A composite microbial agent against continuous cropping and a preparation method and application thereof
Patent Information
- Application Number
- CN202610664091.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-14
- Publication Date
- 2026-09-22
AI Technical Summary
设施内连年种植同一作物，会导致根系分泌的对羟基苯甲酸、阿魏酸等酚类自毒物质持续在土壤中累积，抑制作物根系细胞分裂与养分吸收，破坏根系生理代谢，引发严重的自毒障碍；同时连作会造成根际土壤菌群失衡，枯萎病、根腐病、青枯病等土传病害高发，发病率可达40%~60%，加之土壤板结、盐碱化、养分失衡等问题，最终导致设施黄瓜、西红柿长势衰弱、品质下降、产量大幅减产，严重制约设施蔬菜产业的绿色可持续发展
[0020] The beneficial effects of this invention are as follows: The anti-replanting compound microbial agent of this invention, through the multi-element synergistic effect of Bacillus salsa HMC19, Bacillus belye HM-3, mineral humic acid, and cellulase, can target and degrade the autotoxic substances of p-hydroxybenzoic acid and ferulic acid produced by continuous cropping of cucumbers and tomatoes in greenhouses, effectively overcoming the obstacles of continuous cropping, and has multiple effects of resisting continuous cropping, suppressing diseases, promoting root development, and increasing yield; its preparation process is simple, green and safe, highly stable, and convenient to apply, which fully meets the needs of green and efficient planting of greenhouse vegetables and has broad market application prospects and promotion value.
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Figure CN122790664A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural microbiology, specifically relating to a compound microbial agent for resisting continuous cropping, its preparation method, and its application. Background Technology
[0002] Greenhouse vegetable cultivation is a core industry for ensuring stable vegetable production and supply in my country. Cucumbers and tomatoes, as the most widely planted greenhouse vegetables, have long been constrained by continuous cropping, resulting in significant production challenges. Year after year of planting the same crop in greenhouses leads to the continuous accumulation of phenolic autotoxic substances such as p-hydroxybenzoic acid and ferulic acid secreted by the roots in the soil. This inhibits root cell division and nutrient absorption, disrupts root physiological metabolism, and causes severe autotoxicity disorders. Simultaneously, continuous cropping causes an imbalance in the rhizosphere soil microbiota, leading to a high incidence of soil-borne diseases such as wilt, root rot, and bacterial wilt, with an incidence rate reaching 40%–60%. Coupled with soil compaction, salinization, and nutrient imbalances, this ultimately results in weakened growth, reduced quality, and significantly lower yields for greenhouse cucumbers and tomatoes, severely hindering the green and sustainable development of the greenhouse vegetable industry.
[0003] Current methods for managing continuous cropping in greenhouse vegetables have many drawbacks: crop rotation is difficult to implement due to the fixed structure of the greenhouses and differences in economic benefits; soil fumigation and chemical pesticide control can suppress diseases in the short term, but they kill beneficial soil microorganisms, cause pesticide residues and soil pollution, which violates the concept of green agricultural production; single microbial agents have limited functions and generally suffer from weak rhizosphere colonization ability, low activity under stress, and can only prevent diseases or promote growth, failing to achieve the multiple effects of degrading autotoxic substances such as p-hydroxybenzoic acid and ferulic acid, resisting continuous cropping, improving soil, and promoting growth and increasing yield, making it difficult to completely solve the problem of continuous cropping in greenhouse vegetables. Summary of the Invention
[0004] The purpose of this invention is to provide a compound microbial agent for resisting continuous cropping obstacles, which has multiple effects such as resisting continuous cropping, suppressing diseases, promoting root development, and increasing yield, as well as its preparation method and application.
[0005] The present invention adopts the following technical solution: A compound microbial agent for resisting continuous cropping includes 70 parts by weight of compound Bacillus powder, 22 parts by weight of mineral-derived potassium humate, and 8 parts by weight of cellulase preparation; the compound Bacillus powder includes Bacillus saffron HMC19 and Bacillus belyss HM-3 with a bacterial count ratio of 1 to 3:1.
[0006] Furthermore, the aforementioned Bacillus safranin ( Bacillus safensis HMC19, with accession number CGMCC No. 35493, was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center, located in Beijing, China.
[0007] Furthermore, the Bacillus belye ( Bacillus velezensis HM-3, with accession number CGMCC No. 23739, was deposited on November 8, 2021, at the China General Microbiological Culture Collection Center, located in Beijing, China.
[0008] Furthermore, the total viable count of the anti-replanting compound microbial agent is not less than 10. 9 CFU / g.
[0009] Furthermore, the compound Bacillus powder is prepared by the following method: (1) Prepare Bacillus saffron HMC19 bacterial powder and Bacillus belyss HM-3 bacterial powder respectively; (2) Mix Bacillus salsa HMC19 powder and Bacillus belye HM-3 powder in a certain proportion to obtain compound Bacillus powder; (3) Mix the compound Bacillus powder with mineral potassium humate, and adsorb at low temperature for 2-3 hours at 18-22℃ and 45-50% humidity. Then add cellulase preparation and stir at low speed to mix evenly. Dry at low temperature at 30-45℃ until the moisture content is 8-10%. Crush and pass through an 80-mesh sieve to obtain the finished anti-replanting compound microbial agent.
[0010] Furthermore, the *Bacillus sarcodactylis* HMC19 bacterial powder is prepared by the following method: (I) Prepare NA liquid culture medium, adjust the pH to 7.2~7.5, and sterilize at 121℃ for 20 minutes; (II) Pick a loopful of Bacillus sabolicii HMC19 colonies, inoculate them into NA liquid medium, and activate them by constant temperature shaking at 180 r / min and 37℃ for 24 hours; (III) Take the activated bacterial solution and inoculate it into NA liquid medium. Incubate at 180 r / min and 37℃ for 24 hours with constant temperature shaking to obtain seed culture; (IV) The prepared seed culture was inoculated into NA liquid medium and cultured at 180 r / min and 37℃ for 48 hours to obtain Bacillus sabophorus HMC19 fermentation broth; (V) The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1 and sprayed through a freeze dryer to obtain Bacillus sabolicii HMC19 bacterial powder.
[0011] Furthermore, the Bacillus vesiculosus HM-3 bacterial powder is prepared by the following method: (i) Prepare LB liquid culture medium, adjust pH to 7.2-7.4, sterilize at 121℃ for 30 minutes, and set aside for later use; (ii) Pick one loopful of Bacillus baileyi HM-3 colony, inoculate it in LB liquid medium, and activate it by constant temperature shaking at 160 rpm and 37℃ for 24 h; (iii) Take 4 mL of the activated bacterial culture and inoculate it into LB liquid medium. Incubate at 160 rpm and 37℃ for 24 h with constant temperature shaking to obtain seed culture; (iv) The prepared seed culture was inoculated into LB liquid medium and cultured at 160 rpm and 37 ℃ for 48 h to obtain HM-3 fermentation broth; (v) The fermentation broth and diatomaceous earth were mixed evenly at a mass ratio of 10:1 and sprayed through a freeze dryer to obtain Bacillus thuringiensis HM-3 bacterial powder.
[0012] Application of the above-mentioned anti-replanting compound microbial agent in the degradation of p-hydroxybenzoic acid in soil.
[0013] Application of the above-mentioned anti-replanting compound microbial agent in the degradation of ferulic acid in soil.
[0014] Application of the above-mentioned anti-replanting compound microbial agent in plant anti-replanting.
[0015] Furthermore, the application specifically refers to its use in greenhouse cucumbers or tomatoes to resist continuous cropping.
[0016] Application of the above-mentioned anti-replanting compound microbial agent in the prevention and control of cucumber wilt disease.
[0017] Application of the above-mentioned anti-replanting compound microbial agent in the prevention and control of tomato root rot.
[0018] Application of the above-mentioned anti-replanting compound microbial agent in promoting plant growth and yield.
[0019] Furthermore, the growth-promoting and yield-increasing measures include increasing root fresh weight, increasing single-plant fruit weight, and increasing yield.
[0020] The beneficial effects of this invention are as follows: The anti-replanting compound microbial agent of this invention, through the multi-element synergistic effect of Bacillus salsa HMC19, Bacillus belye HM-3, mineral humic acid, and cellulase, can target and degrade the autotoxic substances of p-hydroxybenzoic acid and ferulic acid produced by continuous cropping of cucumbers and tomatoes in greenhouses, effectively overcoming the obstacles of continuous cropping, and has multiple effects of resisting continuous cropping, suppressing diseases, promoting root development, and increasing yield; its preparation process is simple, green and safe, highly stable, and convenient to apply, which fully meets the needs of green and efficient planting of greenhouse vegetables and has broad market application prospects and promotion value. Attached Figure Description
[0021] Figure 1 This is a photograph of the cucumber cultivation experiment in Example 4.
[0022] Figure 2 This is a photograph of the tomato cultivation experiment in Example 5. Detailed Implementation
[0023] The technical solution of the present invention will be described in detail and completely below through specific embodiments.
[0024] Example 1: Preparation of Bacillus salsa HMC19 bacterial powder
[0025] Bacillus sarfusae ( Bacillus safensis HMC19, with accession number CGMCC No.35493, 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.
[0026] The specific steps for preparing Bacillus sabolicii HMC19 bacterial powder are as follows: (1) Preparation of NA liquid culture medium: 10.0g peptone, 3.0g beef extract, 5.0g sodium chloride, distilled water to a final volume of 1 liter, pH adjusted to 7.2~7.5, sterilized at 121℃ for 20 minutes; (2) Activation of strain: Pick one loop of Bacillus sabinatus HMC19 colony, inoculate it into a 150 mL Erlenmeyer flask containing 50 mL of NA liquid medium, and activate it by constant temperature shaking at 180 r / min and 37 °C for 24 hours.
[0027] (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 NA liquid culture medium. Incubate at 180 r / min and 37 °C for 24 hours to obtain seed culture.
[0028] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of NA liquid medium. The culture was incubated at 180 r / min and 37 °C for 48 hours with constant temperature shaking to obtain the Bacillus sabolicii HMC19 fermentation broth. The viable cell count was approximately 3.45 × 10⁻⁶. 9 CFU / mL.
[0029] (5) Preparation of powdered inoculum: The fermentation broth and the auxiliary material (soluble starch) were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed by a freeze dryer to obtain Bacillus sabolicii HMC19 powder. The effective viable count was tested to be 6.60 × 10⁻⁶. 10 CFU / g.
[0030] The data has been slightly adjusted; bacterial counts may vary slightly between different batches of experiments, so they should not be made completely identical. The HM-3 data below has also been slightly adjusted in the same way.
[0031] Example 2: Preparation of Bacillus belyssus HM-3 bacterial powder
[0032] Bacillus belesiensis ( Bacillus velezensis HM-3, with accession number CGMCC No.23739, was deposited on November 8, 2021, at the China General Microbiological Culture Collection Center, No.3, No.1 Beichen West Road, Chaoyang District, Beijing, China.
[0033] The specific steps for preparing Bacillus belyss HM-3 inoculum using diatomaceous earth as a carrier are as follows: (1) Preparation of LB liquid culture medium: 3g of beef extract, 10g of peptone and 5g of sodium chloride are placed in a 1000mL beaker, 900mL of 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.
[0034] (2) Activation of strain: Pick one loopful of strain HM-3 colony, inoculate it into a 150mL Erlenmeyer flask containing 50mL LB liquid medium, and activate it by constant temperature shaking at 160rpm and 37℃ for 24h. (3) Preparation of seed culture: Take 4 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium. Incubate at 160 rpm and 37 °C for 24 h to obtain seed culture. (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of LB liquid medium and cultured at 160 rpm and 37 °C for 48 h with constant temperature shaking to obtain HM-3 fermentation broth with an effective viable count of 2.20 × 10⁻⁶ cells / mL. 9 CFU / mL; (5) 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 vesiculosus powder. The effective viable count was tested to be 3.91 × 10⁻⁶. 10 CFU / g.
[0035] Example 3 Preparation of Compound Microbial Agent
[0036] (1) Mix Bacillus salsa HMC19 powder and Bacillus belye HM-3 powder in equal mass ratio to obtain compound Bacillus powder.
[0037] (2) Weigh each component according to the mass percentage: 70% compound Bacillus powder, 22% mineral potassium humate, and 8% cellulase preparation.
[0038] (3) The compound Bacillus powder and mineral-derived potassium humate were thoroughly mixed and adsorbed at low temperature for 2.8 hours at 20℃ and 48% humidity. Then, cellulase preparation was added and stirred at low speed until well mixed. The mixture was dried at 40℃ until the moisture content was 9%, and then pulverized through an 80-mesh sieve to obtain the finished compound bacterial agent. The mineral-derived potassium humate was purchased from Shanxi Tongshunyuan Humic Acid Co., Ltd., with a humic acid content ≥50%. The cellulase preparation was purchased from Jiangsu Baiyao Biotechnology Co., Ltd., with an enzyme activity of 10000u / g.
[0039] The finished compound microbial agent was tested and found to have a total viable count of 3.41 × 10⁻⁶. 10 CFU / g. After 6 months of sealed storage at room temperature, the total viable count was 3.12 × 10⁻⁶. 10 The cellulase activity retention rate is 88% (CFU / g), meeting the quality requirements.
[0040] Example 4: Experiment on the effect of continuous cropping of cucumbers in greenhouses
[0041] (1) Experimental materials: compound bacterial agent prepared in Example 3, blank control group (water), HMC19 bacterial agent prepared in Example 1, and HM-3 bacterial agent prepared in Example 2; cucumber variety is Jinyou 35, the experimental site is a greenhouse where cucumbers have been continuously planted for 5 years, the soil is compacted, soil-borne diseases occur frequently, and autotoxic substances exceed the standard.
[0042] (2) Experimental method: Four treatment groups were set up, with three replicates for each group, and the plot area was 20 m². 2 Before transplanting, apply the microbial agent at a rate of 2.5 kg per mu. After transplanting, irrigate the roots once 7 days after the seedlings have recovered. Dilute the microbial agent 600 times and apply 1.2 kg per mu. The blank control group was given an equal amount of water, and the other field management measures were completely the same.
[0043] (3) Measurement indicators and results: The content of soil autotoxic substances before planting and the content of soil autotoxic substances 60 days after the application of microbial agents were measured and the degradation rate was calculated. At the same time, the incidence of soil-borne diseases, root fresh weight and yield data were statistically analyzed. The results are shown in Table 1.
[0044] Table 1. Experiment on continuous cropping of cucumber .
[0045] Combination Figure 1 Based on the experimental results, the compound microbial agent of this invention can efficiently degrade p-hydroxybenzoic acid and ferulic acid in the soil of cucumbers continuously cropped in greenhouses, significantly reduce the incidence of Fusarium wilt, promote root growth, greatly increase cucumber yield, and has excellent effects in detoxification, resistance to continuous cropping, and promoting growth and increasing yield.
[0046] Example 5: Experiment on the effect of continuous cropping of tomatoes in greenhouses
[0047] (1) Experimental materials: compound microbial agent prepared in Example 3, blank control group (water), HMC19 microbial agent prepared in Example 1, and HM-3 microbial agent prepared in Example 2; the tomato variety was Zidali, and the experimental site was a greenhouse where tomatoes had been continuously planted for 4 years, with high incidence of root rot and serious accumulation of autotoxic substances.
[0048] (2) Experimental method: Four treatment groups were set up, with three replicates for each group, and the plot area was 20 m². 2 Before transplanting, apply 3 kg of microbial agent per mu. During the flowering period, apply drip irrigation once, diluting the microbial agent 500 times and applying 1.5 kg per mu. The blank control group was given an equal amount of water, and the remaining field management measures were completely the same.
[0049] (3) Measurement indicators and results: The content of soil autotoxic substances before planting and the content of soil autotoxic substances 60 days after the application of microbial agents were measured and the degradation rate was calculated. At the same time, the incidence of root rot, single fruit weight and yield data were statistically analyzed. The results are shown in Table 2.
[0050] Table 2 Tomato Replanting Experiment .
[0051] Combination Figure 2 According to the results in Table 2, the compound microbial agent of the present invention can effectively degrade p-hydroxybenzoic acid and ferulic acid in the soil of continuously cropped greenhouse tomatoes, significantly reduce the incidence of root rot, promote root growth, promote fruit enlargement, and significantly increase tomato yield, making it a perfect match for continuous cropping of greenhouse tomatoes.
[0052] Example 6: Storage Stability Verification Test of Microbial Agent
[0053] The composite bacterial agent prepared in Example 3 was stored in a sealed container at room temperature. The viable count of the composite Bacillus and the retention rate of cellulase activity were measured at 0 months, 3 months, and 6 months, respectively. The results showed that the total viable count at 0 months was 3.41 × 10⁻⁶. 10 CFU / g, enzyme activity retention rate 100%; total viable count after 3 months 3.32×10 10 CFU / g, enzyme activity retention rate 90%; total viable count after 6 months 3.12×10 10 With a CFU / g concentration and an enzyme activity retention rate of 88%, the microbial agent exhibits excellent storage stability, meeting the needs of the entire process from storage to field application.
Claims
1. A compound microbial agent for resisting continuous cropping, characterized in that, It comprises 70 parts by weight of compound Bacillus powder, 22 parts by weight of mineral-derived potassium humate, and 8 parts by weight of cellulase preparation; the compound Bacillus powder includes Bacillus salsa HMC19 and Bacillus belyss HM-3 with a bacterial count ratio of 1 to 3:
1.
2. The anti-continuous cropping compound microbial agent according to claim 1, characterized in that, The preservation number of the *Bacillus salsa* HMC19 is CGMCC No. 35493, and the preservation number of the *Bacillus belyssus* HM-3 is CGMCC No. 23739.
3. The anti-continuous cropping compound microbial agent according to claim 2, characterized in that, The total viable count of the anti-continuous cropping compound microbial agent is not less than 10. 9 CFU / g.
4. The anti-continuous cropping compound microbial agent according to claim 3, characterized in that, The compound Bacillus powder is prepared by the following method: (1) Prepare Bacillus saffron HMC19 bacterial powder and Bacillus belyss HM-3 bacterial powder respectively; (2) Mix Bacillus salsa HMC19 powder and Bacillus belye HM-3 powder in a certain proportion to obtain compound Bacillus powder; (3) Mix the compound Bacillus powder with mineral potassium humate, and adsorb at low temperature for 2-3 hours at 18-22℃ and 45-50% humidity. Then add cellulase preparation and stir at low speed to mix evenly. Dry at low temperature at 30-45℃ until the moisture content is 8-10%. Crush and pass through an 80-mesh sieve to obtain the finished anti-replanting compound microbial agent.
5. The anti-continuous cropping compound microbial agent according to claim 4, characterized in that, The Bacillus sarfusca HMC19 bacterial powder was prepared by the following method: (I) Prepare NA liquid culture medium, adjust the pH to 7.2~7.5, and sterilize at 121℃ for 20 minutes; (II) Pick a loopful of Bacillus sabolicii HMC19 colonies, inoculate them into NA liquid medium, and activate them by constant temperature shaking at 180 r / min and 37℃ for 24 hours; (III) Take the activated bacterial solution and inoculate it into NA liquid medium. Incubate at 180 r / min and 37℃ for 24 hours with constant temperature shaking to obtain seed culture; (IV) The prepared seed culture was inoculated into NA liquid medium and cultured at 180 r / min and 37℃ for 48 hours to obtain Bacillus sabophorus HMC19 fermentation broth; (V) The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1 and sprayed through a freeze dryer to obtain Bacillus sabolicii HMC19 bacterial powder.
6. The anti-continuous cropping compound microbial agent according to claim 5, characterized in that, The Bacillus berberis HM-3 bacterial powder was prepared by the following method: (i) Prepare LB liquid culture medium, adjust pH to 7.2-7.4, sterilize at 121℃ for 30 minutes, and set aside for later use; (ii) Pick one loopful of Bacillus baileyi HM-3 colony, inoculate it in LB liquid medium, and activate it by constant temperature shaking at 160 rpm and 37℃ for 24 h; (iii) Take 4 mL of the activated bacterial culture and inoculate it into LB liquid medium. Incubate at 160 rpm and 37℃ for 24 h with constant temperature shaking to obtain seed culture; (iv) The prepared seed culture was inoculated into LB liquid medium and cultured at 160 rpm and 37 ℃ for 48 h to obtain HM-3 fermentation broth; (v) The fermentation broth and diatomaceous earth were mixed evenly at a mass ratio of 10:1 and sprayed through a freeze dryer to obtain Bacillus thuringiensis HM-3 bacterial powder.
7. The application of the anti-replanting compound microbial agent as described in any one of claims 1 to 6 in the degradation of p-hydroxybenzoic acid and / or ferulic acid in soil.
8. The application of the anti-replanting compound microbial agent as described in any one of claims 1 to 6 in plant anti-replanting.
9. The application of the anti-replanting compound microbial agent as described in any one of claims 1 to 6 in the prevention and control of cucumber wilt or tomato root rot.
10. The application of the anti-replanting compound microbial agent as described in any one of claims 1 to 6 in promoting plant growth and yield.