Microbial complex microbial inoculant and application thereof in prevention and treatment of plant diseases

Abstract

The invention provides a microbial complex microbial inoculant and application thereof in prevention and treatment of plant diseases, the microbial complex microbial inoculant comprises the following strains by mass: 30%-50% of bacillus subtilis; 20%-40% of bacillus amyloliquefaciens; 10%-30% of pseudomonas fluorescens; 5%-15% of trichoderma harzianum; and 2%-5% of an auxiliary agent. According to the microbial complex microbial inoculant disclosed by the invention, a multiple prevention and control mechanism of'bacteriostasis-competition-parasitism 'is constructed through the synergistic effect of various strains, and meanwhile, the double-layer effect of thallus protection and function enhancement is realized through the assistance of the auxiliaries, so that the prevention and control effect of the prepared microbial complex microbial inoculant is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial agent preparation technology, specifically, it relates to a microbial compound agent and its application in the prevention and control of plant diseases. Background Technology

[0002] Soil-borne plant diseases are a major challenge hindering the sustainable development of modern agriculture. According to FAO statistics, global crop losses due to soil-borne diseases reach 20%-30% annually. While traditional chemical control methods are fast-acting, long-term use leads to drug resistance in pathogens, causing serious environmental pollution and agricultural product safety issues. Microbial control, as an environmentally friendly alternative, has received widespread attention in recent years, but existing microbial agents still face many technical bottlenecks in practical applications.

[0003] Currently, microbial agents on the market suffer from the following technical deficiencies: Regarding strain selection, most products use a single strain, resulting in limited efficacy (field trials show efficacy against Fusarium less than 60%) and susceptibility to environmental factors. In terms of agent stability, conventional preservatives (such as glycerol and skim milk) cannot meet long-term storage requirements. Research data (see *Applied Microbiology and Biotechnology*, 2021) indicates that existing agents generally experience a more than 40% decrease in viable cell count after three months of storage at room temperature. Furthermore, in terms of formulation processes, traditional high-temperature spray drying (>80℃) leads to significant bacterial death, while freeze-drying, although maintaining a high viable cell rate, incurs excessively high production costs (approximately 20,000 RMB per ton), hindering large-scale application.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide a microbial compound inoculant. Through extensive compatibility experiments, the optimal ratio range has been determined, ensuring synergistic effects among various strains. In terms of adjuvants, the protective effect of trehalose, the soil-improving function of humic acid, and the carrier properties of diatomaceous earth are innovatively combined to achieve a dual effect of protecting the bacteria and enhancing their function. Furthermore, by controlling the drying temperature and using dispersants, the viability rate is guaranteed while production efficiency is improved.

[0006] The second objective of this invention is to provide the application of the aforementioned microbial compound agent in the prevention and control of plant diseases. Through the synergistic effect of multiple strains, the efficacy, stability, and application flexibility are comprehensively improved, thereby providing an efficient, economical, and sustainable disease solution for green agriculture.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] A microbial compound inoculant, comprising, by weight percentage, the following strains:

[0009] Bacillus subtilis 30%-50%;

[0010] Bacillus amyloliquefaciens 20%-40%;

[0011] Fluorescent Pseudomonas aeruginosa 10%-30%;

[0012] Trichoderma harzianum 5%-15%;

[0013] Additives: 2%-5%.

[0014] In the microbial compound agent of this invention, a complete "antibacterial-competitive-parasitic" control system is formed by the synergistic effect of four strains: Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens, and Trichoderma harzianum. These four strains can all grow within a suitable pH range and will not inhibit each other due to changes in the environment caused by their metabolites. They can also form a synergistic defense network through different mechanisms of action. At the same time, the different nutritional requirements of these four strains, such as Bacillus subtilis preferentially utilizing proteins, Bacillus amyloliquefaciens specifically decomposing starch, Pseudomonas fluorescens relying on siderophores, and Trichoderma harzianum utilizing cellulose, reduce the nutritional competition among them when used in combination. In addition, the different spatial distributions of these four strains enable the prepared compound agent to achieve comprehensive protection in the control of plant diseases. Based on the above advantages, this invention uses the combination of these four microbial agents to achieve excellent control of plant diseases. Among these four strains, Bacillus subtilis is the dominant strain, mainly by secreting antibacterial substances such as surfactants and iturin to directly destroy the cell membrane of pathogens, thereby achieving the purpose of preventing and controlling pathogens. Bacillus amyloliquefaciens mainly produces amylase and protease to directly decompose the cell wall of pathogens, inhibiting pathogen growth while promoting plant root development. Its combination with Bacillus subtilis can effectively expand the antibacterial spectrum of microbial compound agents, thereby further inhibiting the germination of Fusarium spores. Pseudomonas fluorescens can effectively seize the ecological niche in the rhizosphere by competing for siderophore production and secrete antibiotics to inhibit the growth of bacterial wilt. Trichoderma harzianum achieves parasitism against pathogenic fungi through hyphal entanglement and secretion of chitinase.

[0015] Furthermore, the proportions of the various bacterial strains were determined by the inventors through a series of creative efforts. When the proportion of Bacillus subtilis is below 30%, the concentration of its secreted antibacterial substances is insufficient, thus failing to effectively control pathogens; while a proportion exceeding 50% will inhibit the growth of other strains. Similarly, controlling Trichoderma harzianum at 5%-15% ensures parasitic effects without excessively consuming nutrients in the culture medium. This precise formulation enhances the control effect of the compound microbial agent on common soil-borne diseases such as Fusarium and Botrytis cinerea, improving it by more than 30% compared to single microbial agents.

[0016] Preferably, as a further specific implementation,

[0017] Bacillus subtilis 40%;

[0018] 30% of Bacillus amyloliquefaciens;

[0019] 20% of fluorescent Pseudomonas;

[0020] Trichoderma harzianum 6.5%;

[0021] Additives 3.5%.

[0022] This invention further specifies the preferred ratios between the various strains. When the proportions of *Bacillus subtilis* is 40%, *Bacillus amyloliquefaciens* is 30%, *Pseudomonas fluorescens* is 20%, *Trichoderma harzianum* is 6.5%, and the adjuvant is 3.5%, it exhibits excellent control effects. At this ratio, 40% *Bacillus subtilis* ensures sufficient secretion of antibacterial substances without significantly inhibiting other strains, while 30% *Bacillus amyloliquefaciens* produces a better synergistic effect with *Bacillus subtilis*, and its secreted amylase can... The breakdown of macromolecules into smaller molecules provides nutrition for the organism itself and creates a favorable environment for other strains. Meanwhile, 20% of *Pseudomonas fluorescens* can produce sufficient siderophores, thus more effectively inhibiting the growth of *Ralstonia solanacearum*. After a series of creative efforts, the inventors discovered that when *Trichoderma harzianum* accounts for 6.5%, it has the highest parasitic efficiency. At the same time, the metabolites do not excessively change the pH of the culture medium, and the addition of 3.5% adjuvants can ensure the protective effect of the bacteria without affecting the flowability of the final product.

[0023] Preferably, as a further specific embodiment, the adjuvant includes trehalose, humic acid, and diatomaceous earth carrier;

[0024] The mass ratio of trehalose, humic acid and diatomaceous earth carrier is 1:(0.5-2):(5-15);

[0025] Preferably, the mass ratio of trehalose, humic acid, and diatomaceous earth carrier is 1:1:8.

[0026] In this invention, the system of the adjuvant is specifically defined, wherein the adjuvant includes trehalose, humic acid and diatomaceous earth carrier, and the mass ratio of the three is 1:(0.5-2):(5-15), preferably 1:1:8. This adjuvant system protects the bacteria and enhances their function through multiple mechanisms. Trehalose, as a non-reducing disaccharide, can form a stable protective layer on the surface of the bacteria, thereby significantly enhancing their heat resistance. Humic acid, through its unique carboxyl and phenolic hydroxyl groups, can regulate the soil pH and provide trace elements. At the same time, the diatomaceous earth carrier, with its porous structure, can further realize the slow release of bacteria. Under the preferred ratio of 1:1:8, the survival rate of live bacteria in the compound microbial agent after 6 months of storage at room temperature is still as high as 92%, far exceeding the 70% of conventional adjuvant systems.

[0027] Preferably, as a further specific embodiment, the diatomaceous earth carrier is modified with silver nanoparticles, wherein the silver nanoparticle loading is 0.01%-0.05%.

[0028] In this invention, the comprehensive performance of the composite microbial agent is further enhanced by loading nano-silver onto a diatomaceous earth carrier. The combination of nano-silver and diatomaceous earth employs a special in-situ reduction method. By controlling the concentration of silver nitrate and the amount of reducing agent, the nano-silver particles are evenly distributed in the pores of the diatomaceous earth. This structure ensures the continuous release of nano-silver while avoiding the damage to the composite strains caused by the sudden release of silver ions. Furthermore, the synergistic effect of nano-silver particles and Bacillus subtilis can better disrupt the cell membranes of pathogens, thereby enhancing the control effect of the microbial agent against Fusarium. At the same time, nano-silver can also inhibit the growth of other bacteria by interfering with the electron transport chain and inducing the production of reactive oxygen species. For the microbial agent of this invention, a nano-silver loading of 0.01%-0.05% can inhibit the growth of more than 90% of other bacteria, while having little impact on the functional strains in the composite agent.

[0029] Preferably, as a further specific embodiment, the preparation method of the microbial compound inoculant includes the following steps:

[0030] Each strain was fermented separately, and the cell bodies were collected by centrifugation.

[0031] The bacterial cells were mixed in a certain proportion, and then an adjuvant was added and the mixture was suspended in sterile phosphate buffer to obtain a suspension.

[0032] The suspension is then spray-dried with an inlet temperature ≤60℃ and an outlet temperature ≤40℃ to obtain the final product.

[0033] In this invention, the process parameters in the preparation of the microbial compound inoculant are optimized to ensure the activity of the cells and the stability of the product as much as possible. First, the culture of each strain is carried out by independent fermentation to avoid intermediate inhibition. Then, after the cells are collected, they are mixed with sterile phosphate buffer to stabilize the specific membrane structure. Subsequently, the inlet and outlet temperatures of the spray dryer are controlled to ensure that the water evaporates quickly while avoiding protein denaturation, thus ensuring the survival rate of the strains.

[0034] Preferably, as a further specific embodiment, 0.1wt%-0.3wt% of polyethylene glycol is added as a dispersant before spray drying the suspension.

[0035] In this invention, by adding 0.1wt%-0.3wt% polyethylene glycol as a dispersant before spray drying the obtained suspension, the product quality of the microbial composite agent is further ensured. Polyethylene glycol can reduce the surface tension of the suspension through its surface activity, resulting in more uniform atomized droplets. Simultaneously, the ether bonds in the polyethylene glycol molecule can form hydrogen bonds with the phospholipids on the surface of the bacterial cell membrane, thus maintaining the integrity of the membrane during drying. The highest viability rate after spray drying is achieved when the addition amount is 0.2wt%. Furthermore, the addition of polyethylene glycol can work synergistically with trehalose in the adjuvant to form a glassy matrix and promote the uniform distribution of bacteria within the pores of diatomaceous earth, thus better protecting the bacterial strain.

[0036] This invention also provides the application of the above-mentioned microbial compound inoculant in the prevention and control of plant diseases, including diseases caused by the following pathogens:

[0037] Fungi: Fusarium, Botrytis cinerea;

[0038] Bacteria: Ralstonia solanacearum;

[0039] Soil-borne disease: Rhizoctonia solani.

[0040] The microbial compound agent of this invention can simultaneously and continuously control Fusarium, Botrytis cinerea, Ralstonia solanacearum, and Rhizoctonia solani through targeted control mechanisms and synergistic spatial distribution. Specifically, against fungal diseases such as Fusarium and Botrytis cinerea, the microbial compound agent of this invention works through a triple mechanism: firstly, Surfactin secreted by Bacillus subtilis disrupts the cell membrane integrity of the pathogens; secondly, Iturin produced by Bacillus amyloliquefaciens... A. Inhibiting spore germination, *Trichoderma harzianum* achieves physical parasitism through hyphal entanglement. Through the synergistic effect of these three, fungal diseases are effectively controlled. For bacterial diseases such as bacterial wilt, the microbial compound agent of this invention mainly competes for iron with pyoverdine synthesized by *Pseudomonas fluorescens*, thereby greatly reducing the iron uptake of pathogens. At the same time, it is combined with nano-silver particles to destroy bacterial biofilms, thereby controlling bacterial diseases. For soil-borne diseases, it forms a three-dimensional protective network in the soil through spatial distribution synergy. *Trichoderma harzianum* parasitizes *Rhizoctonia solani* on the surface, while *Pseudomonas fluorescens* occupies the ecological niche of *Rhizoctonia solani* in the rhizosphere. Meanwhile, *Bacillus subtilis* continuously releases antibacterial substances in the deep soil. Through the synergistic effect of these three, soil-borne diseases are controlled.

[0041] Preferably, as a further specific embodiment, the microbial agent is applied by either seed coating or soil drenching.

[0042] Preferably, as a further specific embodiment, when the application method is seed coating, the amount of the microbial agent is (0.5-1) g / kg of seeds;

[0043] The film-forming agent is 0.2% sodium alginate;

[0044] 0.05% nano titanium dioxide was also added as a photoprotectant.

[0045] Preferably, as a further specific embodiment, when the application method is soil drenching, the microbial agent is diluted 500-1000 times, and 100-200 mL is applied to each plant.

[0046] Synergists and adhesives are also added;

[0047] The synergist mentioned above is 0.1% polyglutamic acid;

[0048] The adhesive is 0.02% chitosan.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] (1) This invention provides a microbial compound agent. The optimal ratio range of the microbial compound agent is determined through a large number of compatibility experiments. While ensuring synergistic effects among various strains, it innovatively combines the protective effect of trehalose, the soil improvement function of humic acid and the carrier characteristics of diatomaceous earth in terms of adjuvants to achieve a dual effect of protecting the bacteria and enhancing their function. At the same time, by controlling the drying temperature and using dispersants, the viable bacteria rate is guaranteed while improving production efficiency.

[0051] (2) This invention provides the application of the above-mentioned microbial compound agent in the prevention and control of plant diseases. Through the synergistic effect of multiple strains, the efficacy, stability and application flexibility are comprehensively improved, thus providing an efficient, economical and sustainable disease solution for green agriculture. Detailed Implementation

[0052] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] To more clearly illustrate the technical solutions in this invention, specific embodiments are described below.

[0054] Example 1

[0055] The specific preparation process of the microbial compound inoculant in this embodiment is as follows:

[0056] Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens and Trichoderma harzianum were fermented and cultured respectively, and the cells were collected by centrifugation.

[0057] The percentage of bacterial cells taken is:

[0058] Bacillus subtilis 30%;

[0059] 40% of Bacillus amyloliquefaciens;

[0060] 10% of *Pseudomonas fluorescens*;

[0061] Trichoderma harzianum 15%;

[0062] The four bacterial cells were then mixed in proportion, and 5% of the adjuvant was added and suspended together in sterile phosphate buffer. The mixture was stirred until homogeneous to obtain a suspension.

[0063] The 5% auxiliary agent consists of trehalose, humic acid, and diatomaceous earth carrier loaded with 0.01% nano-silver in a mass ratio of 1:0.5:5.

[0064] Subsequently, 0.1 wt% polyethylene glycol was added to the suspension, and after stirring evenly, the suspension was spray-dried using a centrifugal spray dryer with an inlet temperature of 50°C and an outlet temperature of 30°C to obtain the final product.

[0065] Example 2

[0066] The specific preparation process of the microbial compound inoculant in this embodiment is as follows:

[0067] Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens and Trichoderma harzianum were fermented and cultured respectively, and the cells were collected by centrifugation.

[0068] The percentage of bacterial cells taken is:

[0069] Bacillus subtilis 50%;

[0070] 20% Bacillus amyloliquefaciens;

[0071] Fluorescent Pseudomonas aeruginosa 23%;

[0072] Trichoderma harzianum 5%;

[0073] The four bacterial cells were then mixed in proportion, and 2% of the adjuvant was added and suspended together in sterile phosphate buffer. The mixture was stirred until homogeneous to obtain a suspension.

[0074] The 2% auxiliary agent consists of trehalose, humic acid and diatomaceous earth carrier loaded with 0.05% nano-silver in a mass ratio of 1:2:15.

[0075] Subsequently, 0.3 wt% polyethylene glycol was added to the suspension, and after stirring evenly, the suspension was spray-dried using a centrifugal spray dryer with an inlet temperature of 50°C and an outlet temperature of 30°C to obtain the final product.

[0076] Example 3

[0077] The specific preparation process of the microbial compound inoculant in this embodiment is as follows:

[0078] Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens and Trichoderma harzianum were fermented and cultured respectively, and the cells were collected by centrifugation.

[0079] The percentage of bacterial cells taken is:

[0080] Bacillus subtilis 40%;

[0081] 30% of Bacillus amyloliquefaciens;

[0082] 20% of fluorescent Pseudomonas;

[0083] Trichoderma harzianum 6.5%;

[0084] The four bacterial cells were then mixed in proportion, and 3.5% of the adjuvant was added and suspended together in sterile phosphate buffer. The mixture was stirred until homogeneous to obtain a suspension.

[0085] The 3.5% auxiliary agent consists of trehalose, humic acid, and diatomaceous earth carrier loaded with 0.03% nano-silver in a mass ratio of 1:1:8.

[0086] Subsequently, 0.2 wt% polyethylene glycol was added to the suspension, and after stirring evenly, the suspension was spray-dried using a centrifugal spray dryer with an inlet temperature of 50°C and an outlet temperature of 30°C to obtain the final product.

[0087] Example 4

[0088] The specific implementation steps are the same as in Example 3, except that the mass ratio of trehalose, humic acid and diatomaceous earth carrier in Example 3 is adjusted to 1:1:1.

[0089] Example 5

[0090] The specific implementation steps are the same as in Example 3, except that the mass ratio of trehalose, humic acid and diatomaceous earth carrier in Example 3 is adjusted to 1:5:30.

[0091] Example 6

[0092] The specific implementation steps are the same as in Example 3, except that the loading of nano-silver in Example 3 is adjusted to 0.001%.

[0093] Example 7

[0094] The specific implementation steps are the same as in Example 3, except that the loading of nano-silver in Example 3 is adjusted to 0.2%.

[0095] Example 8

[0096] The specific implementation steps are the same as in Example 3, except that the concentration of polyethylene glycol is adjusted to 0.01 wt%.

[0097] Example 9

[0098] The specific implementation steps are the same as in Example 3, except that the concentration of polyethylene glycol is adjusted to 1 wt%.

[0099] Experiment Example 1: Verification of the Effect of Microbial Compound Inoculant on the Control of Plant Diseases

[0100] 1.1 Experimental Design

[0101] This experimental example verifies the control efficacy of the microbial compound inoculant prepared in Examples 1-9 against Fusarium (fungus), Ralstonia solanacearum (bacteria), and Rhizoctonia solani (soil-borne disease).

[0102] 1.2 Control Experiment

[0103] Positive control: Chemical bactericide

[0104] Negative control: Sterile water treatment

[0105] 1.3 Experimental Methods

[0106] 1.3.1 Pathogen Culture

[0107] Fusarium: PDA medium, incubated at 25℃ for 5 days;

[0108] Bacterial wilt pathogen: NA medium, incubated at 28℃ for 48h;

[0109] Rhizoctonia solani: PDA medium, cultured at 25°C for 7 days;

[0110] 1.4 Microbial Agent Treatment

[0111] 1.4.1 Seed coating (for Fusarium):

[0112] Take 100g of wheat seeds and coat them with the microbial compound inoculant (0.8g / kg seeds) obtained in Examples 1-9, plus 0.2% sodium alginate and 0.05% nano TiO2.

[0113] After drying, sow in seeds containing Fusarium spores (1×10⁻⁶). 5 In sterilized soil (CFU / g soil);

[0114] 1.4.2 Soil drenching (for bacterial wilt and Rhizoctonia solani):

[0115] Tomato seedlings (3-leaf stage) were transplanted into soil inoculated with pathogens (1×10⁶ of bacterial wilt pathogen). 6 CFU / g; Rhizoctonia solani 1×10 4 CFU / g);

[0116] Dilute the microbial compound inoculant obtained in Examples 1-9 800 times (containing 0.1% polyglutamic acid + 0.02% chitosan), and drench the roots of each plant with 150 mL, once every 5 days, for a total of 3 times;

[0117] 1.5 Data Acquisition

[0118] Disease inhibition rate: Inhibition rate (%) = (1 - Incidence rate in the example group / Incidence rate in the negative control) × 100

[0119] Plant growth indicators: plant height (cm), root length (cm), fresh weight (g)

[0120] Soil microbial biomass: Determination of functional strain colonization (CFU / g soil) using the dilution plating method.

[0121] The specific test results are shown in Table 1 below:

[0122] Table 1 Test Results

[0123]

[0124] The results above show that, by adjusting the strain ratio and adjuvant formulation in Examples 1-3, Example 3 was the optimal experimental scheme. Its formulation achieved complementary strain functions while synergistic protection with the adjuvant, resulting in inhibition rates of 92.4%, 94.1%, and 89.7% against Fusarium, Ralstonia solanacearum, and Rhizoctonia solani, respectively, approaching those of chemical pesticides and significantly higher than in Examples 1 and 2. Furthermore, the viable count in Example 3 was 2.5 × 10⁻⁶. 10 The highest levels of CFU / g and 6-month survival rate (93.8%) were observed.

[0125] Comparing Examples 4-5, it can be seen that adjusting the ratio of the adjuvants affects the effect of the final prepared microbial composite agent. In Example 4, when the ratio of the adjuvants was adjusted to 1:1:1, the inhibition rate was significantly lower than that in Example 3. This is because the trehalose ratio was insufficient, which led to a decrease in the heat resistance of the prepared composite microbial agent. In Example 5, when the ratio was adjusted to 1:5:30, the inhibition rate and survival rate were further reduced compared to Example 4. This is because the excessive humic acid in Example 5 led to a decrease in pH, which inhibited the activity of the bacteria. At the same time, the low proportion of diatomaceous earth carrier in Example 5 affected the slow release of the bacteria, while the high proportion would result in an incomplete protective layer, affecting the activity of the bacteria.

[0126] Comparing Examples 6 and 7 reveals that the amount of silver nanoparticles loaded in the diatomaceous earth carrier affects the performance of the final microbial composite agent. In Example 6, when the silver nanoparticle loading was low, the inhibition rate of the prepared microbial composite agent was slightly lower than that in Example 3. This is because insufficient silver nanoparticles led to incomplete inhibition of other microorganisms. However, in Example 7, when the silver nanoparticle loading was too high, the number of viable bacteria suddenly dropped to 1.2 × 10⁻⁶. 10 The CFU / g (survival rate 72.6%) indicates that excessive nano-silver was toxic to the functional strains.

[0127] Comparing Examples 8-9, it can be seen that the concentration of polyethylene glycol also affects the performance of the final prepared microbial composite agent. When the concentration of polyethylene glycol in Example 8 is too low, the uneven atomization of the agent leads to a slight decrease in both the number of viable bacteria and the inhibition rate. When the concentration of polyethylene glycol in Example 9 is too high, the excessive polyethylene glycol will damage the integrity of the bacterial membrane, resulting in a decrease in the number of viable bacteria and a significant decrease in the inhibition rate.

[0128] Experiment Example 2: Screening of Composite Strains

[0129] The specific implementation steps are the same as in Example 3, except that the bacterial strain in Example 3 is replaced.

[0130] An experimental group and a control group were set up respectively, and the control group was the microbial compound inoculant prepared in Example 3;

[0131] The bacterial strain in experimental group 1 was Bacillus subtilis;

[0132] The bacterial strain in experimental group 2 was Bacillus amyloliquefaciens;

[0133] The bacterial species in experimental group 3 was *Pseudomonas fluorescens*.

[0134] The fungal species in experimental group 4 was Trichoderma harzianum;

[0135] The bacterial strains in experimental group 5 were 50% Bacillus subtilis and 50% Bacillus amyloliquefaciens;

[0136] The bacterial strains in experimental group 6 were 50% Bacillus subtilis and 50% Trichoderma harzianum;

[0137] The bacterial strains in experimental group 7 were 50% Bacillus subtilis and 50% Streptomyces.

[0138] The bacterial species in experimental group 8 were 50% Bacillus subtilis, 10% Trichoderma harzianum, and 40% Pseudomonas fluorescens.

[0139] The bacterial species in experimental group 9 were 50% Bacillus subtilis, 10% Agrobacterium actinomycetes and 40% Pseudomonas aeruginosa;

[0140] The bacterial species in experimental group 10 were 50% Bacillus subtilis, 10% Trichoderma harzianum, and 40% arbuscular mycorrhizal fungi;

[0141] Subsequently, the bacterial strains in experimental groups 1-10 were prepared into microbial compound inoculants according to the preparation method in Example 3;

[0142] Finally, the efficacy of the microbial compound inoculants obtained in experimental groups 1-10 and the control group in preventing and controlling plant diseases was verified according to the experimental method in Experiment Example 1.

[0143] The final results are shown in Table 2 below:

[0144]

[0145] The experimental data above show that the solution of this invention, namely the control group containing 40% Bacillus subtilis + 30% Bacillus amyloliquefaciens + 20% Pseudomonas fluorescens + 6.5% Trichoderma harzianum, performed best among all experimental groups. Its inhibition rates against Fusarium, Ralstonia solanacearum, and Rhizoctonia solani were significantly higher than other experimental groups, and the viable cell count also reached the highest level. This result indicates that the single-strain placement effect in experimental groups 1-4 was significantly lower than that in the control group, especially its insufficient broad-spectrum inhibition ability against multiple pathogens. While the improvement of the dual-strain combination in experimental groups 5-7 was better than that of the single-strain combination, it still could not achieve the desired effect. The comprehensive control measures demonstrate that simply adding strains cannot achieve synergistic effects. Among the three-strain combinations in experimental groups 8-10, experimental groups 8 and 10 performed better, but still lower than the control group. This indicates that the synergistic ratio of the four strains has advantages in functional complementarity and niche coverage. Through the synergistic effect of Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens, and Trichoderma harzianum, a multi-layered control network of "antibacterial-competitive-parasitic" was constructed, significantly broadening the antibacterial spectrum. At the same time, the nutritional needs and spatial distribution of the four strains are complementary, avoiding mutual inhibition and achieving efficient colonization.

[0146] Raw material selection in experimental example 3, auxiliaries

[0147] The specific operating steps in this experimental example are the same as in Example 3, the difference being that the raw materials in the adjuvant of Example 3 are adjusted.

[0148] Experimental and control groups were set up separately, with the control group still using the microbial compound inoculant prepared in Example 3;

[0149] The adjuvant in experimental group 12 was trehalose;

[0150] The auxiliary agent in experimental group 13 was humic acid;

[0151] In experimental group 14, the auxiliary agent was a diatomaceous earth carrier without nano-silver particles;

[0152] In experimental group 15, the auxiliary agent was a diatomaceous earth carrier loaded with silver nanoparticles.

[0153] In experimental group 16, the auxiliary agent was glycerin;

[0154] In experimental group 17, the adjuvant was a mixture of glycerol and trehalose, with a mass ratio of 1:1.

[0155] In experimental group 18, the adjuvant was a mixture of trehalose and humic acid, with a mass ratio of 1:1.

[0156] In experimental group 19, the auxiliary agent was a mixture of humic acid and bentonite, with a mass ratio of 1:1.

[0157] In experimental group 20, the auxiliary agent was a mixture of glycerol, bentonite and chitosan, with a mass ratio of 1:1:8.

[0158] In experimental group 21, the adjuvant was a mixture of trehalose, diatomaceous earth and yeast extract, with a mass ratio of 1:1:8.

[0159] In experimental group 22, the auxiliary agent was a mixture of trehalose, humic acid and diatomaceous earth carrier without nano-silver particles, with a mass ratio of 1:1:8.

[0160] Finally, the effect of the microbial compound inoculants obtained in experimental groups 12-22 and the control group on the prevention and control of plant diseases was verified according to the experimental method in Experiment Example 1. The final results are shown in Table 3 below.

[0161] Table 3 Test Results

[0162]

[0163]

[0164] The experimental data above show that the control group performed best in all indicators, with the pathogen inhibition rate leading across the board and the viable count and storage stability significantly higher than other groups. In experimental groups 12-14, the single adjuvant was the least effective. Trehalose alone had insufficient heat resistance, resulting in a low survival rate, while ordinary diatomaceous earth had no antibacterial function. In experimental groups 16-17, when glycerol was used as an adjuvant, its hygroscopicity caused clumping, which reduced the inhibition rate. In experimental group 20, chitosan failed under acidic conditions, and the bentonite carrier had low porosity. The adjuvant system of this invention, through the synergistic design of trehalose-humic acid-nano-silver diatomaceous earth, solves the core problems of poor protective effect, single function, and insufficient stability in the prior art.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microbial compound inoculant, characterized in that, By weight percentage, it includes the following strains: Bacillus subtilis 30%-50%; Bacillus amyloliquefaciens 20%-40%; Fluorescent Pseudomonas aeruginosa 10%-30%; Trichoderma harzianum 5%-15%; Additives: 2%-5%.

2. The microbial compound inoculant according to claim 1, characterized in that, Bacillus subtilis 40%; 30% of Bacillus amyloliquefaciens; 20% of *Pseudomonas fluorescens*; Trichoderma harzianum 6.5%; Additives 3.5%.

3. The microbial compound inoculant according to claim 1, characterized in that, The additives include trehalose, humic acid, and diatomaceous earth carrier; The mass ratio of trehalose, humic acid and diatomaceous earth carrier is 1:(0.5-2):(5-15); The mass ratio of trehalose, humic acid, and diatomaceous earth carrier is 1:1:

8.

4. The microbial compound inoculant according to claim 3, characterized in that, The diatomaceous earth carrier is modified with silver nanoparticles, wherein the silver nanoparticle loading is 0.01%-0.05%.

5. The microbial compound inoculant according to any one of claims 1-4, characterized in that, The preparation method of the microbial compound inoculant includes the following steps: Each strain was fermented separately, and the cell bodies were collected by centrifugation. The bacterial cells were mixed in a certain proportion, and then an adjuvant was added and the mixture was suspended in sterile phosphate buffer to obtain a suspension. The suspension is then spray-dried with an inlet temperature ≤60℃ and an outlet temperature ≤40℃ to obtain the final product.

6. The preparation method according to claim 5, characterized in that, Before spray drying the suspension, 0.1 wt% to 0.3 wt% of polyethylene glycol is added as a dispersant.

7. The application of a microbial compound inoculant as described in any one of claims 1-4 in the prevention and control of plant diseases, characterized in that, The diseases mentioned include those caused by the following pathogens: Fungi: Fusarium, Botrytis cinerea; Bacteria: Ralstonia solanacearum; Soil-borne disease: Rhizoctonia solani.

8. The application according to claim 7, characterized in that, The microbial agent can be applied by either seed coating or soil drenching.

9. The application according to claim 7, characterized in that, When the application method is seed coating, the amount of the microbial agent is (0.5-1) g / kg of seeds; The film-forming agent is 0.2% sodium alginate; 0.05% nano titanium dioxide was also added as a photoprotectant.

10. The application according to claim 7, characterized in that, When the application method is soil drenching, the microbial agent is diluted 500-1000 times, and 100-200 mL is applied to each plant. Synergists and adhesives are also added; The synergist mentioned above is 0.1% polyglutamic acid; The adhesive is 0.02% chitosan.

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