Compound microbial fertilizer suitable for flue-cured tobacco production as well as preparation method and application of compound microbial fertilizer

Abstract

The invention discloses a compound microbial fertilizer suitable for flue-cured tobacco production as well as a preparation method and application thereof. The compound microbial fertilizer is prepared from the following components in percentage by mass: 4-8% of a functional microbial agent, 12-18% of a first modifier, 1-2% of a second modifier, 30-70% of an inorganic material and the balance of pure water, wherein the first modifier is a polyglutamic acid stock solution in which the mass percent of polyglutamic acid is greater than or equal to 3.5%, and the second modifier is a gamma-aminobutyric acid stock solution in which the mass percent of gamma-aminobutyric acid is greater than or equal to 50%; the functional microbial agent is prepared by compounding a bacterial strain XNSD01 and a bacterial strain XNSD02, the bacterial strain XNSD01 is bacillus siamensis with the preservation number of CCTCC (China Center for Type Culture Collection) NO.M 20252934, and the bacterial strain XNSD02 is bacillus mycoides with the preservation number of CCTCC NO.M 20252935. And unification of disease resistance, growth promotion and successive cropping obstacle alleviation is realized.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a compound microbial fertilizer suitable for flue-cured tobacco production, its preparation method, and its application, particularly suitable for areas with continuous tobacco cropping. Background Technology

[0002] Flue-cured tobacco is an important economic crop in my country, but its production faces two major problems: First, continuous cropping leads to soil nutrient imbalance, increased salinization, accumulation of pathogens (such as black shank pathogen), and decreased soil productivity. Second, nutrient requirements are mismatched with fertilization techniques, specifically manifested as follows:

[0003] (1) 10 to 20 days after transplanting, the tobacco plants need to quickly establish their growth structure and have an urgent need for nutrients. However, the utilization rate of soil nutrients in continuous cropping is low, and the existing seedling fertilizers are mostly general-purpose inorganic fertilizers, which lack components that can synergistically improve nutrient efficiency and alleviate the adverse conditions of continuous cropping, resulting in slow growth and weak resistance of tobacco plants.

[0004] (2) In the existing fertilization techniques for flue-cured tobacco production, there is a common cognitive bias of "emphasizing macronutrients and neglecting micronutrients." There is an excessive focus on the ratio and input of nitrogen, phosphorus, and potassium, while seriously neglecting the supplementation of micronutrients such as calcium and magnesium. This directly violates the nutritional requirements of flue-cured tobacco. Long-term application of only nitrogen, phosphorus, and potassium without supplementing micronutrients such as calcium and magnesium will directly lead to nutritional imbalance in tobacco plants, ultimately resulting in reduced flue-cured tobacco yield, uneven color of tobacco leaves after curing, and insufficient oil content, which seriously affects economic value. At the same time, the soil reserves of micronutrients such as calcium and magnesium are continuously depleted, gradually leading to acidification or compaction, further reducing the effectiveness of nitrogen, phosphorus, and potassium, forming a vicious cycle of "fertilization not being effective."

[0005] Currently, there is no technical solution that combines polyglutamic acid and γ-aminobutyric acid with inorganic materials and functional microorganisms to specifically address the obstacles of continuous cropping of flue-cured tobacco and achieve synergistic effects of plant protection and nutrition. Existing technologies either ignore the problem of continuous cropping or cannot accurately match nutrient requirements, resulting in unstable effects. Therefore, the development of compound microbial fertilizers that can promote growth and disease resistance while alleviating the obstacles of continuous cropping is crucial for the green and efficient production of flue-cured tobacco. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a compound microbial fertilizer suitable for flue-cured tobacco production and its preparation method. Through the synergy of "functional microorganisms + secondary metabolites + precise nutrients", it achieves a balance between disease resistance and growth promotion and the alleviation of continuous cropping obstacles, demonstrating the synergistic effect of "plant protection-nutrition".

[0007] The technical solution provided by this invention is as follows:

[0008] This invention provides a compound microbial fertilizer suitable for flue-cured tobacco production, which is composed of the following components by mass percentage: 4%~8% functional microbial agent, 12%~18% first improver, 1%~2% second improver, 30%~70% inorganic materials, and the balance being pure water; The first modifier is a polyglutamic acid stock solution with a mass percentage of ≥3.5% polyglutamic acid, and the second modifier is a γ-aminobutyric acid stock solution with a mass percentage of ≥50% γ-aminobutyric acid; the functional microbial agent is a compound of strain XNSD01 and strain XNSD02, strain XNSD01 is Bacillus sicca with accession number CCTCC NO.M 20252934, and strain XNSD02 is Bacillus mycosis fungoides with accession number CCTCC NO.M 20252935.

[0009] Furthermore, in the functional microbial agent, the ratio of effective viable bacteria counts of strain XNSD01 to strain XNSD02 is 1~3:1, and the effective viable bacteria count in the functional microbial agent is ≥5×10⁻⁶. 9 CFU / mL.

[0010] Furthermore, the inorganic material is composed of calcium ammonium nitrate, magnesium nitrate, and potassium nitrate, and the mass ratio of calcium ammonium nitrate, magnesium nitrate, and potassium nitrate is 1~4 : 0.5~2 : 1~3.

[0011] Furthermore, the inorganic material in the compound microbial fertilizer has a total nutrient content of 8% to 20%, of which nitrogen has a mass fraction of 4% to 12%, potassium has a mass fraction of 4% to 10%, calcium has a mass fraction of 2.5% to 6%, and magnesium has a mass fraction of 0.3% to 6.5%.

[0012] This invention also provides a method for preparing a compound microbial fertilizer suitable for flue-cured tobacco production, comprising the following steps: Ammonium calcium nitrate, magnesium nitrate and potassium nitrate are mixed evenly in a certain proportion to obtain inorganic materials; Inorganic materials are added to a mixture of polyglutamic acid stock solution and γ-aminobutyric acid stock solution, and stirred until homogeneous to obtain a suspension containing polyglutamic acid and γ-aminobutyric acid; wherein the mass percentage of polyglutamic acid in the polyglutamic acid stock solution is ≥3.5%, and the mass percentage of γ-aminobutyric acid in the γ-aminobutyric acid stock solution is ≥50%; After detecting the viable cell counts of bacterial suspensions of strains XNSD01 and XNSD02, they were compounded according to the effective viable cell count ratio to obtain a functional microbial agent; strain XNSD01 is Bacillus sicca with accession number CCTCC NO.M 20252934, and strain XNSD02 is Bacillus mycosis fungoides with accession number CCTCC NO.M 20252935; Under stirring, the functional microbial agent is added to the suspension and stirred evenly. Then, pure water is added to make up the remaining amount, and stirring is continued. The pH value is adjusted to 5.5~6.5 to obtain compound microbial fertilizer.

[0013] Furthermore, the mass ratio of calcium ammonium nitrate, magnesium nitrate, and potassium nitrate is 1-4:0.5-2:1-3; the ratio of effective viable bacteria counts of strain XNSD01 and strain XNSD02 is 1-3:1, and the effective viable bacteria count in the functional microbial agent is ≥5×10⁻⁶. 9 CFU / mL.

[0014] Furthermore, the compound microbial fertilizer contains 4% to 8% by mass of functional microbial agents, 12% to 18% by mass of polyglutamic acid stock solution, 1% to 2% by mass of γ-aminobutyric acid stock solution, and 30% to 70% by mass of inorganic materials.

[0015] The present invention also provides the application of the compound microbial fertilizer described above or the compound microbial fertilizer prepared by the method described above in promoting the growth of flue-cured tobacco, improving the disease resistance of flue-cured tobacco and / or alleviating the obstacles of continuous cropping of flue-cured tobacco.

[0016] Furthermore, the compound microbial fertilizer is diluted 100 to 400 times and applied by root irrigation, with 500 mL applied to each tobacco plant on the 10th and 20th days after transplanting.

[0017] Furthermore, promoting tobacco growth involves increasing plant height, stem circumference, and maximum leaf area; enhancing tobacco disease resistance involves improving tobacco immunity against tobacco black shank pathogens; and alleviating continuous cropping obstacles involves reducing soil bulk density and increasing soil microbial diversity index in continuously cropped tobacco fields.

[0018] Beneficial effects

[0019] The compound microbial fertilizer of this invention, through the synergistic effect of "plant protection-nutrition", reduces the disease index of black shank disease in flue-cured tobacco by 25%-60% compared with the control, increases the plant height by 5%-19%, the stem circumference by 7%-14%, and the maximum leaf area by 6%-14% compared with the control, increases the proportion of top-grade tobacco leaves after curing by 3%-9%, and at the same time reduces the soil bulk density in continuously cropped tobacco fields by 3%-6%, reduces the number of black shank pathogens by 20%-40%, and increases the soil microbial diversity index by 7%-12%, thus alleviating the obstacles of continuous cropping. Attached Figure Description

[0020] Figure 1 The images show the colony (A) and Gram staining results (B) of strain XNSD01.

[0021] Figure 2 This is a phylogenetic tree of strain XNSD01 constructed based on the 16S rDNA gene sequence;

[0022] Figure 3 The images show the colony (A) and Gram staining results (B) of strain XNSD02.

[0023] Figure 4 This is a phylogenetic tree of strain XNSD02 constructed based on the 16S rDNA gene sequence. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] The following are some of the reagent models and manufacturers used in the embodiments of this invention: Inorganic materials: Calcium ammonium nitrate was purchased from Shanxi Jiaocheng Chemical Co., Ltd., with total nitrogen ≥15.5%, of which nitrate nitrogen ≥14.4%, calcium (Ca) ≥18%, calcium oxide ≥26%, moisture ≤0.6%, water-insoluble matter ≤0.2%, particle size 2-4 mm ≥90%, conforming to NY 2269-2012 agricultural grade standard. Magnesium nitrate was purchased from Shanxi Kailan Chemical Co., Ltd., with Mg(NO3)2·6H2O content ≥98%, magnesium oxide (MgO) ≥15%, nitrate nitrogen ≈10.5%, water-insoluble matter ≤0.05%, chlorine ≤0.02%, moisture ≤1.0%, white crystals, conforming to HG / T 4523-2013 agricultural requirements. Potassium nitrate was purchased from Migo Group. It contains KNO3 ≥ 99%, potassium oxide (K2O) ≥ 46%, nitrate nitrogen ≥ 13.5%, moisture ≤ 0.3%, chlorine ≤ 0.02%, water-insoluble matter ≤ 0.01%, and particle size 0.8-2.5 mm, meeting the requirements of GB / T 647-2011 agricultural grade.

[0026] The polyglutamic acid stock solution (polyglutamic acid content ≥12%, implementation standard is QB / T 4587-2013) and the γ-aminobutyric acid stock solution (γ-aminobutyric acid content ≥98%, product standard number is Q / YWF 07-2022) were both purchased from Yunnan Provincial Microbial Fermentation Engineering Research Center Co., Ltd.

[0027] This invention provides a compound microbial fertilizer suitable for flue-cured tobacco production, which is composed of the following components by mass percentage: 4%~8% functional microbial agent, 12%~18% first improver, 1%~2% second improver, 30%~70% inorganic materials, and the balance being pure water; The first improver is a polyglutamic acid stock solution with a mass percentage of ≥3.5% (which has the function of chelating potassium, calcium, and magnesium elements in inorganic materials to improve nutrient utilization, while improving the soil aggregate structure of continuously cropped tobacco fields and reducing soil salinization to alleviate continuous cropping obstacles). The second improver is a γ-aminobutyric acid stock solution with a mass percentage of ≥50% (which has the function of activating the stress-resistant enzyme system of tobacco plants (superoxide dismutase, peroxidase), alleviating transplanting and seedling slow-down and continuous cropping soil stress, and synergistically promoting tobacco plant growth with functional microorganisms). The functional microorganisms are a compound of strains XNSD01 and XNSD02. Strain XNSD01 is Bacillus sicca with the preservation number CCTCC NO.M20252934, and strain XNSD02 is Bacillus mycosis fungoides with the preservation number CCTCC NO.M 20252935. The compound microbial fertilizer achieves a synergistic effect of "plant protection-nutrition" through the synergistic action of polyglutamic acid, γ-aminobutyric acid, functional microbial agents, and inorganic materials, thus solving the problems of high incidence of black shank disease, slow growth of tobacco plants, and soil nutrient imbalance caused by continuous cropping.

[0028] Specifically, the isolation and identification of strain XNSD01:

[0029] (1) Isolation of strain XNSD01: It was previously isolated from the rhizosphere of tobacco in Songming, Kunming. Strain XNSD01 was streaked onto LB solid medium and incubated at 37℃ for 3 days. The colonies were round, pale yellow, opaque, and slightly wrinkled on the surface. Figure 1 ).

[0030] (2) Identification of strain XNSD01: Gram staining and observation under an oil immersion microscope showed that the blue-purple color indicated it was a Gram-positive bacterium. Figure 1 ).

[0031] (3) Molecular identification: Genomic DNA of strain XNSD01 was extracted using the Tiangen DP3032 extraction kit. PCR amplification was performed using universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.3), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.4)). The amplification was performed under the following conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, and a final extension at 72℃ for 5 min after 30 cycles. The amplified products were stored at 4℃. Sequencing was performed by Qingke Biotechnology Co., Ltd., and the sequencing results were compared using BLAST. A phylogenetic tree was constructed using MEGAv7.0.26. The results showed that the 16S rDNA gene of strain XNSD01 is similar to that of Bacillus sicca. Bacillus siamensis cqsM9 clusters in the same branch with a similarity of 96% (see...). Figure 2 The strain XNSD01 is Bacillus sicca.

[0032] Specifically, the isolation and identification of strain XNSD02:

[0033] (1) Isolation of strain XNSD02: It was previously isolated from the rhizosphere of tobacco in Huili, Liangshan. Strain XNSD02 was streaked onto LB solid medium and incubated at 37℃ for 3 days. The colonies were round, pale yellow, opaque, and slightly wrinkled on the surface. Figure 3 ).

[0034] (2) Identification of strain XNSD02: Gram staining and observation under an oil immersion microscope showed that the blue-purple color indicated it was a Gram-positive bacterium. Figure 3 ).

[0035] (3) Molecular identification: Genomic DNA of strain XNSD02 was extracted using the Tiangen DP3032 extraction kit. PCR amplification was performed using universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.3), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.4)). The amplification was performed under the following conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, and a final extension at 72℃ for 5 min after 30 cycles. The amplified products were stored at 4℃. Sequencing was performed by Qingke Biotechnology Co., Ltd., and the sequencing results were compared using BLAST. A phylogenetic tree was constructed using MEGAv7.0.26. The results showed that the 16S rDNA gene of strain XNSD02 is similar to that of Bacillus mycosis fungoides. Bacillus paramycoides JYZ-SD5 clustered in the same branch with a similarity of 94% (see...). Figure 4 Strain XNSD02 is a mycosis fungoides.

[0036] In this embodiment, the ratio of effective viable bacteria counts of strain XNSD01 and strain XNSD02 in the functional microbial agent is 1~3:1, and the effective viable bacteria count in the functional microbial agent is ≥5×10⁻⁶. 9 CFU / mL.

[0037] In this embodiment, the inorganic material is composed of calcium ammonium nitrate, magnesium nitrate and potassium nitrate, and the mass ratio of calcium ammonium nitrate, magnesium nitrate and potassium nitrate is 1~4 : 0.5~2 : 1~3.

[0038] In this embodiment, the inorganic material in the compound microbial fertilizer has a total nutrient mass fraction of 8% to 20%, of which the mass fraction of nitrogen is 4% to 12%, the mass fraction of potassium is 4% to 10%, the mass fraction of calcium is 2.5% to 6%, and the mass fraction of magnesium is 0.3% to 6.5%.

[0039] This invention also provides a method for preparing a compound microbial fertilizer suitable for flue-cured tobacco production, comprising the following steps: Ammonium calcium nitrate, magnesium nitrate and potassium nitrate are mixed evenly in a certain proportion to obtain inorganic materials; Inorganic materials are added to a mixture of polyglutamic acid stock solution and γ-aminobutyric acid stock solution, and stirred evenly (stirred for 80-120 min at 25-30℃ and 160-200 r / min) to obtain a suspension containing polyglutamic acid and γ-aminobutyric acid; the mass percentage of polyglutamic acid in the polyglutamic acid stock solution is ≥3.5%, and the mass percentage of γ-aminobutyric acid in the γ-aminobutyric acid stock solution is ≥50%; in this step, polyglutamic acid fully chelates potassium, calcium, and magnesium elements in the inorganic materials, and γ-aminobutyric acid is uniformly dispersed, laying the foundation for the subsequent synergistic function of microorganisms; After detecting the viable cell counts of bacterial suspensions of strains XNSD01 and XNSD02, they were compounded according to the effective viable cell count ratio to obtain a functional microbial agent; strain XNSD01 is Bacillus sicca with accession number CCTCC NO.M 20252934, and strain XNSD02 is Bacillus mycosis fungoides with accession number CCTCC NO.M 20252935; Under stirring, the functional microbial agent is added to the suspension and stirred evenly. Then, pure water is added to make up the remaining amount, and stirring is continued (stirring for 40-60 minutes at 25-30℃ and 120-150r / min). The pH value is adjusted to 5.5-6.5 to obtain the compound microbial fertilizer.

[0040] In this embodiment, the mass ratio of calcium ammonium nitrate, magnesium nitrate, and potassium nitrate is 1~4 : 0.5~2 : 1~3; the ratio of effective viable bacteria counts of strain XNSD01 and strain XNSD02 is 1~3 : 1, and the effective viable bacteria count in the functional microbial agent is ≥5×10⁻⁶. 9 CFU / mL.

[0041] In this embodiment, the compound microbial fertilizer contains 4% to 8% by mass of functional microbial agents, 12% to 18% by mass of polyglutamic acid stock solution, 1% to 2% by mass of γ-aminobutyric acid stock solution, and 30% to 70% by mass of inorganic materials.

[0042] The embodiments of the present invention also provide the application of the compound microbial fertilizer described above or the compound microbial fertilizer prepared by the method described above in promoting the growth of flue-cured tobacco, improving the disease resistance of flue-cured tobacco and / or alleviating the obstacles of continuous cropping of flue-cured tobacco.

[0043] In this embodiment, the compound microbial fertilizer is diluted 100-400 times and applied by root irrigation, with 500 mL applied to each tobacco plant. The application periods are the 10th and 20th days after transplanting. Specifically, the compound microbial fertilizer is diluted 100-400 times according to the field concentration range, with three gradients of 100 times, 200 times, and 400 times. All are applied by root irrigation, with 500 mL applied to each tobacco plant. The fertilizer is applied once every 10 days after transplanting, for a total of 2 applications. The 200-fold dilution is the optimal field concentration, which can maximize the balance between promoting growth and preventing diseases and cost. In this embodiment, promoting tobacco growth means increasing tobacco plant height, stem circumference, and maximum leaf area; improving tobacco disease resistance means improving tobacco immunity to tobacco black shank pathogen; and alleviating tobacco continuous cropping obstacles means reducing soil bulk density and increasing soil microbial diversity index in continuously cropped tobacco fields.

[0044] The following detailed description, in conjunction with embodiments, illustrates the present invention's provision of a compound microbial fertilizer suitable for flue-cured tobacco production, its preparation method, and its application (Tables 1-10). However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] This invention provides a compound microbial fertilizer suitable for flue-cured tobacco production, with the proportions of each component as shown in Table 1 (the following components are proportioned by mass):

[0047] Table 1. Ingredients of Compound Microbial Fertilizer

[0048]

[0049] Preparation method of Example 1:

[0050] The preparation process of compound microbial fertilizer is designed around "protecting microbial activity and ensuring the function of secondary metabolites," avoiding the inhibition of microorganisms by high concentrations of nutrients, while allowing polyglutamic acid and γ-aminobutyric acid to fully exert their soil-improving and stress-resistance effects. The specific steps are as follows:

[0051] 1. Preparation of compound microbial agent: *Bacillus sicca* and *Bacillus mycosis fungoides* were separately inoculated onto LB agar plates and incubated at 37℃ for 24 h for activation. Then, they were separately inoculated into LB liquid medium and incubated on a shaker at 37℃ and 180 rpm for 24 h to obtain seed culture. The seed culture was then inoculated into fermentation medium (5 g / L glucose, 20 g / L soybean meal, 2.5 g / L bone peptone, 15 g / L corn starch, 2.5 g / L Angel yeast extract, 0.5 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, pH 7.2) and incubated on a shaker at 37℃ for 48 h to obtain fermentation broth. The viable cell counts of the *Bacillus sicca* and *Bacillus mycosis fungoides* broths were tested separately, and then compounded at a 1:1 ratio to obtain a functional microbial agent with a total viable cell count ≥ 5 × 10⁻⁶. 9 CFU / mL.

[0052] 2. Preparation of compound microbial fertilizer: First, prepare inorganic materials by mixing calcium ammonium nitrate, magnesium nitrate and potassium nitrate in a ratio of 1:2:1 to obtain inorganic materials. Inorganic materials are added to a mixture of polyglutamic acid stock solution and γ-aminobutyric acid stock solution, and stirred evenly (stirred for 80-120 min at 25-30℃ and 160-200 r / min) to obtain a suspension containing polyglutamic acid and γ-aminobutyric acid. Under stirring, the functional microbial agent is added to the suspension and stirred evenly. Then, pure water is added to make up the remaining amount, and stirring is continued (stirring for 40-60 minutes at 25-30℃ and 120-150r / min). The pH value is adjusted to 5.5 to obtain the compound microbial fertilizer.

[0053] Example 2

[0054] This invention provides a compound microbial fertilizer suitable for flue-cured tobacco production, with the proportions of each component as shown in Table 2 (the following components are proportioned by mass):

[0055] Table 2 Ingredients of Compound Microbial Fertilizer

[0056]

[0057] Preparation method of Example 2

[0058] The preparation process of compound microbial fertilizer is designed around "protecting microbial activity and ensuring the function of secondary metabolites," avoiding the inhibition of microorganisms by high concentrations of nutrients, while allowing polyglutamic acid and γ-aminobutyric acid to fully exert their soil-improving and stress-resistance effects. The specific steps are as follows:

[0059] 1. Preparation of compound microbial agent: *Bacillus sicca* and *Bacillus mycosis fungoides* were separately inoculated onto LB agar plates and incubated at 37℃ for 24 h for activation. Then, they were separately inoculated into LB liquid medium and incubated on a shaker at 37℃ and 180 rpm for 24 h to obtain seed culture. The seed culture was then inoculated into fermentation medium (5 g / L glucose, 20 g / L soybean meal, 2.5 g / L bone peptone, 15 g / L corn starch, 2.5 g / L Angel yeast extract, 0.5 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, pH 7.2) and incubated on a shaker at 37℃ for 48 h. After viable cell count testing of the *Bacillus sicca* and *Bacillus mycosis fungoides* cultures, they were compounded at a 2:1 ratio to obtain a functional microbial agent with a total viable cell count ≥ 5 × 10⁻⁶. 9 CFU / mL.

[0060] 2. Preparation of compound microbial fertilizer: First, prepare the inorganic material by mixing calcium ammonium nitrate, magnesium nitrate and potassium nitrate in a ratio of 4:0.5:3. Inorganic materials are added to a mixture of polyglutamic acid stock solution and γ-aminobutyric acid stock solution, and stirred evenly (stirred for 80-120 min at 25-30℃ and 160-200 r / min) to obtain a suspension containing polyglutamic acid and γ-aminobutyric acid. Under stirring, the functional microbial agent is added to the suspension and stirred evenly. Then, pure water is added to make up the remaining amount, and stirring is continued (stirring for 40-60 minutes at 25-30℃ and 120-150r / min). The pH value is adjusted to 5.5 to obtain the compound microbial fertilizer.

[0061] Example 3

[0062] This invention provides a compound microbial fertilizer suitable for flue-cured tobacco production, with the proportions of each component as shown in Table 3 (the following components are proportioned by mass):

[0063] Table 3. Ingredients of Compound Microbial Fertilizer

[0064] Preparation method of Example 3:

[0065] The preparation process of compound microbial fertilizer is designed around "protecting microbial activity and ensuring the function of secondary metabolites," avoiding the inhibition of microorganisms by high concentrations of nutrients, while allowing polyglutamic acid and γ-aminobutyric acid to fully exert their soil-improving and stress-resistance effects. The specific steps are as follows:

[0066] 1. Preparation of compound microbial agent: *Bacillus sicca* and *Bacillus mycosis fungoides* were separately inoculated onto LB agar plates and incubated at 37℃ for 24 h for activation. Then, they were separately inoculated into LB liquid medium and incubated on a shaker at 37℃ and 180 rpm for 24 h to obtain seed culture. The seed culture was then inoculated into fermentation medium (5 g / L glucose, 20 g / L soybean meal, 2.5 g / L bone peptone, 15 g / L corn starch, 2.5 g / L Angel yeast extract, 0.5 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, pH 7.2) and incubated on a shaker at 37℃ for 48 h. After viable cell count testing of the *Bacillus sicca* and *Bacillus mycosis fungoides* cultures, they were compounded at an effective viable cell ratio of 1.5:1 to obtain a functional microbial agent with a total viable cell count ≥ 5 × 10⁻⁶. 9 CFU / mL.

[0067] 2. Preparation of compound microbial fertilizer: First, prepare inorganic materials by mixing calcium ammonium nitrate, magnesium nitrate and potassium nitrate in a ratio of 3:1:2 to obtain inorganic materials. Inorganic materials are added to a mixture of polyglutamic acid stock solution and γ-aminobutyric acid stock solution, and stirred evenly (stirred for 80-120 min at 25-30℃ and 160-200 r / min) to obtain a suspension containing polyglutamic acid and γ-aminobutyric acid. Under stirring, the functional microbial agent is added to the suspension and stirred evenly. Then, pure water is added to make up the remaining amount, and stirring is continued (stirring for 40-60 minutes at 25-30℃ and 120-150r / min). The pH value is adjusted to 5.5 to obtain the compound microbial fertilizer.

[0068] Application of the Examples

[0069] Based on the differences in soil fertility in continuously cropped tobacco fields, multiple gradient field concentrations are set to ensure suitability for tobacco areas with different continuous cropping years, while maximizing functionality through differentiated application:

[0070] I. Pot Experiment with Compound Microbial Fertilizer

[0071] 1. Materials and Methods

[0072] 1.1 Experimental Location

[0073] The experiment was conducted in the greenhouse of the Yunnan Provincial Microbial Fermentation Engineering Research Center.

[0074] 1.2 Experimental Materials

[0075] Test soil: Red soil

[0076] Experimental variety: Yunyan 87;

[0077] The tested fertilizer was the compound microbial fertilizer in Example 3, an inorganic fertilizer (excluding trace elements, polyglutamic acid, γ-aminobutyric acid, and microbial agents, with a total nutrient content of 19.88%, of which nitrogen content was 9.95% and potassium content was 9.93%).

[0078] 1.3 Experimental Design

[0079] Experimental group: Three concentration gradients were set up, namely 100 times, 200 times and 400 times dilution of compound microbial fertilizer, which were applied to the roots 10 days and 20 days after transplanting, with 500 mL per plant each time;

[0080] Control group 1: Inorganic fertilizer diluted 200 times was applied, and other procedures were the same as those in the experimental group;

[0081] Control group 2: Water control;

[0082] Five pots were planted for each treatment, and the plants were inoculated with the pathogen of tobacco black shank at the time of transplanting. Growth indicators and black shank disease index were measured 30 days after transplanting.

[0083] Inoculation with the pathogen of black shank disease: The pathogen of tobacco black shank, *Phytophthora tobaccoris* strain YBF-23-003, was provided by the strain bank of the Yunnan Provincial Microbial Fermentation Engineering Research Center. The strain was activated for 5 days by inoculation onto oat solid medium, and then the stipes were inoculated into oat culture medium and incubated at 28℃ and 180 r·min. -1 After shaking culture for 7 days, the bacterial solution containing mycelial fragments was obtained by filtration with sterile gauze. The soil around the rhizosphere was loosened to expose 2 cm of lateral roots, and 20 mL of bacterial solution was injected with a syringe. After covering with soil, the substrate moisture content was maintained at 70%, and the plants were cultured in a greenhouse at 28℃. The incidence of black shank disease was investigated 30 days after transplanting.

[0084] 2. Measurement Results

[0085] 2.1 Measurement of growth indicators

[0086] Table 4. Results of growth indicators measured 30 days after transplanting

[0087]

[0088] The results showed that tobacco plants grown with all concentration gradients of compound microbial fertilizer exhibited better growth than the control. Compared with the inorganic fertilizer control, plant height increased by 15.90%-19.92%, stem circumference increased by 7.13%-13.65%, and maximum leaf area increased by 8.55%-14.73%. This indicates that compound microbial fertilizer can significantly promote tobacco plant growth. The 100-fold and 200-fold dilutions had similar effects, while the 400-fold dilution was slightly less effective.

[0089] 2.2 Determination of Blackleg Disease Severity Index

[0090] Table 5. Results of Black Shank Disease Severity Index and Relative Prevention Efficacy Measurement

[0091]

[0092] The results showed that the black shank disease index of compound microbial fertilizer at all concentration gradients was lower than that of the control. Compared with the inorganic fertilizer control, the black shank disease index decreased by 25.93%–51.85%, and the relative control efficacy increased by 21.21–42.42 percentage points. This indicates that compound microbial fertilizer has a good control effect on black shank disease, with the 100-fold dilution showing the best effect; the 200-fold dilution was the second best, but the control efficacy was still above 50%; the 400-fold dilution was the weakest and suitable for soils with fewer pathogens.

[0093] 3. Summary

[0094] The pot experiment results showed that root irrigation with the compound microbial fertilizer in Example 3 significantly promoted tobacco plant growth and had a good control effect on black shank disease. In promoting tobacco plant growth, the 100-fold and 200-fold dilutions were equally effective, while the 400-fold dilution was slightly less effective. Regarding the control effect on black shank disease, the 100-fold dilution was the most effective, followed by the 200-fold dilution, and the 400-fold dilution was the least effective.

[0095] II. Application of Compound Microbial Fertilizer in Tobacco Production in Huili County, Liangshan City

[0096] 1. Materials and Methods

[0097] 1.1 Test Site

[0098] The experiment was conducted in Lixi Town, Huili City, Liangshan Prefecture, Sichuan Province. The experimental site was at an altitude of 1774.4m, with paddy soil of moderate fertility. It was a plot that had been continuously cropped for 5 years, and black shank disease had been relatively severe in previous years.

[0099] 1.2 Test Materials

[0100] Test variety: Yunyan 121;

[0101] The tested fertilizers were: tobacco-specific compound fertilizer for base (N-P2O5-K2O=10-20-20), tobacco-specific compound fertilizer for top dressing (N-P2O5-K2O=9-0-35), potassium nitrate (N-P2O5-K2O=13.5-0-44.5), and the compound microbial fertilizer in Example 3.

[0102] 1.3 Experimental Design

[0103] This experiment used a plot design with four treatments, three replicates per treatment, and one plot per replicate. Each plot was planted with 60 tobacco plants. The treatments are as follows:

[0104] Table 6 Test Treatment

[0105]

[0106] 1.4 Survey Data and Methods

[0107] (1) After harvesting and baking, a mixed soil sample was taken from each treatment and its soil bulk density, black shank pathogen count and soil bacterial diversity index were determined.

[0108] (2) After the capping, 15 representative tobacco plants were selected for each treatment to measure their plant height, stem circumference and maximum leaf area.

[0109] (3) After harvesting and roasting, the occurrence of black shank disease in each treatment was statistically analyzed.

[0110] (4) After the harvest and curing are completed, the economic traits of each treatment in the experimental area, such as yield per mu, output value per mu, average price, proportion of high-grade tobacco, and proportion of medium-to-high-grade tobacco, are investigated and statistically analyzed.

[0111] 2. Measurement Results

[0112] 2.1 Soil index determination

[0113] Table 7 Soil Nutrients Before and After Tobacco Planting

[0114]

[0115] The results showed that soil indicators at all concentration gradients of compound microbial fertilizer were superior to those of conventional fertilization. Compared with the conventional fertilization control, soil bulk density decreased by 3.15%-6.30%, the number of black shank pathogens decreased by 20.57%-43.35%, and the Sobs index of soil bacterial communities increased by 7.64%-12.96%. This indicates that compound microbial fertilizer can alleviate the continuous cropping obstacles in continuously cropped soils. The 100-fold and 200-fold dilutions had similar effects, while the 400-fold dilution was slightly less effective.

[0116] 2.2 Agronomic Trait Survey

[0117] Table 8 Results of the agronomic traits survey

[0118]

[0119] The results showed that the agronomic traits of flue-cured tobacco treated with compound microbial fertilizer at all concentration gradients were superior to those treated with conventional fertilization. Compared with the conventional fertilization control, plant height increased by 5.33%-10.82%, stem circumference increased by 7.87%-14.61%, and maximum leaf area increased by 6.93%-14.71%. This indicates that compound microbial fertilizer can significantly promote tobacco plant growth. The effects of 100-fold and 200-fold dilutions were comparable, while the effect of 400-fold dilution was slightly weaker.

[0120] 2.3 Investigation into the occurrence of black shank disease

[0121] Table 9. Occurrence of Blackleg

[0122]

[0123] The results showed that the incidence of black shank was better with all concentration gradients of compound microbial fertilizer than with conventional fertilization. Compared with the conventional fertilization control, the disease index decreased by 36.30%-63.70%. This indicates that compound microbial fertilizer has a good control effect on black shank, with the 100-fold dilution showing the best effect, followed by the 200-fold dilution, and the 400-fold dilution showing the weakest effect.

[0124] 2.4 Economic Characteristics of Flue-cured Tobacco

[0125] Table 10 Survey of Economic Characteristics of Flue-cured Tobacco

[0126]

[0127] The results showed that the economic traits of flue-cured tobacco improved by compound microbial fertilizer at all concentration gradients compared to conventional fertilization. Compared with the conventional fertilization control, the yield per mu increased by 3.35%-7.79%, and the proportion of high-quality tobacco increased by 3.55%-9.03%. This indicates that compound microbial fertilizer can improve the economic traits of flue-cured tobacco, with the 100-fold and 200-fold dilutions showing relatively better effects, while the 400-fold dilution was slightly less effective.

[0128] 3. Summary

[0129] Field results showed that root irrigation with the compound microbial fertilizer in Example 3 could alleviate soil continuous cropping obstacles, promote tobacco plant growth, reduce the occurrence of black shank disease, and increase the yield per acre and the proportion of high-quality tobacco. In terms of application, this compound microbial fertilizer adopts a four-in-one synergistic logic of "inorganic nutrients + polyglutamic acid + γ-aminobutyric acid + functional microorganisms": micronutrients are supplemented first, polyglutamic acid chelates nutrients to the roots quickly, γ-aminobutyric acid makes tobacco plants more resistant to adversity, and beneficial microorganisms suppress black shank pathogens, ultimately achieving "disease prevention and growth promotion + continuous cropping repair" simultaneously. The method of use is simple—direct root irrigation allows the effective ingredients to reach the root system directly, quickly overcoming absorption obstacles caused by continuous cropping. Field concentrations follow the principle of "the longer the continuous cropping and the less fertile the soil, the lower the dilution": 100 times dilution is most economical for infertile land with more than 5 years of continuous cropping, 200 times dilution is most economical for medium-fertility land with 3-5 years of continuous cropping, and 400 times dilution is sufficient for relatively fertile land with 1-2 years of continuous cropping, flexibly adapting to different tobacco fields.

[0130] In terms of alleviating continuous cropping obstacles, promoting tobacco plant growth, increasing the yield per acre of flue-cured tobacco, and increasing the proportion of high-quality tobacco, the 100-fold and 200-fold dilutions of the compound microbial fertilizer of this invention are equally effective, while the 400-fold dilution is slightly less effective. Regarding reducing the occurrence of black shank disease, the 100-fold dilution is the most effective, followed by the 200-fold dilution, and the 400-fold dilution is the least effective. Considering both the overall effectiveness and cost, in fields with moderate soil fertility and a continuous cropping period of 3-5 years, it is recommended to use a 200-fold dilution of the compound microbial fertilizer for root irrigation.

[0131] The 16S rDNA sequence of Bacillus sicca XNSD01 (SEQ ID NO.1): 16S rDNA sequence of Bacillus mycoides - like XNSD02 (SEQ ID NO.2): ATGCAAGTCGAGCGAACGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCACATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAATATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTATCCCGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGATAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAAATATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGCTTCCGCCCATTAGTGCTGAAGTTAACGCATTAAGCACTCCGCC TGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCC TTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGTGACAAACCG GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAA CTCCCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTCTTGGAGCCAGCCG Biological Preservation Instructions: Bacillus siamensis XNSD01 was deposited on December 17, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M 20252934. The deposit address is located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University (opposite to the First Affiliated Primary School of Wuhan University).

[0132] Bacillus paramycoides XNSD02 was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M 20252935. The deposit address is located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University (opposite to the First Affiliated Primary School of Wuhan University).

Claims

1. A compound microbial fertilizer suitable for flue-cured tobacco production, characterized in that, It is composed of the following components by mass percentage: 4%~8% functional microbial agent, 12%~18% first improver, 1%~2% second improver, 30%~70% inorganic materials, and the balance is pure water; The first modifier is a polyglutamic acid stock solution with a mass percentage of ≥3.5% polyglutamic acid, and the second modifier is a γ-aminobutyric acid stock solution with a mass percentage of ≥50% γ-aminobutyric acid; the functional microbial agent is a compound of strain XNSD01 and strain XNSD02, strain XNSD01 is Bacillus sicca with accession number CCTCC NO.M 20252934, and strain XNSD02 is Bacillus mycosis fungoides with accession number CCTCC NO.M 20252935.

2. The compound microbial fertilizer suitable for flue-cured tobacco production according to claim 1, characterized in that, In the functional microbial agent, the ratio of effective viable bacteria of strain XNSD01 to strain XNSD02 is 1~3:1, and the effective viable bacteria count in the functional microbial agent is ≥5×10⁻⁶. 9 CFU / mL.

3. The compound microbial fertilizer suitable for flue-cured tobacco production according to claim 1, characterized in that, The inorganic material is composed of calcium ammonium nitrate, magnesium nitrate and potassium nitrate, and the mass ratio of calcium ammonium nitrate, magnesium nitrate and potassium nitrate is 1~4 : 0.5~2 : 1~3.

4. The compound microbial fertilizer suitable for flue-cured tobacco production according to claim 3, characterized in that, The inorganic material in the compound microbial fertilizer has a total nutrient mass fraction of 8% to 20%, of which nitrogen has a mass fraction of 4% to 12%, potassium has a mass fraction of 4% to 10%, calcium has a mass fraction of 2.5% to 6%, and magnesium has a mass fraction of 0.3% to 6.5%.

5. A method for preparing a compound microbial fertilizer suitable for flue-cured tobacco production, characterized in that, Includes the following steps: Ammonium calcium nitrate, magnesium nitrate and potassium nitrate are mixed evenly in a certain proportion to obtain inorganic materials; Inorganic materials are added to a mixture of polyglutamic acid stock solution and γ-aminobutyric acid stock solution, and stirred until homogeneous to obtain a suspension containing polyglutamic acid and γ-aminobutyric acid; wherein the mass percentage of polyglutamic acid in the polyglutamic acid stock solution is ≥3.5%, and the mass percentage of γ-aminobutyric acid in the γ-aminobutyric acid stock solution is ≥50%; After detecting the viable cell counts of bacterial suspensions of strains XNSD01 and XNSD02, they were compounded according to the effective viable cell count ratio to obtain a functional microbial agent; strain XNSD01 is Bacillus sicca with accession number CCTCC NO.M 20252934, and strain XNSD02 is Bacillus mycosis fungoides with accession number CCTCC NO.M 20252935; Under stirring, the functional microbial agent is added to the suspension and stirred evenly. Then, pure water is added to make up the remaining amount, and stirring is continued. The pH value is adjusted to 5.5~6.5 to obtain compound microbial fertilizer.

6. The method for preparing the compound microbial fertilizer suitable for flue-cured tobacco production according to claim 5, characterized in that, The mass ratio of calcium ammonium nitrate, magnesium nitrate, and potassium nitrate is 1-4: 0.5-2: 1-3; the ratio of effective viable bacteria counts of strain XNSD01 and strain XNSD02 is 1-3:1, and the effective viable bacteria count in the functional microbial agent is ≥5×10⁻⁶. 9 CFU / mL.

7. The method for preparing the compound microbial fertilizer suitable for flue-cured tobacco production according to claim 5, characterized in that, The compound microbial fertilizer contains 4% to 8% by mass of functional microbial agents, 12% to 18% by mass of polyglutamic acid stock solution, 1% to 2% by mass of γ-aminobutyric acid stock solution, and 30% to 70% by mass of inorganic materials.

8. The application of the compound microbial fertilizer according to any one of claims 1-6 or the compound microbial fertilizer prepared by the method according to any one of claims 5-7 in promoting the growth of flue-cured tobacco, improving the disease resistance of flue-cured tobacco and / or alleviating the obstacles of continuous cropping of flue-cured tobacco.

9. The application according to claim 8, characterized in that, The compound microbial fertilizer is diluted 100 to 400 times and applied by root irrigation, with 500 mL applied to each tobacco plant on the 10th and 20th days after transplanting.

10. The application according to claim 8, characterized in that, The measures to promote flue-cured tobacco growth include increasing plant height, stem circumference, and maximum leaf area; the measures to improve disease resistance include enhancing the immunity of flue-cured tobacco to the pathogen of tobacco black shank; and the measures to alleviate continuous cropping obstacles include reducing soil bulk density and increasing soil microbial diversity index in continuously cropped tobacco fields.

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