the organic fertilizer carrier, although the number of effective viable bacteria is high in the early stage, the low pH in the later stage inhibits the functional bacteria. Therefore, in the preparation method of bio-organic fertilizer containing rhizosphere growth-promoting bacteria of the present invention, the optimal added mass of amino acid hydrolysate is 20% of the dry weight of the organic fertilizer carrier.

[0062] Experimental Example 3 Shelf-life stability test of bio-organic fertilizer containing rhizosphere growth-promoting bacteria The bio-organic fertilizer containing rhizosphere growth-promoting bacteria from Example 1 was sealed and stored at room temperature. Samples were taken at 1, 2, 3, 4, 5, and 6 months, and the effective viable bacteria count was determined using the dilution plating method.

[0063] The results are as follows Figure 8 As shown, during the first three months of storage, the number of viable bacteria in the bio-organic fertilizer containing rhizosphere growth-promoting bacteria in Example 1 decreased slowly. Although the number of viable bacteria decreased significantly in the sixth month, it was still not less than 2 × 10⁻⁶. 8 The CFU / g value meets the national standards for bio-organic fertilizers, indicating that the bio-organic fertilizer containing rhizosphere growth-promoting bacteria of this invention has good shelf-life stability and can meet the needs of production, storage, and transportation.

Claims

1. A bio-organic fertilizer containing rhizosphere growth-promoting bacteria, characterized in that, The product includes an organic fertilizer carrier, a spore inducer, a spore protectant, an amino acid hydrolysate, and a Bacillus vesiculosus HP26 bacterial solution. Based on the dry weight of the organic fertilizer carrier, the spore inducer accounts for 0.1–0.2% of the dry weight of the organic fertilizer carrier, the Bacillus vesiculosus HP26 bacterial solution accounts for 5–10% of the dry weight of the organic fertilizer carrier, the spore protectant accounts for 5–10% of the dry weight of the organic fertilizer carrier, and the amino acid hydrolysate accounts for 15–20% of the dry weight of the organic fertilizer carrier.

2. The bio-organic fertilizer containing rhizosphere growth-promoting bacteria according to claim 1, characterized in that, The organic fertilizer carrier includes well-rotted pig manure; the moisture content of the organic fertilizer carrier is 25-30%.

3. The bio-organic fertilizer containing rhizosphere growth-promoting bacteria according to claim 1, characterized in that, The spore inducer is a manganese sulfate solution, and the mass fraction of the manganese sulfate solution is 5-10%.

4. The bio-organic fertilizer containing rhizosphere growth-promoting bacteria according to claim 1, characterized in that, The spore protectant includes trehalose, glycerol and fulvic acid, wherein the mass ratio of trehalose to glycerol is (0.8-1.2):1 and the mass ratio of trehalose to fulvic acid is 1:(2.5-3.5).

5. The bio-organic fertilizer containing rhizosphere growth-promoting bacteria according to claim 1, characterized in that, The amino acid hydrolysate has a nitrogen content of 13–18 g / L and a pH of 1.8–2.

0.

6. A method for preparing a bio-organic fertilizer containing rhizosphere growth-promoting bacteria as described in any one of claims 1 to 5, characterized in that, The process includes the following steps: adding the prescribed amount of organic fertilizer carrier to the prescribed amount of amino acid hydrolysate, adjusting the pH to 5.5-6.5, then adding the prescribed amount of Bacillus baileyi HP26 bacterial solution, spore inducer, and spore protectant, followed by fermentation and drying to obtain a bio-organic fertilizer containing rhizosphere growth-promoting bacteria.

7. The method for preparing bio-organic fertilizer containing rhizosphere growth-promoting bacteria according to claim 6, characterized in that, The fermentation process involves standing at 30–35 °C for 5–7 days, with the pile turned over once a day.

8. The application of a bio-organic fertilizer containing rhizosphere growth-promoting bacteria as described in any one of claims 1 to 5 in replacing 15-45% by weight of compound fertilizer, characterized in that, The application rate of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria is 300–500 kg / mu.

9. The application of the bio-organic fertilizer containing rhizosphere growth-promoting bacteria according to claim 8, characterized in that, The bio-organic fertilizer containing rhizosphere growth-promoting bacteria is mixed with 50% by weight of compound fertilizer and applied as a base fertilizer before wheat sowing. The remaining 50% by weight of compound fertilizer is applied as a top dressing during the wheat jointing stage.

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