A complex microbial agent containing bacillus pseudomycoides hmc18 and application thereof
By using a compound microbial agent of Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5, the problem of paclobutrazol residues harming soil and crops has been solved, resulting in improved soil microecological environment and increased crop yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QINHUANGDAO HEMIAO BIOLOGICAL TECH CO LTD
- Filing Date
- 2026-01-07
- Publication Date
- 2026-07-31
AI Technical Summary
Long-term or excessive use of paclobutrazol leads to the accumulation of residues in the soil, affecting plant growth and causing phytotoxicity to subsequent planting. Existing compound microbial agents have poor degradation effects and cannot effectively improve the soil micro-ecological environment.
A compound microbial agent was prepared by using Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5, combined with granular organic fertilizer, binder and anti-caking agent, to degrade paclobutrazol residues and regulate soil microbial community.
It can efficiently degrade paclobutrazol residues, promote crop growth, increase yield, improve the soil micro-ecological environment, and regulate the soil microbial community structure.
Smart Images
Figure CN121874010B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a compound microbial agent containing Bacillus pseudomyophyte HMC18 and its application. Background Technology
[0002] Paclobutrazol, molecular formula C 15 H 20 ClN3O, also known as chlorbuterol, is a synthetic, highly effective, and low-toxic plant growth retardant and broad-spectrum fungicide. It has the effects of delaying plant growth, inhibiting stem elongation, shortening internodes, promoting plant tillering, promoting flower bud differentiation, increasing plant stress resistance, and increasing yield.
[0003] Long-term or excessive use of paclobutrazol can lead to the accumulation of paclobutrazol residues in the soil. These residues may cause phytotoxicity to sensitive plants planted subsequently, and also pose a potential threat to human health through the food chain. Because paclobutrazol affects plant growth, it indirectly impacts soil fertility; under aerobic conditions, the half-life of paclobutrazol in soil ranges from 43 to 618 days (average 182 days). Studies have shown that compared to soils without paclobutrazol application, soil treated with paclobutrazol showed a 58% reduction in bacteria, a 28% reduction in actinomycetes, and a 28% reduction in fungi.
[0004] The application of biotechnology to degrade paclobutrazol residues has the advantages of low cost, simple operation and short remediation cycle, and has great advantages in pollution control. It is an emerging remediation technology, but there are not many compound microbial agents that can be used to degrade paclobutrazol residues at present. Summary of the Invention
[0005] The purpose of this invention is to provide a compound microbial agent that can effectively degrade paclobutrazol residues, improve the soil micro-ecological environment, and increase crop yield.
[0006] The present invention adopts the following technical solution: A compound microbial agent comprising Bacillus pseudomycosis ( Bacillus pseudomycoides HMC18 and Bacillus subtilis ( Bacillus subtilis HM-5.
[0007] Furthermore, the *Bacillus pseudomycosis* ( Bacillus pseudomycoides HMC18, with accession number CGMCC No.35492, was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center, located in Beijing, China.
[0008] Furthermore, the Bacillus subtilis ( Bacillus subtilisHM-5, with accession number CGMCC No. 24999, was deposited on June 2, 2022, at the China General Microbiological Culture Collection Center, located in Beijing, China.
[0009] Furthermore, the total number of viable bacteria in the compound microbial agent is not less than 2 × 10⁻⁶. 8 cfu / g.
[0010] Furthermore, the ratio of viable bacteria counts of *Bacillus pseudomycosis* HMC18 to *Bacillus subtilis* HM-5 is 20-40:25-45.
[0011] Furthermore, the compound microbial agent also includes granular organic fertilizer, binder, and anti-caking agent.
[0012] Preferably, the granular organic fertilizer contains no less than 50% organic matter and no less than 8.5% N+P+K; the binder is caramel color; and the anti-caking agent is a mixture of talc powder and diatomaceous earth in a mass ratio of 1:1.
[0013] Furthermore, the composite microbial agent is prepared by the following method: (1) Prepare Bacillus pseudomycosis HMC18 powder and Bacillus subtilis HM-5 powder respectively.
[0014] (2) Mix Bacillus pseudomycosis HMC18 powder and Bacillus subtilis HM-5 powder to obtain a powder mixture, and then mix the powder mixture with granular organic fertilizer, binder and anti-caking agent evenly.
[0015] Furthermore, the effective viable count of the *Bacillus pseudomycosis* HMC18 powder is not less than 3 × 10⁻⁶. 10 The effective viable count of the Bacillus subtilis HM-5 powder is not less than 2 × 10⁻⁶ cfu / g. 10 cfu / g.
[0016] Furthermore, the mass ratio of the compound microbial inoculant powder mixture, granular organic fertilizer, binder, and anti-caking agent is 5~20:1000:1~10:5~15.
[0017] Application of the above-mentioned compound microbial agent in the degradation of paclobutrazol residues in soil.
[0018] Application of the above-mentioned compound microbial agent in increasing the yield of plants grown in soil with paclobutrazol residue.
[0019] Application of the above-mentioned compound microbial agent in improving the soil microecological environment of paclobutrazol residue.
[0020] The beneficial effects of this invention are as follows: the compound microbial agent of this invention combines Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5, which can efficiently degrade paclobutrazol residues in the soil, promote crop growth and increase yield, and also regulate the soil micro-ecological environment and improve the soil micro-ecological environment. Attached Figure Description
[0021] Figure 1 The colony morphology of Bacillus pseudomycosis HMC18 after 24 hours of culture on solid LB medium.
[0022] Figure 2 The image shows the morphology of Bacillus pseudomycosis HMC18 under an optical microscope after Gram staining.
[0023] Figure 3 Phylogenetic tree of 16S rDNA for Bacillus pseudomycosis HMC18.
[0024] Figure 4 The results of the synergistic experiment between strain HMC18 *Bacillus pseudomycosis* and HM-5 *Bacillus subtilis* are presented. Detailed Implementation
[0025] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents used in the following embodiments are all available from conventional biochemical reagent stores.
[0026] Example 1: Screening and identification of Bacillus pseudomycosis HMC18
[0027] The strain HMC18 was isolated and purified from wheat plant samples collected from a wheat experimental field in Huanghua City, Hebei Province.
[0028] (1) Morphological characteristics The colonies are round, milky white to pale yellow, irregularly raised, with rough edges and a thick texture, like... Figure 1 As shown. The bacteria are rod-shaped, have spores, and are Gram-positive, as indicated. Figure 2 As shown.
[0029] (2) Physiological and biochemical characteristics The physiological and biochemical characteristics of strain HMC18 were identified, and the results are shown in Table 1.
[0030] Table 1. Physiological and biochemical characteristics of strain HMC18 .
[0031] (3) Molecular biological characteristics Genomic DNA was extracted from strain HMC18. Using it as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplified product was recovered and sequenced, yielding a DNA sequence containing 1487 bp (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST alignment analysis. Comparison with the 16S rDNA sequence in the NCBI database revealed that HMC18 showed 99% identity with *Bacillus pseudomycosis*. The phylogenetic tree was constructed as follows: Figure 3 As shown. Based on the morphological, sequencing analysis, and physiological and biochemical detection results, HMC18 was identified as *Bacillus pseudomycosis* (…). Bacillus pseudomycoides ).
[0032] Bacillus pseudomycosis HMC18 was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 35492.
[0033] Example 2: Synergistic test between Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5
[0034] LB solid medium formula: 5g yeast extract, 10g peptone, 10g sodium chloride, and 18g agar are placed in a 1000mL beaker, 900mL distilled water is added and heated to dissolve, the pH is adjusted to 7.2~7.4, and the volume is brought to 1L with distilled water. The mixture is then sterilized at 121℃ for 30 minutes and set aside for later use.
[0035] Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5 were activated using LB medium, respectively.
[0036] A colony of Bacillus subtilis HM-5 was picked up using an inoculation loop and placed in 1 mL of sterile water. The mixture was repeatedly pipetted and stirred to prepare a bacterial suspension. This suspension was then added to LB medium that had not yet solidified but was still warm to the touch. After mixing, the suspension was immediately poured into plates. A Bacillus pseudomycosis HMC18 bacterial cake was placed in the center of the cooled and solidified plate, and the plates were labeled. The process was repeated three times. The plates were incubated at 37°C for 3 days. Results are as follows: Figure 4 As shown, Bacillus subtilis HM-5 and Bacillus pseudomycosis HMC18 co-grow without the formation of inhibition zones, indicating that Bacillus subtilis HM-5 and Bacillus pseudomycosis HMC18 do not inhibit each other.
[0037] Example 3: Degradation test of paclobutrazol by Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5
[0038] Inorganic salt culture medium formula: K2HPO4 0.1g, (NH4)2HPO4 0.1g, MgSO4·7H2O 0.02g, FeCl3 0.01g, CaCl2·2H2O 0.1g, NaCl2 0.1g, distilled water 1000mL, pH 7.0, sterilized at 121℃ for 20 min.
[0039] Paclobutrazol (purity: 98%, standard), purchased from the market.
[0040] Preparation method of inorganic salt culture medium containing paclobutrazol: Prepare a 5 g / L paclobutrazol stock solution with methanol, sterilize it by passing it through a 0.22 μm filter membrane, take a certain amount and place it in a sterile Erlenmeyer flask, and after the methanol has completely evaporated, add sterile inorganic salt culture medium to make the final concentration of paclobutrazol 50 mg / L.
[0041] One loopful of activated *Bacillus pseudomycosis* HMC18 and *Bacillus subtilis* HM-5 were inoculated into 50 mL of LB liquid medium and cultured at 35 °C and 160 rpm for 48 h on a shaker to obtain bacterial suspensions. The bacterial concentration was then adjusted to 1 × 10⁻⁶. 9 cfu / mL, for later use.
[0042] This experiment included the following four treatments: Treatment 1: 1 mL of Bacillus pseudomycosis HMC18 suspension + 50 mL of inorganic salt culture medium containing paclobutrazol; Treatment 2: 1 mL of Bacillus subtilis HM-5 bacterial suspension + 50 mL of inorganic salt culture medium containing paclobutrazol; Treatment 3: 0.5 mL of Bacillus pseudomycosis HMC18 suspension + 0.5 mL of Bacillus subtilis HM-5 suspension + 50 mL of inorganic salt culture medium containing paclobutrazol; Treatment 4: 1 ml of sterile water + 50 mL of inorganic salt culture medium containing paclobutrazol.
[0043] Under aseptic conditions, each treatment was repeated three times and cultured in a shaker at 35°C and 160 rpm for 7 days. The paclobutrazol content was determined by high performance liquid chromatography, and its degradation rate was calculated.
[0044] Table 2. Degradation rate of paclobutrazol in each treatment .
[0045] The results showed that the combination of Bacillus pseudomycosis HMC18 and Bacillus subtilis HM-5 could effectively degrade paclobutrazol, and the degradation effect was better than that of a single species.
[0046] Example 4: Preparation of Bacillus pseudomycosis HMC18 bacterial powder
[0047] (1) LB liquid culture medium: 5g yeast extract, 10g peptone and 10g sodium chloride are placed in a 1000mL beaker, 900mL distilled water is added and heated to dissolve, pH is adjusted to 7.2~7.4, and the volume is adjusted to 1L with distilled water. Sterilize at 121℃ for 30 minutes and set aside.
[0048] (2) Activation of strain: Pick a loopful of Bacillus pseudomycosis HMC18 colonies, inoculate them into a test tube containing 10 mL LB liquid medium, and activate them by constant temperature shaking at 160 rpm and 35 °C for 24 h.
[0049] (3) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. Incubate at 160 rpm and 35 °C for 24 h to obtain seed culture.
[0050] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.6 L of LB liquid medium and cultured at 160 rpm and 35 °C for 48 h with constant temperature shaking to obtain the fermentation broth of Bacillus pseudomycosis HMC18, with an effective viable count of 5.62 × 10⁻⁶ cells / mL. 9 cfu / mL.
[0051] (5) Preparation of bacterial powder: The fermentation broth and diatomaceous earth were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed using a freeze dryer to obtain Bacillus pseudomycosis HMC18 powder. The effective viable count was tested to be 5.22 × 10⁻⁶. 10 cfu / g.
[0052] Example 5: Preparation of Bacillus subtilis HM-5 bacterial powder
[0053] The preparation process was the same as in Example 4. The effective viable count of Bacillus subtilis HM-5 powder was detected to be 4.85 × 10⁻⁶. 10 cfu / g.
[0054] Example 6 Preparation of Compound Microbial Agent
[0055] (1) Materials Adhesive: Caramel color, purchased from the market, dissolved in warm water at a weight ratio of 1:1 before use.
[0056] Granular organic fertilizer: purchased from the market, its content: organic matter 50.0%, N+P+K=8.5%.
[0057] Anti-caking agent: made from talc powder and diatomaceous earth in a 1:1 mass ratio. Both talc powder and diatomaceous earth were purchased from the market.
[0058] (2) Preparation method Take 8g of *Bacillus pseudomycosis* HMC18 bacterial powder (Example 4) and 10.0g of *Bacillus subtilis* HM-5 bacterial powder (Example 5) to obtain a mixture of 18g of bacterial powders. Mix the bacterial powder mixture with 1kg of granular organic fertilizer and stir for 5 minutes. Then add 6g of caramel coloring and stir for 5 minutes. Next, add 8g of anti-caking agent and stir for 5-8 minutes. The prepared product granules do not stick together, do not clump when squeezed, have a black and shiny appearance, and are free of powder, thus obtaining the compound microbial agent. The total viable count of the prepared compound microbial agent was tested to be 8.65 × 10⁻⁶. 8 cfu / g.
[0059] Example 7 Field Trial of Compound Microbial Inoculant
[0060] Experimental Location: A sweet potato field in Lulong County, Qinhuangdao City (soil is heavy clay soil). For many years, this farmer used large amounts of paclobutrazol (15% paclobutrazol wettable powder produced by Sichuan Run'er Technology Co., Ltd.) to increase sweet potato yield, applying 50-75 grams of 15% paclobutrazol per mu (approximately 0.067 hectares) diluted in 50-60 kg of water. The solution was sprayed evenly onto the tops of the sweet potato stems and leaves approximately 50 days after planting, and applied three times during the sweet potato growing season, with a 30-day interval between applications. Ultimately, this resulted in the accumulation of paclobutrazol in the soil, disrupting the soil ecosystem and preventing any increase in sweet potato yield.
[0061] Soil contaminated with paclobutrazol was selected from the region for sweet potato cultivation. Prior to the experiment, the paclobutrazol content in the soil was measured at 0.55 mg / kg. Land preparation was carried out on May 10, 2024. 50 kg of compound fertilizer (N:P2O5:K2O=17:17:17) and 5 kg of the compound microbial inoculant prepared in Example 6 were applied per mu (approximately 0.067 hectares) by furrow application. The experimental area was 3 mu (approximately 0.067 hectares), and the control area was 2 mu (approximately 0.067 hectares) without the inoculant. Planting density: 5000 plants / mu (approximately 0.067 hectares), row spacing 0.6 m, plant spacing 0.2 m, planted variety: Yanshu 25.
[0062] Methods: Approximately 40 days after sweet potato planting, 30 plants were randomly sampled to investigate vine length, stem diameter, and number of branches. Rhizosphere soil samples were collected using a 5-point sampling method. High-performance liquid chromatography (HPLC) was used to determine the paclobutrazol content in the experimental and control fields (average of three replicates for each treatment). Soil microbial communities (fungi, bacteria, and actinomycetes) were investigated using plate count. Sweet potato yields in the experimental and control fields were assessed at harvest.
[0063] Table 3. Statistical table of paclobutrazol content survey .
[0064] The results in Table 3 show that the composite microbial agent prepared in Example 6 can degrade paclobutrazol residues in the soil.
[0065] Table 4. Statistical Table of Soil Microbial Community Survey .
[0066] Table 4 shows that the composite microbial agent prepared in Example 6 increased the number of bacteria and actinomycetes in the soil, decreased the number of fungi, and increased the total number of soil microorganisms. In healthy soil, bacteria account for 70%–90% of the soil. High bacterial abundance combined with an appropriate amount of actinomycetes can enhance functions such as organic matter degradation and nutrient activation, while excessive fungi can easily lead to soil degradation. An increase in the total number of microorganisms, with beneficial bacteria and actinomycetes as the dominant groups, is an important indicator of improved ecological environment. Therefore, the composite microbial agent prepared in Example 6 can achieve the goal of regulating the soil microbial community and improving the soil micro-ecological environment.
[0067] Table 5. Statistical table of sweet potato vine length, number of branches, and yield. .
[0068] The results in Table 5 show that the compound microbial agent prepared in Example 6 can promote sweet potato growth and increase yield.
[0069] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.
Claims
1. A compound microbial agent, characterized in that, It includes Bacillus pseudomycosis ( Bacillus pseudomycoides HMC18 and Bacillus subtilis ( Bacillus subtilis HM-5; the preservation number of Bacillus pseudomyophyte HMC18 is CGMCC No. 35492; the preservation number of Bacillus subtilis HM-5 is CGMCC No. 24999.
2. The compound microbial agent according to claim 1, characterized in that, Its total number of viable bacteria is not less than 2×10 8 cfu / g.
3. The compound microbial agent according to claim 2, characterized in that, The ratio of viable counts of *Bacillus pseudomyophyte* HMC18 to *Bacillus subtilis* HM-5 is 20-40:25-45.
4. The compound microbial agent according to claim 3, characterized in that, It also includes granular organic fertilizer, binders, and anti-caking agents.
5. The compound microbial agent according to claim 4, characterized in that, It is prepared by the following method: (1) Prepare Bacillus pseudomycosis HMC18 bacterial powder and Bacillus subtilis HM-5 bacterial powder respectively; (2) Mix Bacillus pseudomycosis HMC18 powder and Bacillus subtilis HM-5 powder to obtain a powder mixture, and then mix the powder mixture with granular organic fertilizer, binder and anti-caking agent evenly.
6. The compound microbial agent according to claim 5, characterized in that, The effective viable count of the *Bacillus pseudomycosis* HMC18 powder is not less than 3 × 10⁻⁶. 10 The effective viable count of the Bacillus subtilis HM-5 powder is not less than 2 × 10⁻⁶ cfu / g. 10 cfu / g.
7. The compound microbial agent according to claim 6, characterized in that, The mass ratio of the compound microbial agent's bacterial powder mixture, granular organic fertilizer, binder, and anti-caking agent is 5~20:1000:1~10:5~15.
8. The application of a compound microbial agent as described in any one of claims 1 to 7 in the degradation of paclobutrazol residues in soil.
9. The application of a compound microbial agent as described in any one of claims 1 to 7 in increasing the yield of plants grown in soil with paclobutrazol residues and / or improving the soil microecological environment.