Compound bacterial agent and application thereof in prevention and treatment of apple anthrax leaf blight

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound bacterial agent and application thereof to prevention and treatment of apple anthrax leaf blight. The compound bacterial agent disclosed by the invention comprises the following active ingredients: viable bacteria, freeze-dried bacteria and / or a bacterial strain culture of a bacillus velezensis TA-5 strain, and pyraclostrobin, wherein the viable bacteria and the freeze-dried bacteria of the bacillus velezensis TA-5 strain are used as active ingredients of the compound bacterial agent; the bacillus velezensis TA-5 is named as Bacillus velezensis in taxonomy, is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is December 11, 2024, and the preservation number is CGMCC NO.33034. The bacillus velezensis TA-5 is named as Bacillus velezensis and is preserved in the China General Microbiological Culture Collection Center (CGMCC). The compound bacterial agent disclosed by the invention has a remarkable effect on preventing and treating the apple anthracnose leaf blight.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound bacterial agent and its application in the prevention and control of apple anthracnose leaf blight. Background Technology

[0002] Bacillus belesiensis ( Bacillus velezensis As a practical and effective biological control agent, *Bacillus belye* has broad application prospects in the biological control of plant diseases. Bacillus velezensis First isolated from the soil of the Belé River estuary in Spain in 1999, *Bacillus vesiliflorus* possesses advantages such as a broad antibacterial spectrum, rapid growth, strong resistance to adverse conditions, and high biocompatibility. Its mechanism of action primarily involves the secretion of various antibacterial metabolites, such as cell wall degrading enzymes (glucanase and protease), lipopeptide antibiotics (surfactants, iturobrine, and fentanyl), and polyketide antibiotics. These substances can hydrolyze the cell wall of pathogens, inhibit conidial germination and germ tube growth, alter the structure and permeability of the cell membrane, and ultimately lead to the death of pathogen cells. Simultaneously, *Bacillus vesiliflorus* can also secrete plant hormones and volatile compounds, such as IAA and ACC deaminases, promoting plant growth and enhancing the plant's own disease resistance.

[0003] In recent years, anthracnose leaf blight has become widespread in apple-producing areas. Apple anthracnose leaf blight (GLS) is caused by the anthracnose fungus (… Glomerella cingulata This fungal disease, caused by [unclear], can infect leaves and fruits. It is characterized by a short incubation period, high spore production, rapid onset, rapid spread, and strong progression. It causes scorched necrotic lesions on leaves, leading to leaf drop, and 2-3 mm necrotic spots on fruits. Within a short period, it can cause all the leaves to dry up and fall off, affecting fruit development and flower bud formation, severely weakening the tree, and posing a huge threat to the apple industry.

[0004] Currently, chemical control remains the primary method of disease control. However, problems such as pesticide residues, easy development of pathogen resistance, and severe environmental pollution are becoming increasingly serious, leading to the restriction or banning of the use of many chemical fungicides. Pyraclostrobin, a strobilurin fungicide, inhibits mitochondrial respiration in pathogens, interfering with cellular energy metabolism and causing cell death. It is characterized by its broad spectrum, high efficiency, and low toxicity. However, improper application of traditional chemical pesticides has led to increased pathogen resistance and exacerbated ecological risks. Therefore, pesticide reduction and efficiency enhancement technologies have become an important research direction. Pesticide combination is a crucial measure for disease control. The combined use of biological pesticides and chemical agents is considered an environmentally friendly and economical integrated control strategy, which can significantly improve disease control effectiveness, help reduce pesticide dosage, and delay the development of pathogen resistance. Therefore, finding safe and effective control measures to replace chemical fungicides has become an urgent problem to be solved in the prevention and control of apple anthracnose leaf blight, and is also an important measure to reduce the use of chemical pesticides and increase their efficiency, and promote the green development of the fruit industry. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a compound microbial agent and its application in the control of apple anthracnose leaf blight. This compound microbial agent, through the combined use of biological and chemical agents to control apple anthracnose leaf blight, significantly improves the control efficacy compared to single agents, especially demonstrating excellent results in field trials. Simultaneously, it reduces the dosage of chemical agents, making it safe and environmentally friendly, and providing a completely new approach to agricultural disease control.

[0006] To address the above problems, the present invention provides the following solutions.

[0007] In a first aspect, the present invention provides a compound microbial agent, wherein the effective components of the compound microbial agent include live bacteria, freeze-dried bacteria and / or strain culture of Bacillus vesiculosus TA-5 strain, and pyraclostrobin. The taxonomic name of the *Bacillus belyssae* TA-5 strain is: Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 11, 2024, with accession number CGMCC NO.33034.

[0008] In this invention, the Bacillus belye ( Bacillus velezensis The TA-5 strain was isolated from the surface of healthy apple branches in Qingdao, Shandong. Through comparative experiments, the inventors discovered that this *Bacillus belyssioides* (…) Bacillus velezensisThe TA-5 strain inhibited the growth of mycelial growth of *Bacillus ventricosa*, the pathogen causing apple anthracnose leaf blight, resulting in morphological deformities of the mycelium and significantly reducing sporulation and spore germination rates. Furthermore, compound studies revealed that pyraclostrobin can be used as a component of *Bacillus belyssinus*. Bacillus velezensis It is an synergist for TA-5 microbial agent, enhancing its biological control effect.

[0009] Optionally, in the compound bacterial agent, the bacterial strain culture is the *Bacillus belesiensis* (…). Bacillus velezensis Fermentation broth, fermentation supernatant, concentrate and / or dried product of TA-5 strain.

[0010] In a preferred embodiment, the bacterial culture is *Bacillus belesiensis* (B. belesiensis). Bacillus velezensis Fermentation broth of strain TA-5.

[0011] Optionally, the compound microbial agent may be in the form of a liquid or solid preparation.

[0012] Preferably, the liquid preparation is a water dispersant or a bacterial suspension.

[0013] Preferably, the solid preparation is a powder, a wettable powder, or a dry suspension.

[0014] Optionally, in the compound bacterial agent, the Bacillus berberis ( Bacillus velezensis The viable cell count of strain TA-5 was 10. 3 ~10 8 CFU, preferably 10 5 CFU~10 8 CFU.

[0015] Preferably, when the compound bacterial agent is a liquid preparation, the Bacillus belyssus (…) Bacillus velezensis The effective viable bacterial concentration of strain TA-5 is greater than 10. 3 CFU / mL, preferably an effective viable bacteria concentration of 10. 5 CFU / mL, for example 10 5 CFU / mL ~10 8 CFU / mL.

[0016] Optionally, in the compound microbial agent, the pyraclostrobin exists in the form of a formulation; preferably, the pyraclostrobin formulation is an emulsifiable concentrate.

[0017] Optionally, the Bacillus belesiensis ( Bacillus velezensis The ratio of the viable bacterial concentration of strain TA-5 to the concentration of the pyraclostrobin was 10. 3 ~108 A compound of CFU / mL Bacillus vesiculosus TA-5 and 0.5~200 mg / L pyraclostrobin.

[0018] Preferably, the Bacillus belesiensis ( Bacillus velezensis The ratio of the viable bacterial concentration of strain TA-5 to the concentration of the pyraclostrobin was 10. 5 ~10 8 CFU / mL Bacillus vesiculosus TA-5 strain combined with 0.9~166 mg / L pyraclostrobin.

[0019] In the compound bacterial agent, the Bacillus berberis ( Bacillus velezensis The volume ratio of TA-5 strain to the pyraclostrobin is (0.1~10):1.

[0020] Preferably, the Bacillus belesiensis ( Bacillus velezensis The volume ratio of TA-5 strain to the pyraclostrobin is (0.1~9):1 (e.g., 9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 or any ratio therebetween), more preferably (0.5~1.5):1, and even more preferably 1:1.

[0021] In a preferred embodiment, the active ingredient of the compound bacterial agent is derived from the Bacillus berberis (B. berberis). Bacillus velezensis The composition includes live, lyophilized, inactivated bacterial cells or bacterial cultures of TA-5 strain, and pyraclostrobin; preferably Bacillus belesiensis (…). Bacillus velezensis The composition of live bacteria, freeze-dried bacteria, inactivated bacteria or fermentation broth of TA-5 strain with pyraclostrobin.

[0022] Optionally, the compound microbial agent further includes excipients. Preferably, the excipients are acceptable excipients for pesticide-based microbial agents, such as one or more of high-mesh diatomaceous earth, glucose, starch, amino acid powder, sodium dodecyl sulfonate, sodium dodecyl sulfate, and kaolin (e.g., 6000 mesh kaolin).

[0023] Secondly, the present invention provides an application of the compound microbial agent as described in the first aspect in the preparation of biological pesticides.

[0024] Optionally, the biopesticide is used for any one or more of the following purposes: (1) Used to antagonize peritrichous tetroma Glomerella cingulata ; (2) Used to prevent and control diseases caused by *Pterygota cladophyllum* Glomerella cingulata This causes anthracnose leaf blight in apples.

[0025] Thirdly, the present invention provides a compound bacterial agent as described in the first aspect for the preparation of antagonistic agents against *Pterocarya spp.*Glomerella cingulata Uses in pesticides.

[0026] Fourthly, the present invention provides a compound microbial agent as described in the first aspect for the preparation of a treatment for controlling *Pterygota spp.* Glomerella cingulata Its use in pesticides that cause apple anthracnose leaf blight.

[0027] Fifthly, the present invention provides a method for antagonizing *Trichoderma flavonoids* and / or controlling apple anthracnose leaf blight, wherein a compound fungicidal agent as described in the first aspect is sprayed onto apple leaves to control leaf diseases (e.g., caused by *Trichoderma flavonoids*). Glomerella cingulata Caused by apple anthracnose leaf blight.

[0028] Beneficial effects The compound microbial agent in this embodiment of the invention has excellent and efficient antagonistic effect against *Microcystis aeruginosa*. Specifically, it can inhibit the growth of its mycelium, reduce its spore germination rate, and affect the expression of its disease resistance-related genes. In particular, it can achieve a field control efficacy of up to 97.72% for the control of apple anthracnose leaf blight caused by *Microcystis aeruginosa*. Compared with single formulation control, its control effect is improved by at least 20%, playing a key role in the biological control of crops.

[0029] The compound microbial agent in the embodiments of the present invention can effectively reduce the amount of chemical agents used, so as to achieve a better prevention and control effect than chemical reagents, which is in line with the development of green chemistry. Attached Figure Description

[0030] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the implementation. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0031] Figure 1 Colony morphology of Bacillus belyss TA-5 strain on LB medium; Figure 2 Phylogenetic tree of Bacillus belyss TA-5 strain; Figure 3 The effects of fermentation supernatant and bacterial suspension of Bacillus belyss TA-5 on the growth of mycelia of the fungus causing apple anthracnose leaf blight were investigated. Different lowercase letters indicate significant differences between groups. p <0.05); Figure 4 The effect of Bacillus belychnophorus TA-5 fermentation broth on the expression of disease resistance-related genes in apple tissue; as shown in the attached figure. and These indicate that there were significant differences between the treatment group and the control group. p <0.05 and p <0.01); Figure 5 The preventive effects of Bacillus vesiculosus TA-5, pyraclostrobin, and compound inoculants on apple leaves; different lowercase letters indicate significant differences between groups ( p <0.05); A represents the statistics of the disease index, and B represents the statistics of the prevention and control effect as a protective agent; Figure 6 This image shows the field control effect of a compound microbial agent on apple leaves; different lowercase letters indicate significant differences between groups. p <0.05); where A represents the disease index statistics and B represents the prevention and control effect statistics. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0033] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0034] Unless otherwise specified, all reagents used in the experiments of this invention are commercially available.

[0035] The tested biocontrol strain was derived from Bacillus belyssus strain TA-5, which was isolated from the surface of healthy apple branches in Qingdao, Shandong Province, and preserved in the Laboratory of Fruit Tree Disease Epidemiology and Integrated Management at Qingdao Agricultural University. The preservation information is Bacillus belyssus (…). Bacillus velezensis The strain was deposited on December 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33034 and address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] Source of the tested pathogenic strain: The tested apple anthracnose leaf blight pathogen strain GC20190701 was isolated from the Modern Agricultural Demonstration Park of Qingdao Agricultural University in July 2019, and was identified by multi-gene joint identification as... Glomerella cingulata .

[0037] Example 1: Isolation and identification of Bacillus belyss TA-5 strain (1) Isolation and screening of strains In commercial orchards in Qingdao, Shandong Province, healthy branches and fruits of "Fuji" apples were randomly selected. The cortical tissue was ground in 0.1 mmol / L PBS buffer, separated by conventional serial dilution plating method, and cultured on LB medium at 25°C. Single colonies were picked and streaked for inoculation. The streaking was repeated 1-2 times to obtain pure cultures.

[0038] Using *Pseudomonas aeruginosa* as the target pathogen, a bacterial strain with antibacterial activity and significant control effect was obtained and named TA-5.

[0039] (2) Identification of strain TA-5 a. Morphological characteristics like Figure 1 As shown, after streaking on LB plates at 25°C for 24 hours, the bacteria showed good growth, forming opaque, milky-white colonies with irregular edges. The colony surface was rough, with a raised center. Microscopic examination revealed rod-shaped cells. The characteristics exhibited by this strain are consistent with the description of Bacillus in the "Handbook of Systematic Identification of Common Bacteria".

[0040] LB medium: 10g tryptone, 5g yeast extract, 5g NaCl, bring to a final volume of 1000mL with distilled water, and adjust the pH to 7.0.

[0041] b. Physiological and biochemical characteristics Physiological and biochemical tests revealed that this strain was Gram-positive. It can utilize glucose, sucrose, lactose, mannose, sorbitol, arabinose, maltose, and cellobiose as carbon sources. This strain can liquefy gelatin, hydrolyze starch, and form a biofilm. It was positive for catalase and VP tests, and negative for methyl red, urease, and indole production tests (Table 1). The physiological and biochemical reactions exhibited by this strain are consistent with the description of Bacillus in the *Handbook of Systematic Identification of Common Bacteria*.

[0042] Table 1. Physiological and biochemical characteristics of biocontrol strains

[0043] Note: "+" indicates positive; "-" indicates negative.

[0044] c. Genetic identification Experimental methods: Using the strain's DNA as a template, PCR amplification was performed using 16S rDNA primers. The amplified PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequences were analyzed for BLAST homology using NCBI, and multiple sequence comparisons were performed using MEGA11 software to construct a phylogenetic tree.

[0045] Results and Analysis: The obtained sequences were compared with nucleic acid sequences in GenBank using BLAST software from the NCBI database (http: / / www.ncbi.nlm.nih.gov). Strain TA-5 was found to be similar to *Bacillus belye*. Bacillus velezensis (Accession number: OP435757.1) has the highest homology (100%). Figure 2 ) Based on the aforementioned morphological characteristics, physiological and biochemical properties, and sequence analysis results, strain TA-5 was identified as *Bacillus belyesensis*. Bacillus velezensis ).

[0046] Example 2: Study on the control effect of Bacillus belyssus TA-5 1. Effects of Bacillus vesiculosus TA-5 on the mycelial growth of *Bacillus belladonna*, the causal agent of apple anthracnose leaf blight. Bacillus berberis ( Bacillus velezensis The supernatant of the fermentation broth of strain TA-5 (denoted as TA-5 supernatant) and its bacterial suspension (with sterile water as the solvent, denoted as TA-5 bacterial suspension) were added to PDA medium to make the final concentration of the supernatant and bacterial suspension in the medium 1×10⁻⁶. 6 5×10 6 1×10 7 5×10 7 and 1×10 8 CFU / mL was designated as the experimental group. PDA medium without TA-5 strain was used as the control. Activated *Anthracnose leaf blight*, the causal agent of apple anthracnose leaf blight, was inoculated into the experimental and control media (the activation method is a standard procedure in this field: the strain slant was removed from a 4°C freezer, incubated on a PDA plate at 25°C for 3 days, and mycelial pellets were collected using a 5mm diameter sterile punch and inoculated onto another PDA plate; after 3 days, the pellets were ready for use). The plates were then incubated in the dark at 25°C for 4 days, and the colony diameter was observed and measured.

[0047] The fermentation broth of Bacillus belye TA-5 was prepared as follows: 100 μL of glycerol culture was added to a 50 mL Erlenmeyer flask containing 10 mL of LB liquid medium. The flask was incubated at 25°C and 180 rpm for 12 h with shaking. The culture was then streaked onto a solid LB agar plate and incubated at 25°C in the dark for 48 h. A single colony was picked and added to 15 mL of LB liquid medium and incubated at 25°C and 180 rpm with shaking for 12 h. 1 mL of this culture was then added to 100 mL of LB medium and incubated at 25°C and 180 rpm with shaking for 48 h to obtain the fermentation broth of strain TA-5 (5 × 10⁻⁶). 9 The TA-5 fermentation broth was centrifuged at 5000 rpm for 10 min to obtain the fermentation supernatant of strain TA-5. The bacterial cells were resuspended in sterile water to obtain the fermentation broth of strain TA-5.

[0048] The composition of PDA medium is as follows: 200g of peeled potatoes are weighed, cut into small pieces, boiled in water for 15-20 minutes, filtered through eight layers of gauze, and then 20g of glucose and 15g of agar powder are added. The volume is adjusted to 1000mL with distilled water and the pH value is natural. The medium is then autoclaved at 121℃ for 20 minutes.

[0049] The results are as follows Figure 3 As shown in Table 2.

[0050] Table 2. Effects of Bacillus belye TA-5 on mycelial growth of the fungus causing apple anthracnose leaf blight.

[0051] Results analysis: Adding different concentrations of TA-5 supernatant and bacterial solution all had a certain inhibitory effect on apple anthracnose leaf blight pathogens, and the inhibitory effect was significantly improved with increasing concentration.

[0052] 2. Effects of Bacillus belyssus TA-5 treatment on the expression of disease resistance-related genes in apple tissue. Select uniformly sized and healthy Gala / Fuji apple leaves, disinfect them with 75% alcohol, wrap the leaf tips with damp cotton, and spray with TA-5 fermentation filtrate (fermentation filtrate concentration of 10). 8 CFU / mL (prepared using the same method as in Example 2), designated as the Gala / Fuji group, was incubated at 25℃ for 48 hours to retain moisture, and RNA was extracted from samples. Water spraying served as a control (CK group). Disease resistance-related RNA was detected in apple tissue. PR Changes in gene expression.

[0053] See results Figure 4 And Table 3.

[0054] Table 3. Effects of treatment with strain TA-5 on the expression of disease resistance-related genes in apple tissues.

[0055] Results analysis: After spraying TA-5 fermentation filtrate, the following results were detected: MdPR1, MdPR2, MdPR4, MdPR5, MdPR8, MdPR10 All six disease resistance-related genes were significantly upregulated in Gala / Fuji leaves. Figure 4 This indicates that Bacillus belye TA-5 can induce the upregulation of disease-related protein genes in apple tissue, thereby improving the host's resistance to the disease.

[0056] Example 3: Plate virulence determination of chemical reagents against anthracnose leaf blight pathogen. Experimental methods: The mycelial growth rate method was used to determine the indoor toxicity of three agents against the pathogen. PDA medium containing different mass concentrations (0, 0.625, 1.25, 2.5, 5, 12.5, 25, 50 mg / L) of the test agents was prepared, and 5 mm diameter *Bacillus anthracis* leaf blight cakes were inoculated into the medium. The mixture was incubated at 25℃ inverted for 4 days. The colony diameter was then measured, and the inhibition rate (I) was calculated. I=[(D1-5)-(D2-5)] / (D1-5)×100% In the formula, D1 is the diameter (mm) of the colonies in the blank control, and D2 is the diameter (mm) of the colonies in the PDA medium containing the test drug. A regression analysis was performed on the logarithm of the drug concentration (x) to base 10 and the probability value of the corresponding inhibition rate (y) to obtain the toxicity regression equation. The coefficient of determination (R²) of the regression line was then calculated. 2 The effective median concentration (EC50) of the tested agent against anthracnose leaf blight pathogens was calculated. 50 (mg / L): y = a + bx In the formula, a represents the y-intercept of the regression line, and b is the value used to calculate EC. 50 The intermediate value is the absolute value of the slope of the regression line.

[0057] B. Results Analysis: The results of the indoor toxicity tests of the three fungicides against *Hydroxillus chinensis* (Table 4) showed that pyraclostrobin, tebuconazole, and mancozeb all had a certain inhibitory effect on *Hydroxillus chinensis*. Pyraclostrobin showed the strongest toxicity against the pathogen, followed by mancozeb, while tebuconazole showed the weakest toxicity against *Hydroxillus chinensis*, EC50. 50 Higher than 100 mg / L.

[0058] Table 4. Results of indoor toxicity tests of fungicides against *Hydrocotyle spp.*, the causal agent of anthracnose leaf blight.

[0059] Example 4: Effects of compound inoculant on mycelial growth of Anthracnose leaf blight fungus Using the same preparation method as in Example 2, the fermentation supernatant of Bacillus belye TA-5 strain was prepared to a final concentration of 10. 7CFU / mL Preparation of pyraclostrobin formulation: The pyraclostrobin used in this invention is a commercial formulation of 250g / L pyraclostrobin emulsifiable concentrate, produced by BASF Plant Protection (Jiangsu) Co., Ltd. When preparing the formulation, it is diluted with distilled water, and the application concentration in the plate compound inoculant is 0.90mg / L.

[0060] Preparation of compound bacterial agent: The above 1×10 7 The compound microbial agents are prepared by mixing CFU / mL TA-5 microbial agent and 0.9 mg / L pyraclostrobin preparation at volume ratios of 0:10, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1 and 10:0, respectively.

[0061] Experimental methods: The mycelial growth rate method was used. PDA medium containing the compound inoculum was prepared, and 5 mm diameter *Bacillus anthracis* leaf molds were inoculated into the medium. The mixture was incubated at 25℃ inverted for 4 days, and then the colony diameter was measured to calculate the inhibition rate. The Horsfall method was used to calculate the synergistic virulence.

[0062] E th =I1×V1+I2×V2 E th I1 represents the theoretical efficacy of the compound agent, i.e., the theoretical inhibition rate of the compound agent against anthracnose leaf blight pathogen. V1 represents the inhibition rate of the fungicide against anthracnose leaf blight pathogen. I2 represents the inhibition rate of TA-5 against anthracnose leaf blight pathogen. V2 represents the volume ratio of TA-5 in the mixture. I R =E ab / E th I R E represents the efficiency improvement ratio. ab The actual efficacy of the compound agent is its inhibition rate against anthracnose leaf blight pathogens. Based on I... R The value is used to determine the synergistic effect of different formulations. R >1 indicates a synergistic effect; I R =1 indicates an additive effect; I R <1 indicates an antagonistic effect.

[0063] Table 5. Virulence test results of TA-5 combined with pyraclostrobin against *Agrostis chinensis*, the causal agent of leaf blight.

[0064] Results analysis: Based on the above experimental results, 0.90 mg / L pyraclostrobin was mixed with a bacterial cell concentration of 1×10⁻⁶. 7TA-5 at CFU / mL was compounded at different volume ratios for indoor combined toxicity assays. The results (Table 5) show that 0.90 mg / L pyraclostrobin and 1×10⁻⁶ CFU / mL pyraclostrobin were effective in determining the combined toxicity of TA-5. 7 CFU / mL TA-5 showed a synergistic effect in inhibiting anthracnose leaf blight pathogens, with a volume ratio of 1:1 being particularly effective. R With a concentration of 1.45, the actual antibacterial rate was 66.98%, which was higher than the antibacterial rate of other volume ratio treatments, indicating the strongest synergistic effect and the best antibacterial effect.

[0065] Example 5: Preventive effect of Bacillus vesiculosus TA-5 and its compound inoculant on apple anthracnose leaf blight. The fermentation broth of Bacillus belyssus TA-5 strain was prepared using the same preparation method as in Example 2, to a final concentration of 10. 8 The pyraclostrobin formulation was prepared using the same preparation method as in Example 4, with a final concentration of 166 mg / L and a concentration of CFU / mL.

[0066] Preparation of compound bacterial agent: The above 1×10 8 A compound bacterial agent is prepared by mixing CFU / mL TA-5 bacterial agent and 166 mg / L pyraclostrobin preparation at a volume ratio of 1:1.

[0067] To prepare a spore suspension of *Anthracnose leaf blight* pathogen, the pathogen was cultured in Czapek's medium with shaking for 4 days. After filtration through four layers of gauze, the suspension was centrifuged at 8000 rpm for 10 minutes. A conidial suspension was prepared by resuspending the spores in ice dew and adjusting the concentration to 10. 5 Select healthy apple Gala leaves of uniform size per mL, disinfect them with 75% alcohol, and wrap the leaf tips with moist cotton.

[0068] Preventive effect: Spray the prepared TA-5 fermentation broth (10) onto the leaves. 8 The following agents were used as controls: CFU / mL, pyraclostrobin (166 mg / L), TA-5 + pyraclostrobin (1:1), and water. After 48 hours, a spore suspension of *Hypericum anthracnose* was sprayed. After 7 days of incubation at 25°C, the disease index was calculated, and the preventative effect was assessed. The results are summarized in Table 6. Figure 5 As shown.

[0069] The classification is based on the proportion of diseased area to the total leaf area, and the classification criteria are as follows: Grade 0: No lesions; Grade 1: Lesions cover less than 5% of the leaf area; Grade 2: Lesions cover 6% to 30% of the leaf area; Grade 3: Lesions cover 30% to 50% of the leaf area; Grade 4: Lesions cover 51% to 80% of the leaf area; Grade 5: Lesions cover more than 80% of the leaf area or leaf drop.

[0070]

[0071] Table 6. Preventive effect against apple anthracnose leaf blight

[0072] Results analysis: such as Figure 5 The results showed that after 7 days, the leaves of the control group were covered with spots, while the leaves sprayed with TA-5, pyraclostrobin, and the compound inoculant had fewer spots, and the disease index was significantly different from that of the control group. TA-5, pyraclostrobin, and the compound inoculant showed a protective effect of over 80% on apple leaves, indicating that compound inoculants can be used to reduce the amount of pyraclostrobin used while still achieving excellent protective effects.

[0073] Example 6: Field efficacy test of compound bacterial agent against apple anthracnose leaf blight The fermentation broth of Bacillus belyssus TA-5 strain was prepared using the same preparation method as in Example 2, to a final concentration of 10. 8 The pyraclostrobin formulation was prepared using the same preparation method as in Example 4, with a final concentration of 166 mg / L and a concentration of CFU / mL.

[0074] Preparation of compound bacterial agent: The above 1×10 8 A compound bacterial agent is prepared by mixing CFU / mL TA-5 bacterial agent and 166 mg / L pyraclostrobin preparation at a volume ratio of 1:1.

[0075] Preparation of chlorothalonil formulation: The chlorothalonil used in this invention is a commercial formulation of 750g / L chlorothalonil wettable powder, purchased from Syngenta (Suzhou) Crop Protection Co., Ltd. When preparing it, it is diluted with distilled water and the application concentration is 400mg / L.

[0076] Using the same preparation method as in Example 5, a spore suspension of *A. anthracnose* was prepared to a final concentration of 10. 5 per mL.

[0077] Experimental method: Healthy Gala apple trees were selected in the field, and TA-5 fermentation liquid (10) was sprayed on the field. 8 Four groups of fungicides were used: chlorothalonil (400 mg / L), pyraclostrobin (166 mg / L), and TA-5 + pyraclostrobin (1:1). The leaves were sprayed until water droplets fell. The dosage was 150 kg per mu. Spraying with water was used as a control.

[0078] A total of four sprays were applied on June 30, July 7, July 17, and July 28. On August 11, spore suspension was sprayed on samples taken from pruned branches. After 7 days of incubation at 25℃ with the branches covered and kept moist, the disease index was calculated, and the control effect was determined. The results are shown in Table 7. Figure 6 As shown.

[0079] The classification is based on the proportion of diseased area to the total leaf area, and the classification criteria are as follows: Grade 0: No lesions; Grade 1: Lesions cover less than 5% of the leaf area; Grade 2: Lesions cover 6% to 30% of the leaf area; Grade 3: Lesions cover 30% to 50% of the leaf area; Grade 4: Lesions cover 51% to 80% of the leaf area; Grade 5: Lesions cover more than 80% of the leaf area or leaf drop.

[0080]

[0081] Table 7. Field efficacy test of compound bacterial agent against apple anthracnose leaf blight

[0082] Results analysis: such as Figure 6 As shown in Table 7, when the leaf lesions in the control group spread across the entire leaf, the TA-5 treatment group and the compound treatment group exhibited significant control effects. The disease index of the control group sprayed with water reached 95.20%, while the disease index of the pyraclostrobin treatment group reached 82.00%, with a control effect of only 14.23%, presumably due to rainwater erosion. The control effect of the TA-5 treatment group was 70.19%, the control effect of the chlorothalonil treatment group was 66.77%, while the control effect of the TA-5 + pyraclostrobin compound treatment was 97.72%, significantly higher than the control effects of the two single agents (Table 7). This indicates that the combined application of Bacillus belycetamol TA-5 and pyraclostrobin can improve its control effect. Furthermore, the strong adhesion of the compound agent can reduce the impact of weather conditions preventing the effective components of pyraclostrobin from functioning properly.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications 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 protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, The active ingredients of the compound bacterial agent include live bacteria, freeze-dried bacteria and / or bacterial cultures of Bacillus vesiculosus TA-5 strain, as well as pyraclostrobin. The taxonomic name of the *Bacillus belyssae* TA-5 strain is: Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 11, 2024, with accession number CGMCC NO.33034.

2. The compound microbial agent according to claim 1, characterized in that, In the compound bacterial agent, the bacterial strain culture is the Bacillus belesiensis. Bacillus velezensis Fermentation broth, fermentation supernatant, concentrate and / or dried product of TA-5 strain are preferably the Bacillus belesii strain. Bacillus velezensis Fermentation broth or fermentation supernatant of TA-5 strain; And / or, the pyraclostrobin is present in the form of a formulation.

3. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is a liquid or solid preparation. Optionally, the liquid formulation is a water dispersant or a bacterial suspension; Optionally, the solid dosage form is a powder, a wettable powder, or a dry suspension. And / or, in the compound formulation, the Bacillus belye Bacillus velezensis The viable cell count of strain TA-5 was 10. 3 ~10 8 CFU.

4. The compound microbial agent according to claim 3, characterized in that... When the compound bacterial agent is a liquid preparation, the *Bacillus belyssae* is included. Bacillus velezensis The effective viable bacterial concentration of TA-5 strain is greater than 10. 3 CFU / mL, preferably, the effective viable bacteria concentration is 10. 5 CFU / mL, for example 10 5 CFU / mL ~10 8 CFU / mL; Optionally, the Bacillus belesiensis Bacillus velezensis The ratio of the viable bacterial concentration of strain TA-5 to the concentration of the pyraclostrobin was 10:

1. 3 ~10 8 CFU / mL Bacillus vesiculosus TA-5 strain combined with 0.5~200 mg / L pyraclostrobin; Preferably, the Bacillus belesii Bacillus velezensis The ratio of the viable bacterial concentration of strain TA-5 to the concentration of the pyraclostrobin was 10:

1. 5 ~10 8 CFU / mL Bacillus beles Bacillus velezensis The TA-5 strain was combined with 0.9~166 mg / L pyraclostrobin.

5. The compound microbial agent according to claim 4, characterized in that, In the compound bacterial agent, the Bacillus berberis is mentioned. Bacillus velezensis The volume ratio of TA-5 strain to the pyraclostrobin is (0.1~10):1; Preferably, the Bacillus belesii Bacillus velezensis The volume ratio of TA-5 strain to the pyraclostrobin is (0.1~9):1, more preferably (0.5~1.5):1, and even more preferably 1:

1.

6. The compound microbial agent according to claim 1, characterized in that, The active ingredient of the compound bacterial agent is Bacillus belysinus. Bacillus velezensis The composition consists of live bacteria, freeze-dried bacteria, and / or bacterial cultures of TA-5 strain, and pyraclostrobin; preferably Bacillus belesiensis. Bacillus velezensis The TA-5 strain consists of live bacteria, freeze-dried bacteria, inactivated bacteria, or fermentation broth, combined with pyraclostrobin.

7. The compound microbial agent according to any one of claims 1-6, characterized in that, The compound microbial agent also includes excipients, preferably, the excipients are acceptable excipients for pesticide-based microbial agents.

8. The use of a compound microbial agent as described in any one of claims 1-7 in the preparation of biological pesticides.

9. The application according to claim 8, characterized in that, The biopesticide is used for any one or more of the following purposes: (1) Used to antagonize peritrichous tetroma Glomerella cingulata ; (2) Used to prevent and control diseases caused by *Pterygota cladophyllum* Glomerella cingulata This causes anthracnose leaf blight in apples.

10. A method for antagonizing *Trichoderma spp.* and / or controlling apple anthracnose leaf blight, wherein, The compound fungicide as described in any one of claims 1-7 is sprayed onto apple leaves for prevention and control.

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