Preparation method and application of novel liquid compound microbial fertilizer

Abstract

The invention provides a preparation method and application of a novel liquid compound microbial fertilizer, and belongs to the technical field of microbial fermentation. The preparation method of the liquid compound microbial fertilizer comprises the following steps: preparation of a liquid fertilizer, preparation of a compound functional bacterium seed solution and preparation of the liquid compound microbial fertilizer. The liquid compound microbial fertilizer provided by the invention not only can ensure the yield increase of solanaceous vegetables and reduce the occurrence of soil-borne diseases, but also can reduce the use amount of chemical fertilizers and chemical pesticides and reduce the cost; the soil aggregate structure can be improved, the physical and chemical properties of the soil are enhanced, and the soil fertility is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a novel liquid composite microbial fertilizer and a preparation method thereof, as well as application thereof on solanaceous vegetables. Background Art

[0002] Fertilizers are crucial for increasing crop yields. Long-term overuse of chemical fertilizers can lead to a series of problems, including soil compaction, soil fertility decline, and ecological degradation. Microbial fertilizers can improve soil fertility, increase fertilizer utilization, and protect the farmland environment, providing crucial support for sustainable agricultural production.

[0003] Bacillus spores are used in the development of microbial fertilizers and possess the following characteristics: 1) They produce a variety of physiologically active substances that stimulate and regulate plant growth; 2) They produce various organic and inorganic acids, promoting plant absorption of nutrients such as nitrogen, phosphorus, and potassium; 3) They produce iron carriers or iron chelates, antibiotics, and various enzymes to inhibit plant diseases and pests; and 4) The spores they produce are heat-resistant and stress-resistant, facilitating the production, processing, storage, transportation, and survival of microbial agents in the environment. Therefore, Bacillus spores have important application value in agricultural production, and the technology for their use in the development of microbial fertilizers is mature.

[0004] Solanaceous vegetables, primarily including tomatoes, eggplants, and peppers, are important vegetables grown on a large scale. They are high-yield crops requiring significantly higher levels of nitrogen, phosphorus, and potassium than grasses. Each crop removes significant amounts of these nutrients. Without timely replenishment, soil nutrients inevitably become unbalanced, leading to a decline in fertility. Furthermore, Solanaceous vegetable production is often plagued by pests and diseases, with soil-borne bacterial wilt and fusarium wilt being particularly severe and difficult to control. Research has shown that inoculating beneficial bacteria into the vegetable growing medium or rhizosphere can effectively control pests and diseases.

[0005] Liquid fertilizers and their application techniques meet the requirements of "low carbon, energy conservation, high efficiency, and environmental protection." Their nutrients are more easily absorbed by crops, and their absorption and utilization rates are high, making them crucial for reducing chemical fertilizer inputs and mitigating the risk of nitrate contamination in groundwater. The development of liquid compound microbial fertilizers not only increases yields but also improves soil and crop quality, prevents pests and diseases, reduces the use of chemical fertilizers and pesticides, and protects the ecological environment. They have become an important fertilizer source for pollution-free vegetable production. However, key technologies such as fertilizer cost, variety selection, and the scientific determination of nutrient ratios are currently the main factors restricting the further development of modern water-fertilizer coupling irrigation and fertigation technology for greenhouse vegetables. Therefore, developing liquid compound microbial fertilizers with reasonable nutrient ratios, significant yield and efficiency increases, and reduced costs is a prerequisite for implementing integrated water and fertilizer management, achieving controlled fertilizer dosage, and improving fertilizer utilization.

[0006] Based on research and development both domestically and internationally, a successful fertilizer product should demonstrate two key characteristics: first, a formula tailored to the nutritional needs of crop growth, carefully selected raw materials and production processes, and a water solubility of generally above 98%. Second, costs should be effectively controlled, prices reasonable, and the input-output ratio high in the area of ​​use. With the intensification and scale of agriculture and the expansion of drip irrigation and water-saving facilities, water-soluble fertilizers are gaining increasing attention and offer promising development prospects.

[0007] Giant fungus grass (Cenchrus fungigraminus) is a perennial grass of the genus Pennisetum in the Poaceae family. It is a typical C4 plant, with a yield of over 20 tons per mu. It grows rapidly, produces a large biomass, and in the later stages of growth, it reaches a high degree of lignification, with long fibers and high tensile strength, making it an excellent material for board making. Currently, giant fungus grass has been successfully used to produce fungus grass particleboard, fungus grass fiberboard, and fungus grass composite board. However, the fungus grass board-making process produces a large amount of juice. If treated as wastewater and discharged in compliance with standards, the cost is extremely high, seriously affecting the profitability of giant fungus grass board production and hindering the development of the fungus grass industry. Therefore, scientific research on the resourceful utilization of giant fungus grass juice is urgently needed. Previous studies have found that Juncao juice contains 9.19% protein, 7.1% total sugar, 4.03% total amino acids, 15.9mg / kg zinc, 6.2mg / kg manganese, and 32.7mg / kg iron. This indicates that Juncao juice is rich in protein, sugars, amino acids, and various inorganic salts, making it suitable for use as a microbial fermentation medium. Further research has shown that Juncao juice significantly promotes the growth of nightshade vegetables. Summary of the Invention

[0008] In order to improve fertilizer utilization and solve the problem of resource utilization of Juncao juice, the present invention provides a method for producing liquid composite microbial fertilizer using giant Juncao juice, a new agricultural by-product, as the main raw material.

[0009] In order to achieve the above objectives, the inventors provide the following technical solutions:

[0010] A method for preparing a novel liquid compound microbial fertilizer comprises the following steps:

[0011] (1) Preparation of liquid fertilizer: Add 150-250 g / L urea, 40-60 g / L potassium dihydrogen phosphate, 10-30 g / L potassium sulfate, 0.004-0.04 g / L lime, 0.3-0.06 g / L ferrous sulfate, 0.1-0.02 g / L zinc sulfate, 0.01-0.05 g / L manganese sulfate and 0.002-0.02 g / L magnesium sulfate to the waste liquid of giant fungus grass juice, stir well and adjust the pH value to 6.5-7.0;

[0012] (2) Preparation of composite functional bacteria seed solution: The biocontrol bacteria Bacillus licheniformis, the phosphate-solubilizing and nitrogen-fixing bacteria Bacillus subtilis, and the high-yielding indoleacetic acid (IAA) bacteria Bacillus siamese were activated on nutrient agar (NA) plates and inoculated into nutrient broth (NB) liquid culture medium. The culture was cultured at 28-32°C and 150-200 r / min for 20-28 h, and the bacterial solution was diluted to (0.8-1.2)×10 9 cfu / mL; the fermentation broth of Bacillus licheniformis, Bacillus subtilis and Bacillus siamensis was mixed in a volume ratio of 1:(1-3):1 and stored at 2-8°C for later use;

[0013] (3) Preparation of liquid composite microbial fertilizer: Using the liquid fertilizer prepared above as a fermentation medium, the above composite functional bacteria seed liquid is inoculated into the liquid fertilizer at an inoculum rate of 5-10%, and fermented at 28-32°C for 45-50 hours. A preservative is added to the fermentation liquid to finally form a liquid composite microbial fertilizer.

[0014] Furthermore, the Bacillus licheniformis strain is Bacillus licheniformis strain FJAT-41480, the Bacillus subtilis strain is Bacillus subtilis strain FJAT-41479, and the Bacillus siamensis strain is Bacillus siamensis strain FJAT-41481. The three strains were deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with the deposit numbers CGMCC No. 33777, CGMCC No. 33776 and CGMCC No. 33778, respectively, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] Furthermore, the preservative component includes potassium sorbate 0.01-0.5 g / mL, sodium dehydroacetate 0.01-0.5 g / mL and sodium diacetate 0.005-0.01 g / mL.

[0016] Preferably, the preparation method of the novel liquid composite microbial fertilizer comprises the following steps:

[0017] (1) Preparation of liquid fertilizer of giant fungus grass juice: 150 g / L urea, 50 g / L potassium dihydrogen phosphate, 30 g / L potassium sulfate, 0.004 g / L lime, 0.3 g / L ferrous sulfate, 0.02 g / L zinc sulfate, 0.01 g / L manganese sulfate and 0.002 g / L magnesium sulfate were added to the giant fungus grass juice waste liquid, stirred thoroughly, and the pH value was adjusted to 7.0.

[0018] (2) Preparation of composite functional bacteria seed solution: Bacillus licheniformis strain FJAT-41480, Bacillus subtilis strain FJAT-41479 and Bacillus siamese strain FJAT-41481 were activated on nutrient agar plates and inoculated into nutrient broth liquid culture medium. The culture was cultured at 30°C and 180 rpm for 2 days. The bacterial solution was diluted to 1×10 9 cfu / mL. The fermentation broths of strains FJAT-41480, FJAT-41479, and FJAT-41481 were mixed in a volume ratio of 1:2:1 and stored at 4°C for later use.

[0019] (3) Preparation of liquid composite microbial fertilizer: The prepared giant fungus grass juice liquid fertilizer was used as a fermentation medium, and the composite functional bacteria seed liquid was inoculated into the giant fungus grass juice liquid fertilizer at a 10% inoculation rate. The fermentation was carried out at 30°C for 2 days. Preservatives such as potassium sorbate 0.5 g / mL, sodium dehydroacetate 0.5 g / mL, and sodium diacetate 0.01 g / mL were added to the fermentation liquid as preservatives to finally form the liquid composite microbial fertilizer.

[0020] Furthermore, the liquid compound microbial fertilizer is used to promote the germination of nightshade vegetable seeds. The application method is to soak the nightshade vegetable seeds in a 300-800 times diluted liquid of the liquid compound microbial fertilizer for 6-8 hours, wash them, and place them in a germination box at 28-30°C for germination.

[0021] Furthermore, the liquid compound microbial fertilizer is used to promote the growth of Solanaceae plants and prevent soil-borne diseases. The application method is to use a 300-fold diluted solution of the liquid compound microbial fertilizer as a colonization water once when transplanting, 0.5-1 catties per plant, and then irrigate the roots with the liquid compound microbial fertilizer once every 15 days, and continue 2-3 times to consolidate the effect.

[0022] Furthermore, the effective viable bacteria count of the liquid composite microbial fertilizer is 7.3-9.7 billion cfu / mL, and the nutrient (N+P2O5+K2O) content is 20.47%.

[0023] Furthermore, the liquid compound microbial fertilizer is used in soil microecological regulation. The application method is to spray the liquid compound microbial fertilizer on the soil surface at a rate of 80-100 catties / mu during land preparation before crop transplanting, and then plow the soil to effectively improve the soil microecological environment.

[0024] Furthermore, the solanaceous vegetables include tomatoes, peppers or eggplants.

[0025] The present invention has the following advantages:

[0026] (1) The present invention utilizes the waste liquid of giant fungus grass juice, an agricultural by-product, as a resource and uses it as one of the main components of liquid composite microbial fertilizer. This reduces production costs while solving the environmental pollution caused by agricultural by-product waste and achieving sustainable agricultural development.

[0027] (2) The strain of the present invention is a composite strain composed of Bacillus licheniformis FJAT-41480 with broad-spectrum antibacterial activity, Bacillus subtilis FJAT-41479 with phosphorus solubilization and nitrogen fixation properties, and Bacillus siamensis FJAT-41481 with high indoleacetic acid production, which are compounded based on the dose-effect relationship and growth synergy, thereby overcoming the limitations of single strains such as single function, poor environmental adaptability, weak stress resistance, and low nutrient utilization.

[0028] (3) The liquid composite microbial fertilizer provided by the present invention has various components that work synergistically, can effectively improve soil structure, increase soil fertility, enhance soil microbial activity, and enable crops to develop systemic resistance. It can not only reduce the occurrence of soil-borne diseases and ensure increased yields of Solanaceae vegetables, but also reduce the use of chemical fertilizers and chemical pesticides, achieve "fertilizer control and pesticide reduction", protect the ecological environment, and promote sustainable agricultural development.

[0029] (4) The liquid compound microbial fertilizer has a high content of live bacteria, reaching 7.3-9.7 billion cfu / mL, and a nutrient (N+P2O5+K2O) content of 20.47%. Both the bacterial content and nutrient content exceed the national agricultural industry standard for compound microbial fertilizers (NY / T 798-2015). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Identification of growth-promoting and disease-preventing strains described in the specific embodiments. A: Phylogenetic tree of strains FJAT-41479, FJAT-41480, and FJAT-41481 constructed based on 16S rRNA sequences; B, C, and D are the colony morphologies of strains FJAT-41479, FJAT-41480, and FJAT-41481, respectively.

[0031] Figure 2 Figure 1 shows the mixed culture of strains FJAT-41479, FJAT-41480, and FJAT-41481 (A) and the effect of their fermentation liquid on the growth promotion of potted tomato seedlings (B). In Figure A, 1-3 are strains FJAT-41479, FJAT-41480, and FJAT-41481, respectively.

[0032] Figure 3 This is a diagram of the separation of Bacillus sp. in the liquid composite microbial fertilizer according to the embodiment. In the diagram, A, B, and C are random samples.

[0033] Figure 4This is the change in diversity index of rhizosphere microbial community of tomatoes treated with liquid composite microbial fertilizer according to the specific embodiment. In the figure, "*" indicates significant difference, "**" indicates extremely significant difference, and "ns" indicates no significant difference.

[0034] Figure 5 Effects of liquid compound microbial fertilizer treatment on the relative abundance of phyla (A) and genera (B) in tomato rhizosphere soil as described in the specific embodiment.

[0035] Figure 6 This is the principal component analysis of tomato rhizosphere soil samples from the liquid composite microbial fertilizer treatment group and the control group described in the specific implementation manner.

[0036] Figure 7 This is a redundancy analysis of the composition of tomato rhizosphere soil bacterial communities (genus level) and the incidence of bacterial wilt and soil physical and chemical properties as described in the specific implementation method.

[0037] Figure 8 This is a specific implementation method of the liquid compound microbial fertilizer production process and field application effect. DETAILED DESCRIPTION

[0038] To illustrate the technical content, purpose and effect of the technical solution in detail, the following is a detailed description of the specific embodiments and the accompanying drawings. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial channels.

[0039] Example 1: Preparation of liquid compound microbial fertilizer

[0040] 1. Screening of Bacillus spores with growth-promoting and disease-preventing functions

[0041] Screening of Phosphate-Solubilizing and Nitrogen-Fixing Bacillus: Using the molybdenum-antimony anti-spectrophotometric method, five Bacillus strains with the ability to degrade both inorganic and organic phosphorus were screened from our laboratory's Bacillus library. These strains are numbered FJAT-41479, FJAT-41481, FJAT-14464, FJAT-47648, and FJAT-49379. Strain FJAT-41479 showed the strongest degradation of both inorganic and organic phosphorus, with phosphorus increments of 251.48 mg / L and 19.87 mg / L, respectively (Table 1). Furthermore, strain FJAT-41479 exhibited a high nitrogen fixation capacity of 25.72 mg / g (Table 2). Thus, we identified FJAT-41479, a Bacillus strain with excellent phosphate-solubilizing and nitrogen-fixing properties.

[0042] Table 1 Determination of the ability of Bacillus to degrade inorganic phosphorus and organic phosphorus

[0043]

[0044] Table 2 Determination of nitrogen fixation efficiency of Bacillus

[0045]

[0046] Screening of high-yielding indoleacetic acid (IAA) Bacillus: Using the Salkowski colorimetric method, a high-yielding IAA Bacillus strain FJAT-41481 was screened from the Bacillus resource library of our laboratory, with an IAA yield of 19.27 mg / L (Table 3).

[0047] Table 3 Determination of IAA secretion ability of Bacillus

[0048]

[0049] Screening of soil-borne biocontrol agents for bacterial wilt and fusarium wilt: Using Ralstonia solanacearum and Fusarium oxysporum as target bacteria, a strain of Bacillus sp., FJAT-41480, was identified with strong antagonism against both pathogens. Its inhibition diameters against Ralstonia solanacearum and Fusarium oxysporum were 18.32 mm and 21.55 mm, respectively (Table 4). When Bacillus sp. FJAT-41480 was pre-inoculated into potted tomato seedlings and then inoculated three days later with pathogenic Ralstonia solanacearum or Fusarium oxysporum, the strain achieved 81.64% and 82.78% efficacy against bacterial wilt and fusarium wilt, respectively (Table 5).

[0050] Table 4 Antibacterial activity of different Bacillus against Fusarium oxysporum and Ralstonia solanacearum

[0051]

[0052] Table 5 Control effect of strain FJAT-41480 on tomato bacterial wilt and fusarium wilt

[0053]

[0054]

[0055] Identification of growth-promoting and disease-preventing strains: Based on colony morphology and 16S rRNA gene sequence identification, the phosphate-solubilizing and nitrogen-fixing strain FJAT-41479, the high-yielding IAA strain FJAT-41481, and the biocontrol strain FJAT-41480 were Bacillus subtilis, Bacillus siamensis, and Bacillus licheniformis, respectively. Figure 1 ).

[0056] Compatibility study of growth-promoting and disease-preventing strains: strains FJAT-41479, FJAT-41480, and FJAT-41481 were mixed and cultured on LB plates by "well" marking. The results showed that the colonies at the intersection of the strains grew well and there was no mutual inhibition. Figure 2 A), indicating that there is no antagonism between the three functional Bacillus strains and they can be mixed for fermentation. Furthermore, the three strains were inoculated into LB liquid medium in equal volumes for mixed fermentation. It was found that the number of viable bacteria was significantly greater than that of single strain fermentation culture (Table 6). When inoculated into potted tomato seedlings, the mixed fermentation liquid had a significantly greater effect on promoting plant growth than the single strain fermentation liquid ( Figure 2 B).

[0057] Table 6 Number of viable bacteria in separate and mixed cultures of strains FJAT-41479, FJAT-41480 and FJAT-41481

[0058]

[0059] Note: The data in the table are mean ± standard deviation. Different lowercase letters in the same case indicate significant differences at the P < 0.05 level after LSD test.

[0060] Three functional Bacillus strains were screened, namely Bacillus licheniformis strain FJAT-41480, which has antibacterial activity against soil-borne bacterial wilt and Fusarium oxysporum; Bacillus subtilis strain FJAT-41479, which has phosphate-solubilizing and nitrogen-fixing properties; and Bacillus siamensis strain FJAT-41481, which produces high indoleacetic acid. These three strains are compatible and functionally complementary, and are all safe strains that comply with the General Technical Guidelines for Biosafety of Microbial Fertilizers (NY 1109-2006). The three strains can be combined to construct a composite strain that can be used in the research and development of microbial fertilizers.

[0061] 2. Determination of nutritional components of giant grass juice and its application in microbial fermentation

[0062] Giant Juncao juice is nutrient-rich, with total protein content reaching 9.19%, total sugar content reaching 7.1%, total amino acid content reaching 4.03%, zinc content reaching 15.9 mg / kg, manganese content reaching 6.2 mg / kg, and iron content reaching 32.7 mg / kg (Table 7), showing potential for development into liquid fertilizer. Fermentations of giant Juncao juice with Bacillus licheniformis, Bacillus subtilis, Bacillus siamese, Bacillus velezensis, and Bacillus amyloliquefaciens were performed (Table 8). The viable cell counts in the fermentation products were all greater than those in the control Bacillus fermentation cultured in LB medium, demonstrating the feasibility of using Juncao juice for functional Bacillus fermentation.

[0063] Table 7 Nutrients and amino acids detected in giant fungus grass juice

[0064]

[0065] Table 8 Statistics of viable bacteria counts of giant grass juice fermentation Bacillus

[0066]

[0067] Note: The data in the table are mean ± standard deviation. Different lowercase letters in the same case indicate significant differences at the P < 0.05 level after LSD test.

[0068] 3. Orthogonal optimization of adding macroelements to giant fungus grass juice

[0069] Liquid fertilizer was prepared by adding appropriate amounts of nitrogen (urea), phosphorus (potassium dihydrogen phosphate), and potassium (potassium sulfate) to giant fungus grass juice through an orthogonal experiment. The factor level settings are shown in Table 9, and the orthogonal experiment settings are shown in Table 10. Each nutrient was added to 1 L of giant fungus grass juice according to the designed formula ratio. Heat and stir until completely dissolved. After cooling to room temperature, store at room temperature until ready for use.

[0070] Table 9 Factor levels of orthogonal experimental design for macroelement ratios

[0071]

[0072] Table 10 Orthogonal experimental design of macroelement ratio

[0073]

[0074] Plant growth determination: Tomato was used as the test object, and 30-day-old tomato (pink crown variety) potted seedlings with consistent growth were selected. The liquid fertilizer with different treatment levels was diluted 300 times and then watered with tomato seedlings, 100 mL / pot. Commercial compound microbial fertilizer (purchased from Hebei Qianbaiji Agricultural Technology Co., Ltd.) was used as the positive control (CK + ), with water as negative control (CK - ), with 3 replicates per treatment and 15 days of treatment, the plant height, stem diameter, and total leaf number were measured.

[0075] The results are shown in Table 11. The plant height growth of treatment levels 1, 3, 7 and 8 was greater than that of CK. + and CK - ; The stem diameter growth of treatment levels 4, 7, 8 and 9 was greater than that of CK + and CK - The total leaf number growth of treatment levels 6, 7, 8 and 9 was greater than that of CK + and CK -Taking into account the plant height, stem diameter and total leaf number, treatment level 8 had the best effect, so the addition amount of macroelements was determined to be 150 g / L urea, 50 g / L potassium dihydrogen phosphate and 30 g / L potassium sulfate.

[0076] Table 11 Plant growth determination of different ratios of macroelements in giant fungus grass juice liquid fertilizer

[0077]

[0078] 4. Orthogonal optimization of adding trace elements to giant grass juice

[0079] Solanaceous vegetables need trace elements including calcium, magnesium, iron, manganese, zinc, etc., with calcium carbonate (calcium), magnesium sulfate (magnesium), zinc sulfate (zinc), manganese sulfate (manganese), ferrous sulfate (iron) as the influencing factors, using L18 (5 3 ) orthogonal table for a 5-factor, 3-level orthogonal experiment. The factor level settings are shown in Table 12, and the orthogonal experiment settings are shown in Table 13. Add each micronutrient according to the designed formula ratio to 1 L of giant fungus grass juice fertilizer (containing 150 g / L urea, 50 g / L potassium dihydrogen phosphate, and 30 g / L potassium sulfate). Heat and stir until completely dissolved. After cooling to room temperature, store at room temperature until ready for use.

[0080] Table 12 Orthogonal levels of trace elements

[0081]

[0082] Table 13 Orthogonal experimental design of trace elements

[0083]

[0084] Plant growth determination: Tomato was used as the test object, and 30-day-old tomato (pink crown variety) potted seedlings with consistent growth were selected. The liquid fertilizer with different treatment levels was diluted 300 times and then watered with tomato seedlings, 100 mL / pot. Commercial compound microbial fertilizer (purchased from Hebei Qianbaiji Agricultural Technology Co., Ltd.) was used as the positive control (CK + ), with water as blank control (CK - ), with 3 replicates per treatment. Fifteen days after treatment, the plant height, stem diameter, and total leaf number of tomatoes were counted.

[0085] The results are shown in Table 14. The plant height increases of treatment levels 3, 8, 9, 10, 11, 12, 14, 15, 16, 17 and 18 were all greater than those of the positive and blank controls, among which treatment 12 had the largest plant height increase of 41.96%; the stem diameter increases of treatment levels 1, 2, 4, 5, 7, 8, 9, 10, 12, 13, 15, 16, 17 and 18 were all greater than those of the positive and blank controls, among which treatment 7 had the largest increase of 19.55%, followed by treatment 12 with a stem diameter increase of 17.58%; except for treatments 1, 4 and 8, the total leaf number increases of tomato in other treatments were greater than those of the positive and blank controls, among which treatment 12 had the largest increase of 22.61%. Considering the plant height, stem diameter and total leaf number indicators, treatment level 12 had the best effect. Therefore, the addition amount of trace elements was determined to be 0.004g / L lime (calcium element), 0.3g / L ferrous sulfate (iron element), 0.02g / L zinc sulfate (zinc element), 0.01g / L manganese sulfate (manganese element) and 0.002g / L magnesium sulfate (magnesium element).

[0086] Table 14 Plant growth determination in different proportions of trace elements in giant fungus grass juice liquid fertilizer

[0087]

[0088]

[0089] 5. Screening of the ratio of growth-promoting and disease-preventing functional bacteria in liquid compound microbial fertilizers

[0090] Preparation of composite functional bacteria seed solution: The Bacillus licheniformis strain FJAT-41480, Bacillus subtilis strain FJAT-41479 and Bacillus siamensis strain FJAT-41481 were activated on nutrient agar plates and inoculated into nutrient broth liquid culture medium. The culture was cultured at 30°C and 180 rpm for 24 h. Each bacterial solution was diluted to 1.0 × 10 9 The seed solutions of FJAT-41480, FJAT-41479, and FJAT-41481 were mixed at different ratios of 1:1:1 (MIX1), 1:2:1 (MIX2), 1:1:2 (MIX3), and 2:1:1 (MIX4) to prepare seed solutions of composite functional bacteria with different ratios.

[0091] Preparation of liquid composite microbial fertilizer: The seed liquid of the above-mentioned single strain and composite strains with different ratios were inoculated into giant grass juice liquid fertilizer (containing 150 g / L urea, 50 g / L potassium dihydrogen phosphate, 30 g / L potassium sulfate, 0.004 g / L lime, 0.3 g / L ferrous sulfate, 0.02 g / L zinc sulfate, 0.01 g / L manganese sulfate and 0.002 g / L magnesium sulfate) at a 10% inoculation rate, and cultured at 30°C and 180 rpm for 2 days to obtain giant grass juice liquid single and composite microbial fertilizers, respectively.

[0092] Statistics of viable bacteria: dilute the above-mentioned single and compound microbial fertilizers in a 10-fold series gradient, apply them to NA culture medium, and culture at 30℃ for 2 days. Count the number of colonies on the NA plate and calculate the number of viable bacteria in different ratios of giant grass juice single and compound microbial fertilizers.

[0093] Growth promotion test: The above single and compound microbial fertilizers were diluted 300 times and inoculated into 30-day-old leaf tomatoes (Jinshiwang No. 1, purchased from Xiamen Ruyi Seedlings High-Tech Co., Ltd.) at 100 mL / pot. A commercial compound microbial fertilizer (purchased from Hebei Qianbaiji Agricultural Technology Co., Ltd.) was used as a positive control (CK + ), with water as blank control (CK - ), 15 days after treatment, the plant height, stem diameter and total leaf number of tomatoes were counted.

[0094] Disease prevention test: Taking the control effect of liquid compound microbial fertilizer on soil-borne bacterial wilt of tomatoes as an example, the above-mentioned single and compound microbial fertilizers were diluted 300 times and pre-inoculated into 30-day-old tomatoes. Three days later, the highly pathogenic strain FJAT-91 of the bacterial wilt pathogen was inoculated. The commercial compound microbial fertilizer (purchased from Hebei Qianbaiji Agricultural Technology Co., Ltd.) was pre-treated and then inoculated with FJAT-91 three days later as a positive control. The plant was pre-treated with clean water for three days and then inoculated with FJAT-91 as a blank control. The disease condition of the plants was observed every day after inoculation, and the incidence rate and prevention effect were calculated.

[0095] The results showed that the number of viable bacteria in the composite microbial fertilizers with different ratios was higher than that in the microbial fertilizers prepared with a single strain. Among the composite microbial fertilizers, MIX2, which was prepared by mixing strains FJAT-41480, FJAT-41479, and FJAT-41481 in a ratio of 1:2:1 and then inoculating, had the highest number of viable bacteria, at 7.25×10 9cfu / mL (Table 15). Compared with the water control, both single and compound microbial fertilizers containing giant grass juice promoted the growth of tomato plants. The increases in plant height, stem diameter, and leaf number of tomato plants using the microbial fertilizers containing the single strain FJAT-47169 and the compound microbial fertilizers were greater than those using the positive control. MIX2 had the largest increase, increasing plant height, stem diameter, and total leaf number by 42.73%, 18.62%, and 218.17%, respectively (Table 16). Ten days after inoculation with the highly pathogenic strain FJAT-91 of Ralstonia solanacearum, the blank control group experienced a 100% disease incidence rate. Inoculations with FJAT-41480, FJAT-41479, FJAT-41481, MIX1, MIX2, MIX3, and MIX4 resulted in disease incidence rates of 34.65%, 69.52%, 71.84%, 32.58%, 30.84%, 35.85%, and 33.84%, respectively, with control efficacies of 65.35%, 30.48%, 28.16%, 67.42%, 69.16%, 64.15%, and 66.16%, respectively (Table 17). This indicates that different compound microbial fertilizer ratios all improved control efficacy, with MIX2 showing the best results, reaching 69.16%.

[0096] Table 15 Live bacteria count of single and compound microbial fertilizers

[0097]

[0098] Table 16 Effect of compound microbial fertilizer on tomato growth promotion

[0099]

[0100]

[0101] Table 17 Disease prevention effect of compound microbial fertilizer on tomato bacterial wilt

[0102]

[0103] 6. Optimization of fermentation conditions for growth-promoting and disease-preventing functional bacteria in liquid compound microbial fertilizers

[0104] In view of the four influencing factors of inoculation volume, pH value, temperature and fermentation time, the effective viable bacteria count was used as the investigation index. 4 ) Orthogonal table was used to conduct a four-factor three-level orthogonal experiment to optimize the fermentation conditions of the growth-promoting and disease-preventing composite bacteria. The factor level settings are shown in Table 18, and the orthogonal experiment settings are shown in Table 19. The effective live bacteria counts of composite functional bacteria in different orthogonal optimization combinations were counted.

[0105] Table 18 Factor level design of orthogonal experiment for optimization of fermentation conditions of composite functional strains

[0106]

[0107] Table 19 Orthogonal experimental design for optimization of solid-state fermentation conditions

[0108]

[0109] The results are shown in Table 20. For treatment level 9, the inoculation amount was 10%, the initial pH value was 7, the fermentation temperature was 30°C, and the fermentation time was 2 days. The effective viable bacteria count was the largest, which was 9.70×10 9 cfu / L, which was significantly different from the other treatment levels (P<0.01); followed by treatment levels 4 and 7, where the effective viable counts were 2.83×10 9 cfu / L and 3.22×10 9 cfu / L; treatment level 1, the effective viable bacteria count was the lowest, which was 1.17×10 8 cfu / L. Therefore, the optimal fermentation conditions for the composite functional bacteria were an inoculum size of 10%, an initial pH of 7, a fermentation temperature of 30°C, and a fermentation time of 2 days.

[0110] Table 20 Results of orthogonal test for optimization of solid fermentation conditions

[0111]

[0112]

[0113] Note: The data in the table are mean ± standard deviation. Different capital letters in the same case indicate significant differences at the P < 0.01 level after LSD test.

[0114] In summary, the optimal formula of liquid compound microbial fertilizer is to add 150g / L urea, 50g / L potassium dihydrogen phosphate, 30g / L potassium sulfate, 0.004g / L lime, 0.3g / L ferrous sulfate, 0.02g / L zinc sulfate, 0.01g / L manganese sulfate and 0.002g / L magnesium sulfate to giant fungus grass juice, with the ratio of FJAT-41480:FJAT-41479:FJAT-41481 being 1:2:1, the inoculum size being 10%, the pH value being 7.0, the fermentation temperature being 30℃ and the fermentation time being 2d.

[0115] 7. Screening of preservatives in liquid compound microbial fertilizers

[0116] After activation on nutrient agar (NA) plates, FJAT-41480, FJAT-41479, and FJAT-41481 were inoculated into nutrient broth (NB) liquid culture medium and cultured at 30°C and 180 rpm for 24 h. Each bacterial solution was diluted to 1.0×10 9CFU / mL, mixed at a ratio of 1:2:1, and inoculated with giant fungus grass juice liquid fertilizer (giant fungus grass juice contains 150g / L urea, 50g / L potassium dihydrogen phosphate, 30g / L potassium sulfate, 0.004g / L lime, 0.3g / L ferrous sulfate, 0.02g / L zinc sulfate, 0.01g / L manganese sulfate and 0.002g / L magnesium sulfate). The culture was shaken at 30°C and 180rpm for 2 days. The fermentation broth was used as a liquid compound microbial fertilizer. Through orthogonal experiments, the optimal proportion of compound preservatives was added to the liquid compound microbial fertilizer. Advantages of compound preservatives: On the one hand, they can overcome the defect of a single preservative having a narrow antibacterial spectrum; on the other hand, the combined use of preservatives can enhance the preservation effect and reduce production costs. A three-factor three-level orthogonal experiment was conducted with potassium sorbate, sodium dehydroacetate and sodium diacetate as influencing factors. The orthogonal experiment settings are shown in Table 21. Samples were taken at room temperature for 7 days, 30 days, 90 days and one year, and the number of viable bacteria in the liquid compound microbial fertilizer was counted for different orthogonal optimization combinations.

[0117] The results showed that, except for treatment levels 3 and 6, the bacterial counts of the other treatment levels and the control group changed significantly with storage time (P<0.05). The viable bacterial counts of the four control groups after 90 days of storage were significantly lower than the initial bacterial count (day 0). The bacterial counts of treatment levels 1, 2, 4, and 8 showed a trend of first increasing and then decreasing with storage time, while the bacterial count of treatment level 9 showed a significant downward trend with storage time. The bacterial counts of treatment levels 3 and 6 remained relatively stable with storage time and did not change significantly. Combined with cost accounting, the optimal preservative addition amounts (final concentrations) were determined to be 0.5 g / mL potassium sorbate, 0.5 g / mL sodium dehydroacetate, and 0.01 g / mL sodium diacetate, respectively.

[0118] Table 21 Orthogonal experimental design for optimization of adding preservatives to liquid compound microbial fertilizer

[0119]

[0120] Note: Different lowercase letters in the same row indicate significant differences at the P < 0.05 level as determined by LSD test.

[0121] The effective viable bacteria count of the prepared compound microbial fertilizer is 7.3-9.7 billion cfu / mL, with a long shelf life. After being stored at room temperature for one year, there is no significant difference in the effective viable bacteria count.

[0122] Example 2: Quality testing of liquid compound microbial fertilizer

[0123] The quality of the liquid compound microbial fertilizer was tested using the effective viable bacterial count, nutrient content, seed germination of solanaceous vegetables, plant growth, and the control effect of soil-borne bacterial wilt as indicators. The preparation method of the giant grass juice liquid compound microbial fertilizer was similar to that of Example 1.

[0124] (1) Randomly select 3 portions of liquid compound microbial fertilizer, dilute them 10 times with sterile water, apply them to NA culture medium, culture them at 30℃ for 48h, and count the number of effective living bacteria. Figure 3 As shown, the viable bacteria counts of the three samples were 7.30×10 9 cfu / g( Figure 3 A), 9.32×10 9 cfu / g( Figure 3 B) and 9.70×10 9 cfu / g( Figure 3 C).

[0125] (2) Nutrient content determination: Three samples of liquid compound microbial fertilizer were randomly selected and sent to the Institute of Agricultural Quality Standards and Testing Technology of Fujian Academy of Agricultural Sciences for determination of physical and chemical properties and nutrient composition, including pH (according to NY / T 1121.2-2006), total nitrogen content (according to NY / T 525-2021), total phosphorus (according to NY / T 2541-2014) and total potassium content (NY / T 2540-2014), and the average value was calculated.

[0126] Physical and chemical properties and nutrient composition measurements showed the developed compound microbial fertilizer had a pH of 6.10±0.26, total nitrogen content of 139.00±4.58g / L, total phosphorus content of 30.60±5.13g / L, total potassium content of 35.10±3.15g / L, calcium content of 26.20±4.01mg / L, magnesium content of 15.4±2.92mg / L, iron content of 7.44±0.20mg / L, manganese content of 2.68±0.15mg / L, and zinc content of 7.78±0.31mg / L. The total nutrient content (N+P2O5+K2O) was 20.47%, exceeding the national standard for microbial fertilizers (NY / T 798-2015).

[0127] (3) Determination of seed germination index: Tomato, pepper and eggplant were used as test materials. Liquid compound microbial fertilizer was diluted 100, 300, 500 and 800 times respectively and stored at 4℃ for later use. Using the paper germination method, tomato, pepper and eggplant seeds were rinsed twice in 55℃ warm water, removed and drained, and soaked in different dilution times of the extract for 8 hours. Sterile water soaking was used as negative control (CK). - ), commercial compound microbial fertilizer (purchased from Hebei Qianbaiji Agricultural Science and Technology Co., Ltd.) was used as the positive control (CK +After soaking, transfer the seeds to 9-cm transparent Petri dishes with two layers of filter paper on the bottom. Place 15 seeds per dish, with three replicates. Place the dishes in a 27°C constant-temperature artificial climate chamber with 16 h of light and 8 h of darkness. Observe seed germination and record the number of germinations. If no new seeds germinate for three consecutive days, measure the seed germination rate, germination index, and seed vigor index.

[0128] Germination rate (%) = (number of germinated seeds in a specified number of days / number of test seeds) × 100%

[0129] Germination index (GI) = Σ(Gt / Dt), Gt is the number of seeds germinated on the tth day, and Dt is the corresponding number of germination days.

[0130] Vitality index (VI) = germination index × radicle length (cm)

[0131] Seed germination tests showed that, compared with the control, a 100-fold dilution of the liquid compound microbial fertilizer significantly inhibited tomato seed germination, while a 300-fold dilution significantly increased the germination index and seed vigor index (P < 0.05). A 500-fold dilution significantly increased the germination rate of tomato seeds. For pepper seed germination, an 800-fold dilution of the liquid compound microbial fertilizer significantly increased the germination rate, germination index, and seed vigor index of pepper seeds compared to the water control, while other dilutions inhibited pepper seed germination. For eggplant seed germination, an 800-fold dilution of the liquid compound microbial fertilizer promoted seed germination and significantly increased the germination index and seed vigor index (P < 0.05), while 100- and 300-fold dilutions significantly inhibited seed germination (Table 22).

[0132] Table 22 Effects of liquid compound microbial fertilizer on seed germination of solanaceous vegetables

[0133]

[0134] Note: Different lowercase letters in the same case in the table indicate significant differences at the P<0.05 level by LSD test.

[0135] (4) Plant growth determination: Tomato, pepper and eggplant were used as research objects. Liquid compound microbial fertilizer was diluted 100, 300, 500 and 800 times and irrigated into 25-day-old tomato, pepper and eggplant seedlings, 100 mL / pot. Commercial compound microbial fertilizer was used as positive control (CK + ), clean water was used as blank control (CK - ), with 3 replicates per treatment. Fifteen days after treatment, the plant height, stem diameter, and total leaf number of tomatoes were counted.

[0136] The results of the growth promotion test showed that 300- and 500-fold dilutions of the liquid compound microbial fertilizer could significantly increase the plant height, stem diameter and total leaf number of tomato plants (P<0.05); 500- and 800-fold dilutions of the liquid compound microbial fertilizer could significantly promote the growth of pepper and eggplant plants (P<0.05); while 100-fold dilution of the liquid compound microbial fertilizer significantly inhibited the growth of tomato, pepper and eggplant plants (Table 23).

[0137] Table 23 Effects of liquid compound microbial fertilizer on the growth of tomato, pepper and eggplant plants

[0138]

[0139] Note: Different lowercase letters in the same case in the table indicate significant differences at the P<0.05 level by LSD test.

[0140] (5) Disease prevention test: Taking the control effect of giant fungus grass juice compound microbial fertilizer on soil-borne bacterial wilt as an example, the fertilizer prepared above was diluted 100, 300, 500 and 800 times, and pre-inoculated into 25-day-old tomato, pepper and eggplant seedlings, 100 mL / pot, and inoculated with the highly pathogenic bacterial wilt pathogen FJAT-91 3 days later. The negative control (CK) was inoculated with FJAT-91 after pre-treatment with clean water for 3 days. - ), commercial compound microbial fertilizer (purchased from Hebei Qianbaiji Agricultural Science and Technology Co., Ltd.) was pretreated for 3 days and then inoculated with FJAT-91 as a positive control (CK + ), with 3 replicates per treatment, and the disease conditions of the plants were observed every day after inoculation, and the incidence rate and control efficacy were calculated.

[0141] The results showed that in the control group, tomato, pepper, and eggplant plants began to develop disease on days 4, 7, and 5 after inoculation, respectively, reaching 100% disease on days 10, 12, and 10. The efficacy of the compound microbial fertilizer against bacterial wilt in tomatoes, peppers, and eggplant decreased with increasing dilution ratios. The 100- and 300-fold dilutions of the compound microbial fertilizer each achieved over 70% efficacy against bacterial wilt in tomatoes, while the 100-, 300-, and 500-fold dilutions also achieved over 70% efficacy against bacterial wilt in peppers and eggplant (Table 24). Combined with the results of plant growth measurements, the recommended application concentrations of the compound microbial fertilizer for tomatoes, peppers, and eggplant are 300-, 500-, and 500-fold dilutions, respectively.

[0142] Table 24 Control effect of compound microbial fertilizer on bacterial wilt of tomato, pepper and eggplant

[0143]

[0144] Note: Different lowercase letters in the same case in the table indicate significant differences at the P<0.05 level by LSD test.

[0145] Example 3: Field application effect of liquid compound microbial fertilizer on greenhouse tomatoes

[0146] From September 2023 to February 2024, a study on the field application effect of giant fungus grass juice liquid compound microbial fertilizer on facility tomatoes was carried out in the tomato planting greenhouse of Jiaxing Farm, Hui'an County, Quanzhou, Fujian (for 6 consecutive years) and the tomato planting greenhouse of Daheng Town, Yanping District, Fujian (for 3 consecutive years). Liquid compound microbial fertilizer was prepared with reference to Example 1. Before transplanting the tomato plants, a 300-fold diluted solution of compound microbial fertilizer was sprayed on the soil surface, 50L / mu, and plowed. When transplanting tomatoes, the roots were irrigated with 300mL / plant, and irrigated once every 15d for 2 times. The roots of tomatoes were irrigated with clear water as a control. Three greenhouses were used for treatment and control, and 600 tomatoes were planted in each greenhouse. 1) The incidence rate, plant height and stem thickness of the plants were adjusted during the tomato vegetative growth period (20d after transplanting) and the reproductive growth period (90d after transplanting). 2) Collect tomato rhizosphere soil samples as follows: Dig up the entire plant, shake off the loose matrix soil, and collect the soil attached to the roots, removing gravel and plant debris. 100 g of soil was collected for each treatment. After passing through a 2 mm sieve, a portion was used to determine soil physical and chemical properties, and a portion was used to detect soil microbial diversity.

[0147] Determination of soil physical and chemical properties: Soil pH was determined by the potentiometric method (water: soil ratio was 2.5:1); soil organic matter was determined by the potassium dichromate-sulfuric acid method; soil total ammonia (TN) was determined using an automatic nitrogen analyzer; soil total phosphorus was determined using the alkali fusion-molybdenum antimony anti-spectrophotometry method; total potassium was determined using the NaOH fusion-flame photometer method.

[0148] Soil microbial diversity was assessed by Beijing Aoweisen Gene Technology Co., Ltd. Bacterial 16S rDNA V3-V4 regions were sequenced using the Illumina Miseq PE300 high-throughput sequencing platform. The 16S rRNA primers were 520F: 5'-AYTGGGYDTAAAGNG-3' and 806R: 5'-TACNVGGGTATCTAATCC-3' (Claesson et al., 2009). The PCR reaction system (25 μL) consisted of 12.5 μL KAPA2G Robust Hot Start ReadyMix, 1 μL each of the upstream and downstream primers (5 μM), 5 μL template DNA, and 5.5 μL sterile water. The PCR protocol was 95°C for 5 min, followed by 28 cycles of denaturation at 95°C for 45 s, annealing at 55°C for 50 s, and annealing at 72°C for 45 s, followed by a final extension at 72°C for 10 min.

[0149] Paired-end sequencing was performed on the Illumina MiSeq platform. Raw data were filtered, spliced, and chimeras removed. Sequences with scores below 20, ambiguous bases, primer mismatches, or sequence lengths less than 150 bp were removed. Sequence information from each treatment group was clustered into OTUs for species classification based on barcodes. The corresponding species taxonomic information for each OTU was then compared to the Silva database. Alpha diversity analysis (including Shannon, Simpson, and Chao1) was then performed using Mothur software (v1.31.2).

[0150] (1) Effects of liquid compound microbial fertilizer on the growth of tomato plants in the field

[0151] The results, as shown in Table 25, show that application of liquid compound microbial fertilizer to tomato greenhouses in Hui'an and Yanping promoted tomato plant growth and reduced the incidence of bacterial wilt. Except for the vegetative growth period in the Hui'an test site, where plant height differences between the liquid compound microbial fertilizer-treated and control tomatoes were not significant, liquid compound microbial fertilizer treatment significantly increased plant height and stem diameter and reduced bacterial wilt in all other treatments. Compared with the control, during the vegetative growth period, the liquid compound microbial fertilizer increased the plant height and stem diameter of tomatoes in the Hui'an test site by 8.02% and 18.65% respectively, and reduced the incidence of bacterial wilt by 81.50%; the plant height and stem diameter of tomatoes in the Yanping test site increased by 10.94% and 11.51% respectively, and reduced the incidence of bacterial wilt by 94.36%; during the reproductive growth period, the liquid compound microbial fertilizer increased the plant height and stem diameter of tomatoes in the Hui'an test site by 27.92% and 18.63% respectively, and reduced the incidence of bacterial wilt by 76.38%; the plant height and stem diameter of tomatoes in the Yanping test site increased by 124.98% and 110.73% respectively, and reduced the incidence of bacterial wilt by 84.46%.

[0152] Table 25 Effects of liquid compound microbial fertilizer on growth and wilt incidence of tomato plants in the field

[0153]

[0154]

[0155] Note: HA and YP represent the experimental sites of Hui'an and Yanping, respectively; TR and CK represent the compound microbial fertilizer treatment group and the control group, respectively; V and R represent the vegetative growth period and the reproductive growth period, respectively, the same below.

[0156] (2) Effects of liquid compound microbial fertilizer on the physical and chemical properties of tomato rhizosphere soil

[0157] The results are shown in Table 26. Liquid compound microbial fertilizer treatment had little effect on soil pH, but significantly increased soil organic matter (SOC), total nitrogen (TN), and total phosphorus (TP) contents (P < 0.05). At the Hui'an site, liquid compound microbial fertilizer treatment significantly increased total potassium (TK) content, but the effect on TK content at the Yanping site was not significant. Furthermore, SOC, TN, TP, and TK contents in the tomato root soil during the reproductive growth phase were lower in both the liquid compound microbial fertilizer treatment and control groups than in the root soil during the vegetative growth phase, indicating that plant growth consumes a significant amount of nutrients.

[0158] Table 26 Effects of liquid compound microbial fertilizer on physical and chemical properties of soil in field tomato root system

[0159]

[0160] Note: SOC, TN, TP and TK are organic matter, total nitrogen, total phosphorus and total potassium respectively.

[0161] (3) Effects of liquid compound microbial fertilizer on bacterial community structure in tomato rhizosphere soil

[0162] like Figure 4 As shown in the results, liquid compound microbial fertilizer treatment can improve the diversity of tomato rhizosphere microbial community structure. In the Hui'an experimental site, the Chao1 and Shannon index of the tomato rhizosphere microbial community diversity index in the vegetative and reproductive growth stages of the tomato treated with liquid compound microbial fertilizer were significantly higher than those in the control (P < 0.05). In the Yanping experimental site, the Shannon index of the tomato rhizosphere soil treated with liquid compound microbial fertilizer was significantly higher than that in the control during both the vegetative and reproductive growth stages. However, Chao1 was higher only in the vegetative growth stage; there was no significant difference in the reproductive growth stage.

[0163] At the phylum level, the bacterial phylum with the highest relative content in the tomato rhizosphere soil of the liquid compound microbial fertilizer treatment and the control group was Proteobacteria, whose relative abundance in each sample was greater than 25% ( Figure 5 A). Compared with the control, the liquid compound microbial fertilizer treatment significantly increased the relative abundance of Actinobacteriota and significantly decreased the relative abundance of Acidobacteriota and Myxococcota. In addition, the liquid compound microbial fertilizer treatment significantly increased the relative abundance of Dependentiae in the rhizosphere soil of tomatoes during the reproductive growth period ( Figure 5 A).

[0164] At the genus level, compared with the control, the liquid compound microbial fertilizer treatment significantly increased the relative abundance of Bacillus, Saccharimonadales, Bauldia, and BIRII41, and significantly decreased the relative abundance of Pseudolabrys, Ralstonia, and Vicinamibacteraceae ( Figure 5 B). In addition, the liquid compound microbial fertilizer treatment significantly increased the relative abundance of Actinoplanes (except for the rhizosphere soil of tomatoes during the reproductive growth period in the Hui'an test site), and significantly reduced the relative abundance of Flavisolibacter and Acidibacter (except for the rhizosphere soil of tomatoes during the vegetative growth period in the Yanping test site) ( Figure 5 B).

[0165] Principal component analysis showed that the first principal component (PC1) and the second principal component (PC2) explained 65.19% of the changes in bacterial community structure ( Figure 6 ), indicating that they are the main factors causing the differences in sample communities. PC1 can clearly distinguish the tomato rhizosphere soils from the Yanping and Hui'an test sites, indicating that principal component 1 is the main factor causing the differences in the bacterial community structure of the rhizosphere soil samples from the two test sites. PC2 can clearly distinguish the tomato rhizosphere soils from the compound microbial fertilizer treatment group and the control group, indicating that principal component 2 is the main factor causing the changes in the bacterial community structure of the tomato rhizosphere soil caused by compound microbial fertilizer treatment.

[0166] The RDA analysis of tomato rhizospheric bacterial communities, soil physical and chemical properties, and bacterial wilt incidence (DI) showed that ( Figure 7 ), DI was negatively correlated with soil pH and SOC, TN and TP contents, and positively correlated with TK content; DI was positively correlated with the relative abundance of Ralstonia and Vicinamibacter, with a correlation coefficient (r 2 ) were 0.87 and 0.63, respectively, indicating that the increase in the relative abundance of bacteria in these two genera may cause bacterial wilt; DI was negatively correlated with Streptomyces, Nocardioides, Bacillus, and BIRii41, r 2 The values ​​were -0.47, -0.54, -0.49 and -0.38, respectively, indicating that the increase in the relative abundance of bacteria in these genera would inhibit the outbreak of bacterial wilt.

[0167] In summary, the effective viable bacteria count of liquid compound microbial fertilizer can reach 7.3-9.7 billion cfu / mL, the nutrient (N+P2O5+K2O) content is 20.47%, and it has a long shelf life and can be stored at room temperature for 1 year. Liquid compound microbial fertilizer can promote the germination of seeds of fruits and vegetables such as tomatoes, peppers, and eggplants, as well as the growth of plants. It can also effectively prevent and control bacterial wilt by improving the physical and chemical properties of rhizosphere soil and soil microecology. Figure 8 ).

[0168] It should be noted that although the above embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the present invention's specification and drawings, and direct or indirect application of the above technical solutions to other related technical fields, are all included in the scope of patent protection of the present invention.

Claims

1. A method for preparing a novel liquid composite microbial fertilizer, comprising the following steps: Preparation of liquid fertilizer: Add 150-250g / L urea, 40-60g / L potassium dihydrogen phosphate, 10-30g / L potassium sulfate, 0.004-0.04g / L lime, 0.3-0.06g / L ferrous sulfate, 0.1-0.02g / L zinc sulfate, 0.01-0.05g / L manganese sulfate and 0.002-0.02g / L magnesium sulfate to the giant grass juice waste liquid, stir thoroughly, and adjust the pH value to 6.5-7.0; Preparation of composite functional bacteria seed solution: After activating Bacillus licheniformis, Bacillus subtilis and Bacillus siamese on nutrient agar plates, they were inoculated into nutrient broth liquid culture medium, cultured at 28-32°C and 150-200 r / min for 20-28 h, and the bacterial solution was diluted to (0.8-1.2)×10 9 cfu / mL; the fermentation broth of Bacillus licheniformis, Bacillus subtilis and Bacillus siamensis was mixed in a volume ratio of 1:(1-3):1 and stored at 2-8°C for later use; Preparation of liquid compound microbial fertilizer: using the liquid fertilizer prepared above as a fermentation medium, inoculating the above compound functional bacteria seed liquid into the liquid fertilizer at an inoculation rate of 5-10%, fermenting and culturing at 28-32°C for 45-50h, adding a preservative to the fermentation liquid, and finally forming a liquid compound microbial fertilizer.

2. The preparation method of the novel liquid composite microbial fertilizer according to claim 1, characterized in that: The licheniformis is Bacillus licheniformis strain FJAT-41480, the subtilis is Bacillus subtilis strain FJAT-41479, and the siamensis is Bacillus siamensis strain FJAT-41481. The three strains were deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with the deposit numbers CGMCC No. 33777, CGMCC No. 33776 and CGMCC No. 33778, respectively. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

3. The preparation method of the novel liquid composite microbial fertilizer according to claim 1, characterized in that: The preservative component comprises 0.01-0.5 g / mL of potassium sorbate, 0.01-0.5 g / mL of sodium dehydroacetate and 0.005-0.01 g / mL of sodium diacetate.

4. Use of the novel liquid composite microbial fertilizer according to any one of claims 1 to 3 in promoting seed germination of solanaceous vegetables.

5. Use of the novel liquid compound microbial fertilizer according to any one of claims 1 to 3 in the prevention and treatment of soil-borne diseases of solanaceous vegetables.

6. Use of the novel liquid composite microbial fertilizer according to any one of claims 1 to 3 in promoting the growth of solanaceous vegetable plants.

7. Use of the novel liquid compound microbial fertilizer according to any one of claims 1 to 3 in soil microecological regulation.

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