Compound microbial agent with disease prevention and growth promotion functions and application thereof

Abstract

The application discloses a compound microbial agent with disease prevention and growth promotion functions and application thereof, and belongs to the technical field of microbial application. Pseudomonas taiwanensis The compound microbial agent comprises Pseudomonas taiwanensis Bacillus velezensis , Bacillus velezensis Pseudomonas plecoglossicida , Pseudomonas mutabilis Calidifontibacillus erzurumensis , and Bacillus elzermanii, and the viable cell number ratio of the Pseudomonas taiwanensis Bacillus velezensis , the Bacillus velezensis Pseudomonas plecoglossicida , the Pseudomonas mutabilis Calidifontibacillus and the Bacillus elzermanii is (1-3):(1-3):(1-3):(1-3). The compound microbial agent can inhibit the growth of pathogenic bacteria and promote the growth of Altingia sieboldii.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a compound microbial agent with both disease prevention and growth promotion functions and its application. Background Technology

[0002] Shantongzi( Idesia polycarpa *Vernicia fordii* (Malus macrantha) is a fast-growing tree species with high economic value. Its fruit can be pressed for oil, earning it the reputation of "oil depot on the tree." Major diseases affecting *Vernicia fordii* include anthracnose, rust, powdery mildew, stem rot, and root rot. It is also susceptible to various insect pests such as lepidopteran moths, bark beetles, and longhorn beetles. Current control methods primarily rely on chemical control (such as spraying carbendazim and deltamethrin), supplemented by physical and biological control. However, existing methods have significant drawbacks: excessive use of chemical pesticides disrupts the ecological balance and endangers natural enemies, and the timing and dosage of control are difficult to control precisely. Seedling diseases are poorly controlled and result in low seedling survival rates due to inadequate management techniques. Wood-boring insects (such as wood-boring moths) live in seclusion, and their adults are highly dispersible, making conventional spraying ineffective.

[0003] Currently, single microbial agents in agricultural production suffer from drawbacks such as narrow antibacterial spectrum, unstable efficacy, and limited growth-promoting ability, making it difficult to simultaneously meet the dual needs of crop disease prevention and quality improvement. Microbial compound agents, with their advantages of complementary strain functions, long-lasting effects, and green safety, have become the mainstream development direction of green agriculture. However, existing compound agents still have room for optimization in terms of strain ratio, storage stability, and field adaptability. Therefore, developing a broad-spectrum, highly efficient, and stable general-purpose biological agent is of great practical significance. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, one objective of this invention is to provide a composite microbial agent, wherein the composite microbial agent includes *Pseudomonas taiwanensis* (…). Pseudomonas taiwanensis ), Bacillus belesi ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis ).

[0005] The second objective of this invention is to provide a method for preparing the aforementioned composite microbial agent, the specific steps of which are as follows: (1) The strain of Pseudomonas taiwanensis ( Pseudomonas taiwanensis Bacillus belesiensis ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis ) was activated and then subjected to liquid fermentation to obtain *Pseudomonas taiwanensis* ( Pseudomonas taiwanensis Bacterial suspension, Bacillus belesiensis ( Bacillus velezensis ) bacterial suspension, Pseudomonas proteus (Pseudomonas plecoglossicida ) bacterial suspension and Bacillus elzuru ( Calidifontibacillus erzurumensis The bacterial suspension was prepared, and the number of viable bacteria in the bacterial suspension was determined.

[0006] (2) According to the proportion of viable bacteria, mix *Pseudomonas taiwanensis* ( Pseudomonas taiwanensis Bacterial suspension, Bacillus belesiensis ( Bacillus velezensis ) bacterial suspension, Pseudomonas proteus ( Pseudomonas plecoglossicida ) bacterial suspension and Bacillus elzuru ( Calidifontibacillus erzurumensis The bacterial suspension was mixed to obtain a compound bacterial solution.

[0007] (3) Add a microbial agent protectant to the obtained compound bacterial solution and mix to prepare a compound microbial agent.

[0008] Preferably, in step (2) of the present invention, *Pseudomonas taiwanensis* (… Pseudomonas taiwanensis Bacterial suspension, Bacillus belesiensis ( Bacillus velezensis ) bacterial suspension, Pseudomonas proteus ( Pseudomonas plecoglossicida ) bacterial suspension and Bacillus elzuru ( Calidifontibacillus erzurumensis The live bacteria ratio of ) is (1-3):(1-3):(1-3):(1-3).

[0009] Preferably, the microbial agent protectant in step (3) of the present invention contains sucrose, glycerol, monosodium glutamate and skim milk powder; wherein the final concentration of sucrose is 5-15 g / L, the final concentration of glycerol is 3-7 mL / L, the final concentration of monosodium glutamate is 3-7 g / L and the final concentration of skim milk powder is 5-15 g / L.

[0010] As a further preferred embodiment of the present invention, the final concentration of sucrose in the microbial agent protectant in step (3) of the present invention is 10 g / L, the final concentration of glycerol is 6 mL / L, the final concentration of monosodium glutamate is 4 g / L, and the final concentration of skim milk powder is 12.5 g / L.

[0011] Preferably, in step (3) of the present invention, the volume ratio of compound bacterial solution to bacterial agent protectant in the compound microbial agent is 1:(5-10).

[0012] The third objective of this invention is to provide the application of the composite microbial agent in the following two aspects: (1) The role of the compound microbial agent in inhibiting the survival of pathogens.

[0013] (2) Application of the compound microbial agent in promoting the growth of *Vernicia fordii*.

[0014] The beneficial effects of this invention are: (1) The compound microbial fertilizer obtained by the present invention has good antibacterial effect and can control a variety of common crop diseases. It is highly versatile.

[0015] (2) The components of the compound microbial fertilizer obtained by the present invention have synergistic functions. Compared with a single strain, the disease prevention effect is more prominent. At the same time, it can effectively promote the growth of tung oil trees and realize multiple uses of one strain.

[0016] (3) The microbial agent protectant described in this invention can improve the stability of the microbial agent, extend the shelf life, and have a high live bacteria retention rate.

[0017] (4) The compound microbial agent described in this invention can be used directly or in combination with organic fertilizer. The application method is flexible and adaptable to different planting patterns.

[0018] (5) The product is a microbial preparation, green and free of chemical residues, safe for crops, humans and animals and the environment, and meets the requirements of ecological agriculture development; the preparation process is simple, the raw materials are readily available, and it is easy to industrialize and promote on a large scale. Attached Figure Description

[0019] Figure 1 This is the result of the plate antibacterial test in Example 1 of the present invention.

[0020] Figure 2 This is a graph showing the statistical results of the antibacterial rate of the optimal combination of bacterial solutions in Example 2 of the present invention.

[0021] Figure 3 This is a graph showing the results of a single-factor test of the protective agent in Example 3 of this invention.

[0022] Figure 4 This is the disease incidence of *Vernicia fordii* in different groups in Example 1 of the present invention.

[0023] Figure 5 This describes the effect of different treatment groups on the growth-promoting effect of *Vernicia fordii* in Example 1 of this invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described therein; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and all raw materials used in the present invention can be purchased through conventional commercial channels.

[0025] The *Pseudomonas taiwanensis* used in this invention ( Pseudomonas taiwanensis Purchased from the German Microbial Culture Collection (DSMZ), accession number DSM 21245; Bacillus belesi ( Bacillus velezensis Purchased from the China Industrial Microbial Culture Collection Center (CICC), preservation number (product number) CICC 23325; *Pseudomonas proteus* ( Pseudomonas plecoglossicidaPurchased from the China Industrial Microbial Culture Collection Center (CICC), accession number (product number) CICC 22994; *Bacillus elzuru* ( Calidifontibacillus erzurumensis Purchased from the German Microbial Culture Collection (DSMZ), accession number (product number) is DSM 107530.

[0026] The PDA culture medium and seedling substrate used in this invention are all commercially available products that are commonly used in the field, and those skilled in the art can choose them as needed.

[0027] Example 1 The specific steps for the plate inhibition test of bacterial strains are as follows: The plate confrontation method was used to select various common crop pathogens as target bacteria; the activated test strains were co-cultured with the target bacteria, and the diameter of the inhibition zone was measured after the culture was completed to evaluate *Pseudomonas fortatum*. Pseudomonas taiwanensis Bacillus belesiensis ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis The antibacterial activity of single strains was evaluated by comparing the pathogenic bacterium Rhizoctonia solani with that of the single strain. Rhizoctonia solani ), Citrus intercalation pathogen ( Diaporthe citri (HS Fawc.) FA Wolf), Clostridium difficile ( Neofusicoccum parvum Alternaria ( Alternaria alternata ), Microsporum simulans ( Pestalotiopsis microspora Botrytis cinerea ( ) Botrytis cinerea Fusarium oxysporum ( Fusarium oxysporum ), Fusarium wilt of pepper ( Fusarium oxysporum f. sp. capsici ), tobacco gray mold ( Botrytis cinerea ), Staphylococcus aureus ( Botryosphaeria dothidea ), Sclerotium sclerotiorum ( Sclerotinia sclerotiorum ), Anthrax bacillus ( Colletotrichum acutatum Rice sheath blight pathogen ( Thanatephorus cucumeris ), Pseudomonas macrosporum stem spot ( Diaporthe sojae ), wheat scab ( Gibberella zeae ), tomato early blight pathogen ( Alternaria nightshade Verticillium wilt of eggplant ( Verticillium dahliae ) and collodion ( Colletotrichum gloeosporioides The bacteria were inoculated onto PDA medium and cultured for 7 days for activation. Uniformly sized mycelial cakes were then punched out from the edge of the colony using a 0.5 cm diameter sterile punch. The antagonistic bacteria *Pseudomonas taiwanensis* were determined using the plate confrontation method. Pseudomonas taiwanensis Bacillus belesiensis ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus from Erzurum Antibacterial activity against the above pathogenic bacteria: The prepared pathogenic bacteria mycelium was picked and placed in the center of the PDA medium. Then, the cross-shaped method was used to position the mycelium. A single colony of antagonistic bacteria was inoculated at a position 3 cm away from the center of the mycelium. Two antagonistic bacteria inoculation points were inoculated at the symmetrical position of the mycelium on each plate as the experimental group. At the same time, plates without antagonistic bacteria inoculation were set up as the control group. After inoculation, all the treated plates were inverted and placed in an incubator with a constant temperature of 28℃ for incubation. After 5 days of incubation, the antibacterial rate of the antagonistic bacteria against the pathogenic bacteria was calculated according to formula (2-1).

[0028] (2-1) In the formula: DCK is the colony diameter of the control group (cm); DT is the colony diameter of the experimental group (cm); 0.5 is the diameter of the mycelial cake (cm).

[0029] The results are as follows Figure 1 As shown, the inhibition rates of each antagonistic bacterium were between 10% and 60%, and all exhibited inhibitory activity against pathogenic bacteria. However, their inhibitory abilities showed significant differentiation. *Bacillus belyssus* (…) Bacillus velezensis High activity was most frequent, repeatedly showing the highest antibacterial rate across the entire event, with the best broad-spectrum antibacterial efficacy; *Pseudomonas taiwanensis* ( Pseudomonas taiwanensis The upper limit is the highest, with some pathogens showing an inhibition rate exceeding 54%, and the peak value is the strongest, but its stability is weaker than that of Bacillus belye ( ). Bacillus from Velez ); El Zulu-Bacillus ( Calidifontibacillus erzurumensis Overall, the level is moderate; *Pseudomonas proteus* ( Pseudomonas plecoglossicida Among the four strains, it had the lowest overall antibacterial effect, and its antibacterial rate against multiple pathogens was significantly lower than that of the other three.

[0030] Example 2 The specific steps for screening the optimal combination ratio of bacterial strains are as follows: (1) Activation and fermentation of strains: Pseudomonas taiwanensis ( Pseudomonas taiwanensis Bacillus belesiensis ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis The strain was streaked on PDA slant medium and activated at 36°C. The activated strain was then picked and placed in LB liquid medium and fermented at 36°C and 180 rpm until the OD of the bacterial suspension reached a certain value. 600 =1.10, and the viable bacterial count was 1.335×10. 9cfu / mL.

[0031] (2) Based on the viable count obtained in step (1), according to the *Pseudomonas taiwanensis* (… Pseudomonas taiwanensis Bacillus belesiensis ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis Five compounding schemes were set for the ratio of live bacteria in the bacterial suspension: 2:2:2:2, 3:1:1:1, 1:3:1:1, 1:1:3:1 and 1:1:1:3, with a total volume of 6 mL for the compounded bacterial suspension.

[0032] (3) Performance testing of compound bacterial solutions: Plate inhibition tests were conducted again on the compound bacterial solutions of each group in step (2). The target bacteria used were *Citrus intercalar pathogen* (Citrus intercalar pathogen). Citrus diaportha (HS Fawc.) FA Wolf), Clostridium difficile ( Neofusicoccus minor Alternaria ( Alternaria alternata ), Microsporum simulans ( Pestalotiopsis microspora Botrytis cinerea ( ) Botrytis cinerea ), Staphylococcus aureus ( Botryosphaeria dothidea ), Sclerotium sclerotiorum ( Sclerotinia sclerotiorum ), Anthrax bacillus ( Colletotrichum acutatum ), Pseudomonas macrosporum stem spot ( Diaporthe sojae ), Colloidal anthracnose bacteria ( Colletotrichum gloeosporioides Fusarium oxysporum ( Fusarium oxysporum ), Fusarium wilt of pepper ( Fusarium oxysporum f. sp. capsicum ), tobacco gray mold ( Botrytis cinerea ), tomato early blight pathogen ( Alternaria solani Verticillium wilt of eggplant ( Verticillium dahliae ), wheat scab ( Gibberella Zea Rhizoctonia solani ( ) Rhizoctonia solani ), pepper anthracnose bacteria ( Colletotrichum capsici ), Sorghum cocci ( Epicoccum sorghum ), stone fruit black rot skin fungus ( White-throated loach (Pers.) Fr.), Staphylococcus aureus ( Colletotrichum vinifera ), Magnolia niger ( Nigrospora spherica ) and rice sheath blight pathogen ( Cucumber beetle ).

[0033] The statistical results of the antibacterial rate are as follows: Figure 2 As shown, the compound bacterial solution exhibited antibacterial effects against all target bacteria, and when *Pseudomonas taiwanensis* (…), it showed an antibacterial effect. Pseudomonas taiwanensis Bacillus belesiensis ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya (Calidifontibacillus erzurumensis When the ratio of viable bacteria in the bacterial suspension is 1:3:1:1, the antibacterial effect on each target bacteria is the best; the optimal ratio of bacterial strains is determined based on the antibacterial effect.

[0034] Example 3 The formulation and specific steps for the microbial preservative are as follows: (1) Single-factor experiments: Commonly used microbial preservative raw materials were selected, and the effects of sucrose, glycerol, monosodium glutamate and skim milk powder at different concentrations on the viable count of compound microbial solutions were verified by single-factor experiments. Microbial preservatives with sucrose concentrations of 5, 7.5, 10, 12.5 and 15 g / L were prepared, with a final concentration of glycerol of 5 mL / L, monosodium glutamate of 5 g / L and skim milk powder of 10 g / L. Microbial preservatives with glycerol concentrations of 3, 4, 5, 6 and 7 mL / L were prepared, with a final concentration of sucrose of 10 g / L, monosodium glutamate of 5 g / L and skim milk powder of 10 g / L. Microbial preservatives with monosodium glutamate concentrations of 3, 4, 5, 6 and 7 g / L were prepared, with a final concentration of glycerol of 5 mL / L. The final concentrations of sucrose and skim milk powder were 10 g / L. Microbial preservatives were prepared at concentrations of 5, 7.5, 10, 12.5, and 15 g / L of skim milk powder, with final concentrations of glycerol (5 mL / L), sucrose (10 g / L), and monosodium glutamate (MSG) (5 g / L). The samples were treated using a moist heat sterilization process at 121℃ for 20 minutes. After the sterilization process was completed, the sterilized samples were removed and allowed to cool naturally to room temperature to obtain the microbial preservatives. The bacterial solution and preservatives were mixed at a volume ratio of 8:1 and dried in a freeze dryer for 48 hours. After drying, the viable cell count was determined using the plate count method to determine the optimal single-factor concentration. Specific results are shown below. Figure 3 As shown, the bacterial preservative produced the highest number of viable bacteria in the compound bacterial solution when the sucrose concentration was 10 g / L, the glycerol concentration was 6 mL / L, the monosodium glutamate concentration was 4 g / L, and the skim milk powder concentration was 12.5 g / L.

[0035] (2) Orthogonal experiment: Based on the results of single-factor experiments, an orthogonal experiment was designed, and the number of viable bacteria of the strain stored at room temperature was used as the evaluation index to optimize the best combination and ratio of protective agents.

[0036] ①Based on the optimal range of each factor obtained from the single-factor experiment results in step (1), an orthogonal experiment was conducted in SPSS software, and the sucrose concentration was coded as A, the glycerol concentration as B, the monosodium glutamate concentration as C, and the skim milk powder concentration as D.

[0037] (2) Set the low level, high level, and center point value for each factor, design the experiment, and obtain the experimental conditions as follows: Sucrose concentration (A): 5, 7.5 and 10 g / L.

[0038] Glycerol concentration (B): 5, 6 and 7 mL / L.

[0039] Monosodium glutamate (C): 4, 5 and 6 g / L.

[0040] Skim milk powder concentration (D): 7.5, 10 and 12.5 g / L.

[0041] (3) The preparation of the microbial agent protectant and the determination of the viable number of the strain were carried out according to the designed conditions. The results are shown in Table 1.

[0042] Table 1 Results of the orthogonal experiment for optimizing freeze-drying protectant Note: The experiment was conducted in three independent replicates. Data are presented as mean ± standard deviation. One-way ANOVA was used. Superscripts "a", "b", "c", and "d" for viable counts indicate multiple comparison markers. The same multiple comparison marker in the same column indicates no significant difference between groups, while different multiple comparison markers in the same column indicate significant differences between groups. p <0.05); where K1, K2 and K3 represent the sum of viable bacteria counts in all experimental groups at each level (low level, high level and central point value) of a certain factor, respectively; k1, k2 and k3 represent the average viable bacteria counts in all experimental groups at each level (low level, high level and central point value) of a certain factor, respectively.

[0043] Table 1 shows that when the glycerol concentration is 6 mL / L (A2), the monosodium glutamate concentration is 4 g / L (B1), the sucrose concentration is 10 g / L (C3), and the skim milk powder concentration is 12.5 g / L (D3), the theoretical optimal viable count of the compound bacterial solution can reach 17.33 (±0.58) × 10⁻⁶. 9 cfu / mL.

[0044] (4) Preparation of compound microbial inoculant: The optimal compound bacterial solution and the optimized protective agent are thoroughly mixed at a volume ratio of 5:1 and dried in a freeze dryer for 48 hours to obtain the compound microbial inoculant.

[0045] Example 1 The specific steps for performance testing of the finished compound microbial agent are as follows: (1) Preparation of disease-preventing and growth-promoting microbial organic fertilizer: The compound microbial agent prepared in step (4) of Example 3 is uniformly mixed into sterilized tung oil meal fertilizer (Guizhou Mingwei Fertilizer Co., Ltd.) at a solid-liquid ratio of 3:1, and stirred and mixed evenly to obtain disease-preventing and growth-promoting microbial organic fertilizer.

[0046] (2) Potted verification experiment: 32 normal *Vernicia fordii* seedlings with consistent growth were selected and divided into 4 groups. 8 seedlings were selected as the blank control group (CK), which was only watered. The remaining seedlings were inoculated with *Colletotrichum gloeosporioides*. The seedlings inoculated with *Colletotrichum gloeosporioides* were divided into 3 groups. One group was not treated and served as the negative control group (T1). One group was treated with *Bacillus subtilis* fertilizer (Shandong Dehe Mingxing Biotechnology Co., Ltd.) and served as the positive control group (T2). The last group was treated with the disease-preventing and growth-promoting microbial organic fertilizer prepared in step (1) and served as the organic fertilizer group (T3). The amount of microbial fertilizer applied to each seedling was 2 kg. Seedlings of *Vernicia fordii* were cultured at 25±1℃ for 40 days. Disease incidence was observed and recorded in each group of seedlings. Disease incidence and disease index were regularly calculated for each group, and relative control efficacy was determined. Simultaneously, plant height, root length, and root weight were measured. Seedlings were graded according to lesion area: Grade 0 - disease-free; Grade 1 - mild infection (lesion area 1%–25% of plant); Grade 2 - moderate infection (lesion area 26%–50% of plant); Grade 3 - severe infection (lesion area 51%–75% of plant); Grade 4 - extremely severe or complete plant death (lesion area over 75% or plant death). Results are shown in Table 2. Figure 4 As shown: Table 2 The incidence rate in the CK group (blank control) was 0, while the incidence rate in the T1 group (inoculated only with collodion anthracnose spores) was as high as 100%, with 62.5% of the plants being severely infected at grades 3 and 4, and the disease index reaching 71.88%. This indicates that the collodion anthracnose fungus is highly pathogenic and the inoculation effect is good, providing a reliable reference for evaluating the control efficacy of each treatment group. Compared with the T1 group, the disease index of the *Vernicia fordii* bio-organic fertilizer treatment group (T3) and the positive control group (T2) decreased to 21.88% and 18.75%, respectively, with incidence rates of 37.5% and 25%, and control rates of 69.56% and 73.91%, respectively. The treatment effects of the T3 group and the T2 group were comparable, with a difference of approximately 4.35 percentage points in their control rates. Nevertheless, all control efficacy indicators of the T3 group were significantly lower than those of the T1 group, indicating that the *Vernicia fordii* bio-organic fertilizer has a significant control effect on *Vernicia fordii* collodion anthracnose and has application value in actual production.

[0047] (3) Select 15 seedlings of *Vernicia fordii* with similar growth. The potted plant experiment was set up in three groups, with 5 parallels in each group. Group K1: planted in seedling substrate and watered with clean water every day; Group K2: planted in 50% seedling substrate with 50% Bacillus subtilis fertilizer (Shandong Dehe Mingxing Biotechnology Co., Ltd.); Group K3: planted in 50% seedling substrate with 50% disease-preventing and growth-promoting microbial organic fertilizer and watered with clean water every day. After 60 days of planting, the height, root length, above-ground fresh weight and underground fresh weight of the *Vernicia fordii* seedlings were measured.

[0048] The results are as follows Figure 5 As shown, the disease-preventing and growth-promoting microbial organic fertilizer prepared in this embodiment has a significant effect on promoting seedling growth. Compared with the blank control group (K1), the K3 treatment significantly improved various growth indicators of seedlings; among them, plant height increased from 17.05cm to 26.98cm, an increase of 58.24%; aboveground fresh weight increased from 3.05g to 8.43g, an increase of 176.39%, showing the most significant growth effect; underground root weight increased from 6.98g to 11.53g, an increase of 65.19%, showing outstanding root biomass accumulation. In contrast, although the K2 treatment also promoted seedling growth, it did not significantly improve plant height and aboveground fresh weight. The above-ground fresh weight and underground root weight were all lower than those of the K3 treatment, increasing by 38.01%, 96.07%, and 26.07%, respectively. This indicates that the disease-preventing and growth-promoting microbial organic fertilizer is more effective than commercial microbial fertilizer in promoting above-ground growth. This may be closely related to its microbial composition, nutrient composition ratio, and synergistic effect with the rhizosphere microorganisms of *Vernicia fordii*. By improving the nutritional status of plants and enhancing their systemic resistance, the disease-preventing and growth-promoting microbial organic fertilizer increases the growth potential and biomass of seedlings, achieving an organic unity of plant protection and growth promotion, and providing effective technical support for the green and sustainable production of *Vernicia fordii*.

[0049] Example 4 A 10 g / L sucrose solution, a 6 mL / L glycerol solution, a 4 g / L monosodium glutamate solution, and a 12.5 g / L skim milk powder solution were prepared and treated using a moist heat sterilization process. The sterilization temperature was set at 121°C and the sterilization time was 20 min. After the complete sterilization process was completed, the sterilized samples were removed and allowed to cool naturally to room temperature. The bacterial solution and the preservative were mixed at a volume ratio of 1:10 to obtain a compound bacterial solution. The compound bacterial solution had the same effect as the compound bacterial solution in Example 3.

[0050] Example 5 A 10 g / L sucrose solution, a 6 mL / L glycerol solution, a 4 g / L monosodium glutamate solution, and a 12.5 g / L skim milk powder solution were prepared and treated using a moist heat sterilization process. The sterilization temperature was set at 121°C and the sterilization time was 20 min. After the complete sterilization process was completed, the sterilized samples were removed and allowed to cool naturally to room temperature. The bacterial solution and the preservative were mixed at a volume ratio of 1:8 to obtain a compound bacterial solution. The compound bacterial solution had the same effect as the compound bacterial solution in Example 3.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound microbial agent, characterized in that: The compound microbial agent includes *Pseudomonas taiwanensis* (… Pseudomonas taiwanensis ), Bacillus belesi ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis ).

2. The compound microbial agent according to claim 1, characterized in that: The compound microbial agent also includes an agent protectant.

3. The method for preparing the composite microbial agent according to claim 1 or 2, characterized in that, The specific steps are as follows: (1) The strain of Pseudomonas taiwanensis ( Pseudomonas taiwanensis ), Bacillus belesi ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis ) was activated and then subjected to liquid fermentation to obtain *Pseudomonas taiwanensis* ( Pseudomonas taiwanensis Bacterial suspension, Bacillus belesiensis ( Bacillus velezensis ) bacterial suspension, Pseudomonas proteus ( Pseudomonas plecoglossicida ) bacterial suspension and Bacillus elzuru ( Calidifontibacillus erzurumensis Bacterial suspensions were prepared, and the number of viable bacteria in the bacterial suspensions was determined. (2) Based on the proportion of viable bacteria, Pseudomonas taiwanensis ( Pseudomonas taiwanensis Bacterial suspension, Bacillus belesiensis ( Bacillus velezensis ) bacterial suspension, Pseudomonas proteus ( Pseudomonas plecoglossicida ) bacterial suspension and Bacillus elzuru ( Calidifontibacillus erzurumensis The bacterial suspension was mixed to obtain a compound bacterial solution; (3) Add a microbial agent protectant to the obtained compound bacterial solution and mix to prepare a compound microbial agent.

4. The method for preparing the composite microbial agent according to claim 3, characterized in that: The Taiwan Pseudomonas ( Pseudomonas taiwanensis ), Bacillus belesi ( Bacillus velezensis ), Pseudomonas proteus ( Pseudomonas plecoglossicida ) and El Zulu-Menya ( Calidifontibacillus erzurumensis The ratio of live bacteria was (1-3):(1-3):(1-3):(1-3).

5. The method for preparing the composite microbial agent according to claim 3, characterized in that, The microbial agent protectant in step (3) contains sucrose, glycerol, monosodium glutamate and skim milk powder; wherein the final concentration of sucrose is 5-15 g / L, the final concentration of glycerol is 3-7 mL / L, the final concentration of monosodium glutamate is 3-7 g / L and the final concentration of skim milk powder is 5-15 g / L.

6. The method for preparing the composite microbial agent according to claim 3, characterized in that, In step (3), the volume ratio of compound bacterial solution to bacterial agent protectant in the compound microbial agent is (5-10):

1.

7. The application of the compound microbial agent according to claim 1 in inhibiting the survival of pathogenic bacteria.

8. The application of the compound microbial agent according to claim 1 in promoting the growth of *Vernicia fordii*.

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