Compound microbial agent as well as preparation and application methods thereof

Abstract

The invention provides a compound microbial agent as well as a preparation method and an application method thereof, aiming at the problems of environmental pollution, drug resistance, unstable biocontrol effect of a single strain and the like which are easily caused by existing chemical control, and belongs to the technical field of microbial preparations and biological control of plant diseases. The microbial agent is composed of bacillus aryabhattai MES85801 (CGMCC No.25404), bacillus velezensis MES861 (CGMCC No.16857), bacillus subtilis MES810 (CGMCC No.14514) and bacillus amyloliquefaciens MES81165, liquid fermentation culture is performed till the spore formation rate is larger than or equal to 90%, glycerin, skim milk powder, sodium alginate and a PVP composite stabilizer are added, spray drying is performed to prepare a powder preparation, and the effective viable count of the product is larger than or equal to 1.0 * 10 CFU / g. The complex microbial inoculant has a remarkable antagonistic effect on pathogenic bacteria of peach tree canker, has the functions of promoting growth, improving quality and increasing yield, and is suitable for the sustainable development requirement of green agriculture.

Description

A composite microbial agent and its preparation and application method Technical Field

[0001] This invention relates to the field of microbial agents and biological control of plant diseases, specifically to a compound microbial agent and its preparation and application methods. Background Technology

[0002] In the field of fruit tree disease control, peach canker, a serious fungal disease, has long been controlled primarily through agricultural management practices and chemical agents. Currently, conventional control methods include selecting resistant varieties, optimizing cultivation management (such as proper fertilization, irrigation, and pruning), reducing mechanical damage to plants to lower the risk of infection, and regularly spraying or applying chemical fungicides, such as benzimidazole pesticides like carbendazim. These methods can control the spread of the disease to some extent and have become relatively mature and widely used technical approaches in agricultural production. Furthermore, with the development of green agriculture, strategies utilizing single or multifunctional microorganisms to inhibit pathogens through competitive colonization, inducing resistance, or producing antimicrobial substances are gradually gaining attention and have been implemented in the field of some crop systems.

[0003] However, long-term reliance on chemical pesticides not only easily leads to drug resistance in pathogens, but also may cause environmental residues, disrupt the soil microecological balance, and affect the quality and safety of agricultural products, making it difficult to meet the development needs of sustainable agriculture. Meanwhile, existing microbial control products generally suffer from limited antagonistic activity of strains, weak environmental adaptability, poor formulation stability, or unstable efficacy, limiting their large-scale promotion in actual production. In particular, for peach canker, a specific disease, there is currently a lack of a compound microbial agent that combines highly effective antibacterial activity, promotes plant growth, improves fruit quality, and can function stably. Therefore, developing a novel compound microbial agent based on a combination of functional Bacillus strains with synergistic effects, clear storage information, and a reproducible production process has become an urgent technical challenge to be solved. Summary of the Invention

[0004] The core technical problem to be solved by this invention is to provide a compound microbial agent that has a highly effective biocontrol effect on peach tree canker, while also promoting peach tree growth, improving fruit quality and yield, overcoming the environmental pollution and drug resistance risks brought about by existing chemical control, as well as the defects of unstable biocontrol effect and narrow antibacterial spectrum of single strains.

[0005] To solve this problem, the core technical solution adopted by the present invention is: a compound microbial agent containing an effective amount of active microbial components and pharmaceutically acceptable excipients. The active microbial components include four functional strains of the genus Bacillus. The functional strains are Bacillus aryabhattai MES85801, Bacillus velezensis MES861, Bacillus subtilis MES810, and Bacillus amyloliquefaciens MES81165, and the total effective viable count in the compound microbial agent is not less than 1.0 × 10¹⁰ CFU / g.

[0006] Furthermore, in the compound microbial agent, the ratio of viable bacteria of Bacillus aryabhattai MES85801, Bacillus velezensis MES861, Bacillus subtilis MES810 and Bacillus amyloliquefaciens MES81165 is (1~3):(1~3):(1~3):(1~4).

[0007] Furthermore, the compound microbial agent is a solid preparation, and the preparation method of the agent includes the following steps: each strain is inoculated into a liquid fermentation medium after primary seed culture, and fermented for 25-40 hours at a temperature of 35°C and a tank pressure of 0.02-0.07 MPa, controlling the spore formation rate at the end of fermentation to be ≥90%; after fermentation, a compound stabilizer composed of glycerol, skim milk powder, sodium alginate and PVP is added, and then spray-dried to obtain a powdered preparation.

[0008] Furthermore, the liquid fermentation medium comprises the following components: corn steep liquor 0.4-0.8%, (NH4)2SO4 0.3-0.7%, CaCO3 0.8-1.2%, glucose 2-4%, KH2PO4 0.05-0.09%, and the pH is adjusted to 7.0-7.5.

[0009] Furthermore, in the composite stabilizer, by weight percentage, the amount of glycerin added is 1.0~2.5%, the amount of skim milk powder added is 0.1~0.4%, the amount of sodium alginate added is 1.5~2.5%, and the amount of PVP added is 0.5~2.0%.

[0010] Furthermore, the process parameters for spray drying are: inlet temperature 160~200°C, outlet temperature 75~90°C, and feed rate 15~30 L / h.

[0011] Furthermore, the compound microbial agent is a liquid preparation containing fermentation broth, stabilizer and water, and can be stably stored for more than 6 months at 4°C, with the effective viable count decreasing by no more than 15%.

[0012] Furthermore, the compound microbial agent is used to prevent and control peach canker caused by Dothiorellagregaria Sacc. or Botryosphaeria ribis (Tode).

[0013] Furthermore, the application method of the microbial agent is to dilute the compound microbial agent 500 times and then apply it by root irrigation, foliar spraying, or / and by smearing it on and around the diseased area.

[0014] The beneficial effects of the present invention are as follows: (1) The four Bacillus strains used are all clearly identified and independently preserved functional strains with stable genetic characteristics and a reproducible production basis; (2) The combination of multiple strains shows a significant synergistic effect in inhibiting the pathogens of peach tree canker (Dothiorella gregaria and Botryosphaeria ribis), with an inhibition rate of up to 82.5% or more; (3) By optimizing the fermentation process parameters (such as temperature, pH, tank pressure, and culture time) and the formula of the stabilizer before spraying and drying, the activity of the bacteria and the stability of the preparation are ensured, and the consistency of the field application effect is improved; (4) The bacterial agent not only has a strong ability to antagonize pathogens, but also has multiple functions such as promoting growth, improving soil, and degrading pesticide residues, realizing the integration of "disease prevention + yield increase + quality improvement", fundamentally solving the problems of traditional control methods relying on chemical pesticides, high ecological risks, and low efficacy and poor stability of existing microbial preparations, providing a safe, efficient and sustainable technical path for the green control of peach tree canker. Attached Figure Description

[0015] Figure 1. Inhibitory effects of four strains against peach tree canker pathogen: from left to right, the antagonistic effects of Bacillus aureus, Bacillus subtilis, Bacillus belye, and Bacillus amyloliquefaciens on peach tree canker pathogen. Detailed Implementation

[0016] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0017] The *Bacillus aryabhattai* MES85801 involved in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 26404, deposit date: January 6, 2023; *Bacillus velezensis* MES861 is deposited at the CGMCC, accession number: CGMCC No. 16857, deposit date: December 3, 2018; *Bacillus subtilis* MES810 is deposited at the CGMCC, accession number: CGMCC No. 14514, deposit date: August 10, 2017; and *Bacillus amyloliquefaciens*... The strain MES81165 (Bacillus amyloliquefaciens) was purchased from the China Agricultural Microbiological Culture Collection Center (ACCC) in March 2023. Its accession number at ACCC is ACCC 10225. Bacillus amyloliquefaciens belongs to the genus Bacillus, is Gram-positive, catalase-positive, oxidase-positive, and has smooth colonies measuring 5-10 mm.

[0018] Example 1 Preparation of Microbial Compound Agent The preparation method includes the following steps: (1) Primary seed culture: Bacillus oryzae MES85801, Bacillus belye MES861, Bacillus subtilis MES810, and Bacillus amyloliquefaciens MES81165 strains activated by test tube slant culture were cultured in ampoules. The ampoule culture medium was NA medium. The culture medium formula was: 12 g peptone, 4 g beef extract, 5 g sodium chloride, 20 g agar, 1000 ml ultrapure water, and pH 7.0. The culture conditions were: temperature 30℃, culture for 24 h.

[0019] (2) Fermentation: at 30m 3 The fermentation tank contained 20L of liquid fermentation medium. After being sterilized by moist heat at 120℃ for 40min, it was cooled to 35℃. The primary seed was inoculated into the liquid fermentation medium. The tank pressure was controlled at 0.05 MPa, and the culture was carried out at 35℃ for 30h. The raw materials and dosage (W / V) of the fermentation medium were: corn steep liquor 0.6%, (NH4)2SO4 0.5%, CaCO3 1.0%, glucose 3%, KH2PO4 0.06%, and pH value 7.0.

[0020] (3) When the spore formation rate is greater than or equal to 90%, stop the fermentation and put the product into a tank; (4) Combine the above fermentation liquid with a stabilizer to make a liquid preparation. The stabilizer consists of glycerol, skim milk powder, sodium alginate, and PVP, with addition amounts (W / V) of 2%, 0.25%, 2%, and 1.5%, respectively; (5) Concentrate and spray dry the liquid fermentation liquid into powder in a spray drying tower and pack it into a packaging bag; (6) Detect and count the effective viable bacteria count (CFU) of the product to reach ≥1.0×10 10 CFU / g.

[0021] Example 2 Antibacterial test of compound microbial agent against the pathogen of peach tree canker (I) Test time and location: May 2024 in the laboratory of the company's R&D center.

[0022] (II) Experimental methods: (1) Source of test strains The pathogen causing peach tree canker was isolated from Taoyuan, Fengrun District, Tangshan City, Hebei Province. After being isolated, purified and cultured by the company's R&D personnel, the purified strain was identified by 16S rDNA molecular identification and finally identified as Dothiorellagregaria Sacc. It was identified as highly pathogenic by pathogenicity determination.

[0023] Bacillus amyloliquefaciens, Bacillus vesalis, Bacillus subtilis, and Bacillus amyloliquefaciens were prepared by fermentation and spray drying of the strains described in Example 1.

[0024] (2) For the plate antagonism experiment, PDA solid plates were prepared. A small amount of *Trichoderma clump-forming* pathogen cells and *Bacillus aureus*, *Bacillus belye*, *Bacillus subtilis*, and *Bacillus amyloliquefaciens* MES81165 cells were picked up with a sterile inoculation needle and placed at both ends of the petri dish. A blank control was set up by inoculating only *Trichoderma clump-forming* without functional antagonistic bacteria. Each treatment was repeated three times. The plates were incubated at 28℃. When the hyphae of the blank control covered the entire petri dish, the colony radius of the control colony inoculated only with *Trichoderma clump-forming* and the colony radius of the pathogens inoculated with *Bacillus aureus*, *Bacillus belye*, *Bacillus subtilis*, and *Bacillus amyloliquefaciens* were measured and expressed as inhibition rate (inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100).

[0025] (3) Antagonistic Effect: This experiment showed that *Bacillus aureus* MES85801, *Bacillus subtilis* MES810, *Bacillus belyss* MES861, and *Bacillus amyloliquefaciens* had inhibition rates of 66.7%, 68.89%, 71.11%, and 75.56% against peach canker, respectively. The average colony radius of the control group was 4.5 cm, while the average colony radius of *Bacillus aureus* inoculated with the control group was 1.5 cm, *Bacillus subtilis* 1.4 cm, *Bacillus belyss* 1.3 cm, and *Bacillus amyloliquefaciens* 1.1 cm (as shown in Figure 1). This indicates that *Bacillus aureus*, *Bacillus subtilis*, and *Bacillus amyloliquefaciens* have significant inhibitory effects on peach canker and possess biocontrol potential for controlling peach canker.

[0026] Example 3 Test on the effect of compound microbial agent on peach tree canker: (1) Compound microbial agent: the compound microbial agent solid preparation prepared in Example 1, diluted with water 500 times; (2) Chemical agent: 800 times dilution of 50% carbendazim wettable powder (50 ml of medicine mixed with 40 kg of water) (Sichuan Guoguang Agrochemical Co., Ltd.); (3) Blank control agent: water.

[0027] Experiment time and location: The experiment was conducted in an orchard in Tangshan, Hebei Province in August 2024.

[0028] Experimental method: Diseased plants were divided into 3 groups and treated with 3 different experimental agents respectively: (1) water; (2) chemical agent; (3) compound microbial agent. The specific experimental method was as follows: each treatment agent was measured, diluted with water to the corresponding concentration, and evenly poured on the roots of the plant and applied to the diseased parts of the plant and the surrounding area. The effect was observed after 3-30 days.

[0029] Experimental Results: See Table 2. The results show that the disease index of the treatment with the compound microbial agent (9.85) was significantly lower than that of the blank control (86.43), but slightly higher than that of the treatment with chemical agents (8.16). This indicates that the compound microbial agent containing Bacillus subtilis, Berry's bacillus, amyloliquefaciens, and Bacillus argentea can be used as a fungicide for controlling peach tree canker and has broad application value. Disease Investigation Method: Peach tree canker was classified into four grades according to the transverse length of the lesions. The grading indicators are shown in Table 1.

[0030] Table 1. Grading of Peach Tree Canker Disease Severity Disease Grade | Severity Representative Values ​​| I. Normal bark color, no lesions | 0 | II. Lesion length less than 1 / 4 of the affected periderm | 1 | III. Lesion length 1 / 4 to 1 / 2 of the affected periderm | 2 | IV. Lesion length more than 1 / 2 of the affected periderm, weakened or dying tree | 3 Disease index = ∑(number of diseased plants at each level × corresponding level value) / (total number of plants surveyed × maximum level value) × 100; Control effect (%) = (disease index of blank control - disease index of treatment) / disease index of blank control × 100. The severity levels are as follows: Mild: Disease index below 10 (or disease incidence ≤ 10%, mortality ≤ 5%) Moderate: Disease index 10–30 (or 10%) <感病株率≤30％，5％<死亡率<10％）重度：病情指数30以上（或感病株率＞30％，死亡率≥10％）表2不同处理对柑橘黄化病的防治效果处理病情指数防效（%）多菌灵800倍液8.1690.56复合微生物菌剂500倍9.8588.60空白对照86.430.00The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.< / 感病株率≤30％，5％<死亡率<10％）重度：病情指数30以上（或感病株率＞30％，死亡率≥10％）表2不同处理对柑橘黄化病的防治效果处理病情指数防效（%）多菌灵800倍液8.1690.56复合微生物菌剂500倍9.8588.60空白对照86.430.00

Claims

1. A compound microbial agent, comprising an effective amount of active microbial components and pharmaceutically acceptable excipients, characterized in that, The active microbial components include four functional strains of the genus Bacillus; these functional strains are Bacillus aryabhattai MES85801, Bacillus velezensis MES861, Bacillus subtilis MES810, and Bacillus amyloliquefaciens MES81165, and the total effective viable count in the compound microbial agent is not less than 1.0 × 10⁻⁶. 10 CFU / g.

2. The compound microbial agent according to claim 1, characterized in that, In the compound microbial agent, the viable count ratio of Bacillus aryabhattai MES85801, Bacillus velezensis MES861, Bacillus subtilis MES810, and Bacillus amyloliquefaciens MES81165 is (1~3):(1~3):(1~3):(1~4).

3. The method for preparing the composite microbial agent according to claim 1, characterized in that, Includes the following steps: After primary seed culture, each strain was inoculated into liquid fermentation medium and fermented for 25-40 hours at 35°C and 0.02-0.07 MPa, with the spore formation rate controlled at ≥90% at the end of fermentation. After fermentation, a composite stabilizer composed of glycerol, skim milk powder, sodium alginate and PVP was added, and then spray-dried to obtain a powdered composite microbial agent.

4. The method for preparing the composite microbial agent according to claim 3, characterized in that, The liquid fermentation medium contains the following components: corn steep liquor 0.4-0.8%, (NH4)2SO4 0.3-0.7%, CaCO3 0.8-1.2%, glucose 2-4%, KH2PO4 0.05-0.09%, and the pH is adjusted to 7.0-7.

5.

5. The method for preparing the composite microbial agent according to claim 3, characterized in that, In the composite stabilizer, by weight percentage, the amount of glycerin added is 1.0~2.5%, the amount of skim milk powder added is 0.1~0.4%, the amount of sodium alginate added is 1.5~2.5%, and the amount of PVP added is 0.5~2.0%.

6. The method for preparing the composite microbial agent according to claim 3, characterized in that, The process parameters for spray drying are: inlet temperature 160~200°C, outlet temperature 75~90°C, and feed rate 15~30 L / h.

7. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is a liquid preparation containing fermentation broth, stabilizer and water, and can be stably stored at 4°C for more than 6 months with an effective viable count decrease of no more than 15%.

8. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is used to prevent and control peach canker caused by Dothiorella gregaria Sacc. or Botryosphaeria ribis (Tode).

9. The compound microbial agent according to claim 1, characterized in that, The application method of the microbial agent is to dilute the compound microbial agent 500 times and then apply it by root irrigation, foliar spraying, or / and by smearing on the diseased area and its surroundings.

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