Bacillus subtilis, composite microbial agent, preparation method and application thereof

Abstract

This invention provides a strain of Bacillus subtilis, a compound bacterial agent, its preparation method, and its application, belonging to the field of plant disease biological control technology. This invention involves expanding the culture of Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05, followed by mixed culture to obtain a compound bacterial solution. The pot control efficacy of the compound bacterial solution reaches 42.70%. A wettable powder is prepared by selecting suitable carriers, dispersants, wetting agents, and protectants. The wettable powder was applied in pot and field trials to control leaf spot disease of honeysuckle berries caused by Alternaria spp., achieving control efficiencies of 58.34% and 46.46%, respectively. The biocontrol strains in the compound bacterial solution and wettable powder of this invention enhance the control effect of both on honeysuckle berry leaf spot disease through synergistic action.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases, and particularly relates to a strain of Bacillus subtilis, a compound bacterial agent, its preparation method and application. Background Technology

[0002] In recent years, leaf spot disease has occurred in many honeysuckle berry growing areas in my country, leading to leaf wilting, reduced honeysuckle berry yield, and severe economic losses. Currently, the control of honeysuckle berry leaf spot disease mainly relies on chemical pesticide spraying, but the control effect is not significant, and chemical pesticide residues cause serious food safety and environmental pollution problems. Biological control utilizes naturally occurring beneficial microorganisms in the environment to combat plant pathogens, offering advantages such as environmental friendliness and no secondary pollution. Its green, safe, and highly efficient control process and effects are gaining increasing acceptance. Bacillus is a commonly used biocontrol bacterium in production, possessing strong resistance, stable colonization, strong antagonistic effects, and a good safety profile. However, research on biocontrol bacteria for controlling honeysuckle berry leaf spot disease is currently limited. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a strain of Bacillus subtilis, a compound microbial agent, its preparation method and application. Based on the biocontrol characteristics of Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 and the advantages of using wettable powder, a wettable powder compound microbial agent for controlling leaf spot disease of honeysuckle berries is prepared. Through the synergistic effect of the two strains and the better adsorption and effectiveness of the wettable powder on honeysuckle berry leaves, its control effect on leaf spot disease of honeysuckle berries can be enhanced.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a strain of Bacillus subtilis NEAU-L03, which was deposited at the China General Microbiological Culture Collection Center on December 19, 2024, with the accession number CGMCC No. 33129.

[0006] The present invention also provides a compound microbial agent, comprising Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05;

[0007] The Bacillus velezensis NEAU-L05 was deposited at the China General Microbiological Culture Collection Center on December 19, 2024, with accession number CGMCC No. 33130.

[0008] The present invention also provides a liquid formulation containing the aforementioned compound microbial agent, wherein the preparation method of the liquid formulation includes the following steps:

[0009] 1) Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 were inoculated into LB broth and cultured to obtain Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 bacterial suspensions, respectively.

[0010] 2) Mix Bacillus subtilis NEAU-L03 bacterial suspension and Bacillus velezensis NEAU-L05 bacterial suspension to obtain a mixed bacterial suspension. Inoculate the mixed bacterial suspension into a fermentation medium and culture it to obtain a liquid preparation containing the aforementioned compound bacterial agent.

[0011] Preferably, in step 1), the inoculum amount of Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 is 0.5-1.5% of the LB culture medium volume; the culture temperature is 25-30°C, the culture rotation speed is 150-200 rpm, and the culture time is 18-30 h.

[0012] Step 2) The volume ratio of Bacillus subtilis NEAU-L03 bacterial suspension and Bacillus velezensis NEAU-L05 bacterial suspension is 2-6:4-8; the inoculation amount of the mixed bacterial suspension is 3-5% of the fermentation medium volume; the fermentation medium comprises the following components at the following concentrations: maltose 15-25 g / L, yeast extract 30-40 g / L, magnesium sulfate 5-8 g / L, and the balance being water; the culture temperature is 28-32℃, and the culture time is 42-54 h.

[0013] The present invention also provides a wettable powder containing the aforementioned compound microbial agent, wherein the preparation method of the wettable powder includes the following steps:

[0014] 1) The liquid preparation and kaolin are mixed according to the saturated adsorption capacity and dried to obtain the mother powder;

[0015] 2) Mix the masterbatch powder, dispersant, wetting agent and protective agent, crush and sieve to obtain a wettable powder containing the compound microbial agent.

[0016] Preferably, the mass ratio of the masterbatch powder, dispersant, wetting agent, and protective agent in step 2) is 85-90:6-7:2-3:0.01-0.02;

[0017] The dispersant includes sodium lignosulfonate, the wetting agent includes sodium dodecylbenzenesulfonate, and the protective agent includes sodium alginate.

[0018] The present invention also provides the application of the aforementioned Bacillus subtilis NEAU-L03 in inhibiting Alternaria spp.

[0019] This invention also provides the application of the aforementioned compound microbial agent, liquid formulation, and wettable powder in the prevention and control of plant leaf spot disease.

[0020] Preferably, the plant includes honeysuckle fruit.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention involves expanding the culture of biocontrol strains (Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05), and mixing the bacterial solutions of the two strains in a certain proportion to obtain a composite bacterial solution. The antibacterial rate of the composite bacterial solution reached 59.44%, which is significantly higher than the antibacterial rate of the two individual biocontrol strains.

[0023] This invention selects suitable carriers, dispersants, wetting agents, and protectants to prepare a wettable powder containing biocontrol strains (Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05). This wettable powder features strong leaf adsorption, convenient storage and transportation, low cost, minimal environmental pollution, and good compatibility. It can be used to control leaf spot disease of honeysuckle berries caused by Alternaria tenuissima. In pot experiments and field trials, the control efficacy of this wettable powder was 58.34% and 46.46%, respectively, significantly superior to commercially available microbial inoculants.

[0024] The biocontrol strains in the compound bacterial solution and wettable powder of this invention work synergistically to enhance the control effect of the compound bacterial solution and wettable powder on leaf spot disease of honeysuckle berries. This overcomes the shortcomings of single biocontrol bacteria, such as a single biocontrol mechanism and poor adaptability to the environment. It has great application value for the cultivation of economic crops and helps to reduce the use of chemical agents, making it green and safe. Attached Figure Description

[0025] Figure 1 Streak plates of strains NEAU-L03 and NEAU-L05 (where A is strain NEAU-L03 and B is strain NEAU-L05);

[0026] Figure 2 The images show the plate inhibition effects of strains NEAU-L03 and NEAU-L05 (where A is the control group, i.e., no biocontrol strain was inoculated; B is strain NEAU-L03; C is strain NEAU-L05; and D is the combined control effect of strains NEAU-L03 and NEAU-L05).

[0027] Figure 3 The image shows the 16S rDNA identification results of strains NEAU-L05 and NEAU-L03 (where A is strain NEAU-L03 and B is strain NEAU-L05).

[0028] Figure 4 This is a schematic diagram of a wettable powder (where A is the solid state and B is the liquid state dissolved in water).

[0029] Figure 5 These are the control efficacy results of potted plant experiments after spraying;

[0030] Figure 6 These are images of leaf diseases in potted plants from different treatment groups (where A is the control group, B is treatment group 1, C is treatment group 2, D is treatment group 3, E is treatment group 4, F is treatment group 5, and G is treatment group 6).

[0031] Biological Preservation Instructions

[0032] The Bacillus subtilis NEAU-L03 provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33129, deposited on December 19, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] The Bacillus velezensis NEAU-L05 provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33130, deposited on December 19, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0034] This invention provides a strain of Bacillus subtilis NEAU-L03, which was deposited at the China General Microbiological Culture Collection Center on December 19, 2024, with the accession number CGMCC No. 33129.

[0035] The present invention also provides a compound microbial agent, comprising Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05;

[0036] The Bacillus velezensis NEAU-L05 was deposited at the China General Microbiological Culture Collection Center on December 19, 2024, with accession number CGMCC No. 33130.

[0037] The present invention also provides a liquid formulation containing the aforementioned compound microbial agent, wherein the preparation method of the liquid formulation includes the following steps:

[0038] 1) Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 were inoculated into LB broth and cultured to obtain Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 bacterial suspensions, respectively.

[0039] 2) Mix Bacillus subtilis NEAU-L03 bacterial suspension and Bacillus velezensis NEAU-L05 bacterial suspension to obtain a mixed bacterial suspension. Inoculate the mixed bacterial suspension into a fermentation medium and culture it to obtain a liquid preparation containing the aforementioned compound bacterial agent.

[0040] In this invention, *Bacillus subtilis* NEAU-L03 and *Bacillus velezensis* NEAU-L05 are respectively inoculated into LB culture medium and cultured to obtain *Bacillus subtilis* NEAU-L03 and *Bacillus velezensis* NEAU-L05 bacterial suspensions. The inoculation amount of *Bacillus subtilis* NEAU-L03 and *Bacillus velezensis* NEAU-L05 is preferably 0.5-1.5% of the LB culture medium volume, more preferably 0.8-1.2%; the culture temperature is preferably 25-30°C, more preferably 26-29°C; the culture rotation speed is preferably 150-200 rpm, more preferably 160-190 rpm; and the culture time is preferably 18-30 h, more preferably 21-27 h.

[0041] In this invention, Bacillus subtilis NEAU-L03 bacterial suspension and Bacillus velezensis NEAU-L05 bacterial suspension are mixed to obtain a mixed bacterial suspension. The mixed bacterial suspension is then inoculated into a fermentation medium and cultured to obtain a liquid preparation containing the aforementioned compound bacterial agent. The preferred volume ratio of the Bacillus subtilis NEAU-L03 bacterial suspension and the Bacillus velezensis NEAU-L05 bacterial suspension is 2–6:4–8, more preferably 3–5:5–7; the preferred inoculation amount of the mixed bacterial suspension is 3–5% of the fermentation medium volume, more preferably 3.5–4.5%; the fermentation medium comprises the following components at the following concentrations: maltose 15–25 g / L, yeast extract 30–40 g / L, magnesium sulfate 5–8 g / L, and the balance being water; the preferred concentration of the maltose is 18–22 g / L; the preferred concentration of the yeast extract is 32–38 g / L; the preferred concentration of the magnesium sulfate is 6–7 g / L; the preferred culture temperature is 28–32°C, more preferably 29–31°C; the preferred culture time is 42–54 h, more preferably 45–51 h.

[0042] The present invention also provides a wettable powder containing the aforementioned compound microbial agent, wherein the preparation method of the wettable powder includes the following steps:

[0043] 1) The liquid preparation and kaolin are mixed according to the saturated adsorption capacity and dried to obtain the mother powder;

[0044] 2) Mix the masterbatch powder, dispersant, wetting agent and protective agent, crush and sieve to obtain a wettable powder containing the compound microbial agent.

[0045] In this invention, the liquid preparation and kaolin are mixed according to the saturated adsorption capacity and dried to obtain a mother powder. During the mixing process, stirring is performed until the mixture is moist, uniform, free of lumps, and free of running water. The drying temperature is preferably 25–35°C, more preferably 28–32°C. The drying time is preferably 8–16 hours, more preferably 10–14 hours.

[0046] In this invention, a masterbatch powder, dispersant, wetting agent, and protective agent are mixed, pulverized, and sieved to obtain a wettable powder containing the aforementioned compound microbial agent. The preferred mass ratio of the masterbatch powder, dispersant, wetting agent, and protective agent is 85–90:6–7:2–3:0.01–0.02, more preferably 86–89:6.2–6.8:2.2–2.8:0.01–0.02; the dispersant includes sodium lignosulfonate, the wetting agent includes sodium dodecylbenzenesulfonate, and the protective agent includes sodium alginate; the preferred sieve mesh size is 300–350 mesh, more preferably 310–340 mesh.

[0047] The present invention also provides the application of the aforementioned Bacillus subtilis NEAU-L03 in inhibiting Alternaria spp.

[0048] This invention also provides the application of the aforementioned compound microbial agent, liquid formulation, and wettable powder in the prevention and control of plant leaf spot diseases. In this invention, the plant includes honeysuckle berries.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Isolation, antibacterial activity determination and identification of strains NEAU-L03 and NEAU-L05

[0052] 1. Isolation of strains NEAU-L03 and NEAU-L05

[0053] Rhizosphere soil from healthy honeysuckle berry plants at the Xiangyang Base of Northeast Agricultural University was collected from the surface of the plants (1–10 cm). This soil was stored at 4℃ for later use. 0.3 g of each soil sample was added to 30 mL of sterile water and incubated at 30℃ in a shaker for 30 min. Then, 30 μL of the culture was spread onto LB agar (5 g / L yeast extract, 10 g / L tryptone, 5 g / L NaCl, 20 g / L agar, natural pH) and incubated at 30℃ for 48 h. After single colonies grew on the plates, colonies of different morphologies were selected using a sterile toothpick and streaked onto LB agar (5 g / L yeast extract, 10 g / L tryptone, 5 g / L NaCl, 20 g / L agar, natural pH) for purification. The purified colonies were then incubated at 30℃ until uniform single colonies grew and stored at 4℃ for later use. Finally, strains NEAU-L03 and NEAU-L05 were isolated from the rhizosphere soil of healthy honeysuckle berries.

[0054] Morphological identification: Streak plates of strains NEAU-L03 and NEAU-L05 are shown below. Figure 1 As shown. Among them, strain NEAU-L03, after being streaked on LB agar plates for 24 hours, produced yellowish-white, round or irregularly round, opaque colonies with slightly wavy edges, a dry and rough surface, obvious wrinkles, a convex center, and flattened edges. After 24 hours of incubation, the colony diameter was approximately 2-4 mm. Figure 1 (A) In the text, strain NEAU-L05 was streaked on LB agar plates for 24 hours. The colonies were milky white, round, opaque, with neat edges, smooth and dry surfaces, and slight wrinkles. After 24 hours of incubation, the colony diameter was approximately 2-5 mm. Figure 1 (B in the middle).

[0055] 2. Determination of antibacterial activity of strains NEAU-L03 and NEAU-L05

[0056] Target strain tested: Alternaria tenuissima LD-12 pathogenic bacteria see (YanHaohao, MiYaozu, Man Zijian, Zang Hailian, Guo Liangchuan, Huo Junwei, Li Yuxuan, Chen Zexu, Zhang Bing, Sang Mingyu, Li Chunyan*, Cheng Yi*. First report of leafspot disease caused by Alternaria tenuissima on Lonicera caerulea L. in Heilongjiang Province, China. Plant Disease, 2023.).

[0057] Using Alternaria tenuissima LD-12 as the target, the antibacterial activity of strains NEAU-L03 and NEAU-L05 was determined by the plate confrontation method.

[0058] Using a sterile punch with a diameter of 5 mm, the pathogen colonies were punched into holes, and the mycelial cakes were transferred to the center of a PDA plate. Using a cross-hatching method, single colonies of NEAU-L03, NEAU-L05, and a combination of NEAU-L03 and NEAU-L05 were inoculated at four opposite points 3 cm from the center of the plate. No inoculation was performed in the control group. The experiment was conducted in triplicate, incubated at 28°C, and the pathogens on the uninoculated plates were observed growing to the edge of the plate. The diameters of the control and treated colonies were measured and expressed as the inhibition rate. Inhibition rate (%) = [(average diameter of control colonies – diameter of treated colonies) / (average diameter of control colonies – 5 mm)] × 100.

[0059] Experimental results: as shown in Tables 1-3 and Figure 2 As shown.

[0060] Table 1. LD-12 of NEAU-L03 against Alternaria tenuissima

[0061]

[0062] Table 2. LD-12 of NEAU-L05 against Alternaria tenuissima

[0063]

[0064] Table 3. LD-12 of NEAU-L03 and NEAU-L05 against Alternaria tenuissima

[0065]

[0066] The results showed that bacteria NEAU-L03 and NEAU-L05 had strong inhibitory effects on *Alternaria tenuissima* LD-12, with inhibition rates of 51.07% and 53.69%, respectively. The combined use of NEAU-L03 and NEAU-L05 resulted in an inhibition rate of 59.44% against *Alternaria tenuissima* LD-12. Bacteria NEAU-L03 and NEAU-L05 were able to inhibit the mycelial growth of *Alternaria tenuissima*.

[0067] 3. Strain identification

[0068] (1) 16S rDNA sequencing

[0069] Genomic DNA was extracted from strains NEAU-L03 and NEAU-L05. Using these DNA samples as templates, PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplified products were recovered and sequenced.

[0070]

[0071]

[0072] (2) Submit the sequence obtained from sequencing to GeneBank for homology comparison.

[0073] Experimental results: such as Figure 3 As shown in Table 4, strain NEAU-L03 showed 100.00% homology with Bacillus subtilis, and strain NEAU-L05 showed 100.00% homology with Bacillus velezensis. Based on Gram staining and physiological and biochemical characteristics (Table 4), strain NEAU-L03 was identified as Bacillus subtilis and named Bacillus subtilis NEAU-L03. Strain NEAU-L05 was identified as Bacillus velezensis and named Bacillus velezensis NEAU-L05.

[0074] Table 4 Gram staining and physiological and biochemical characteristics of strains NEAU-L03 and NEAU-L05

[0075]

[0076] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0077] Example 2

[0078] Preparation of compound bacterial solution and wettable powder

[0079] (1) Take purified Bacillus subtilis NEAU-L03 and Bacillus belye NEAU-L05 and inoculate them into LB culture medium at an inoculation rate of 1% of the volume of LB culture medium. Incubate at 28℃ and 180rpm for 24h to obtain Bacillus subtilis NEAU-L03 bacterial suspension and Bacillus belye NEAU-L05 bacterial suspension, respectively.

[0080] (2) Mix Bacillus subtilis NEAU-L03 bacterial solution and Bacillus belye NEAU-L05 bacterial solution at a volume ratio of 4:6. Inoculate the bacterial solution into the fermentation medium at an inoculation rate of 4% of the fermentation medium volume. Incubate at 30℃ for 2 days to obtain a compound bacterial solution. The fermentation medium consists of 20.0g maltose, 36.7g yeast extract, 6.4g magnesium sulfate, 1L water, and pH 6.

[0081] (3) Add kaolin to the composite bacterial solution in (2) according to the saturated adsorption capacity, stirring with a glass rod while adding, and mix thoroughly. Stop when the mixture is moist, uniform, and free of lumps, and there is no water flowing in the beaker. Dry in a forced-air drying oven at 30°C for 12 hours to make mother powder.

[0082] (4) Mix the masterbatch powder, sodium lignosulfonate, sodium dodecylbenzenesulfonate, and sodium alginate in a mass ratio of 90g:7g:3g:0.01g, pulverize the mixture using a pulverizer, and pass it through a 325-mesh sieve to obtain a wettable powder (such as...). Figure 4 (As shown).

[0083] Example 3

[0084] Pot experiment on the control effect of compound bacterial solution and wettable powder on leaf spot disease of honeysuckle berries

[0085] A control efficacy experiment was conducted in a greenhouse at the Horticulture Station of Northeast Agricultural University. The greenhouse temperature was maintained at 25±5℃, and the plants grown were *Honeysuckle berries* (Blue Elf variety). This experiment used foliar spraying at a concentration of 10... 7 A CFU / mL compound bacterial solution / wettable powder was sprayed onto the leaves of honeysuckle berries. 24 hours after inoculation with the biocontrol bacteria, a suspension of pathogen spores was evenly sprayed onto all leaves of each plant using a handheld sprayer, ensuring the liquid droplets did not fall off. An average of 10 mL was inoculated per plant. After inoculation, the severity of leaf tissue disease was continuously observed based on the actual situation, and the plants were graded. The following treatments were performed:

[0086] Control group: sterile water;

[0087] Treatment group 1: Pathogenic fungus (Alternaria tenuissima LD12);

[0088] Treatment group 2: Fermentation broth (uninoculated with biocontrol bacteria: Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05) + pathogenic fungi (Alternaria tenuissima LD12);

[0089] Treatment group 3: The compound bacterial solution prepared in Example 2 + pathogenic fungus (Alternaria tenuissima LD12);

[0090] Treatment group 4: Wettable powder (uninoculated biocontrol bacteria: Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05) + pathogenic fungus (Alternaria tenuissima LD12);

[0091] Treatment group 5: Wettable powder prepared in Example 2 + pathogenic fungus (Alternaria tenuissima LD12);

[0092] Treatment group 6: Commercially available microbial agent (Bacillus polymyxa wettable powder, purchased from Beihai Yeshengwang Biotechnology Co., Ltd.) + pathogenic fungus (Alternaria tenuissima LD12).

[0093] Each treatment had three replicates, with 10 leaves investigated in each replicate. After the control group had developed sufficient disease, the disease index of each treatment was investigated, and the control effect was calculated.

[0094] Disease severity is rated on a scale of 0 to 5 (0 = no symptoms; 1 = lesion area <5% of leaf area; 2 = lesion area 5 to 10% of leaf area; 3 = lesion area 10 to 25% of leaf area; 4 = lesion area 25 to 50% of leaf area; 5 = lesion area ≥50% of leaf area).

[0095] Disease index percentage = Σ(number of diseased leaves of honeysuckle under each grade × relative grade) / (total number of leaves surveyed × highest disease grade) × 100%.

[0096] Prevention and control effect = (disease index of treatment group 1 - disease index of treatment) / disease index of treatment group 1 × 100%.

[0097] Experimental results: as shown in Tables 5 and 6. Figure 5 and Figure 6 As shown.

[0098] Table 5. Control efficacy of each treatment in pot experiments after spraying in control group and treatment groups 1-2.

[0099]

[0100] Table 6 shows the control efficacy of each treatment group (3-6) in pot experiments after spraying.

[0101]

[0102] From Tables 5-6, Figure 5 and Figure 6 It can be seen that the compound bacterial solution has a good control effect on honeysuckle leaf spot disease, reaching 42.70%; the wettable powder has the best control effect on honeysuckle leaf spot disease, reaching 58.34%.

[0103] Example 4

[0104] Field trial of the efficacy of wettable powder in controlling leaf spot disease of honeysuckle berries

[0105] To investigate the field efficacy of wettable powder in controlling leaf spot disease in honeysuckle berries, a field trial was designed. Honeysuckle berries (Blue Elf variety) were used, with a plant spacing of 0.5 m and a row spacing of 0.5 m. The experiment was conducted from June to August 2024 at the Honeysuckle berry demonstration garden in Harbin, Heilongjiang Province, specifically at: 126.55°E; 45.45°N; altitude 179.8 m. Two treatments were included (control group: sterile water; treatment group 1: application of wettable powder; treatment group 2: application of commercially available microbial agent, purchased from Beihai Yeshengwang Biotechnology Co., Ltd.). Each treatment consisted of three plots, each containing 15 honeysuckle berry plants, with each plot covering an area of ​​33.75 m². 2 Use a pesticide sprayer to evenly spray a 100-fold diluted wettable powder / commercially available microbial agent onto the plant leaves, ensuring the leaves are evenly moistened with the pesticide solution and a few drops drip off. Apply 0.67L per plant per application. Use an equal amount of water as a control.

[0106] Application timing: Based on previous spraying practices regarding the annual onset time of leaf spot disease in this area, the disease gradually appears around the harvest season (around June 16th). Therefore, the application dates were set for June 5th, June 15th, and June 25th, 2024, for a total of three applications, with each application spaced ten days apart. Five sampling points were fixed in each plot using a diagonal method, with one randomly selected honeysuckle berry plant at each point. Each treatment consisted of three plots, resulting in a total of 15 honeysuckle berry plants surveyed per treatment. Fifty leaves were randomly selected from each plant. The disease index and control effect were calculated seven days after the last application.

[0107] Disease severity is rated on a scale of 0 to 5 (0 = no symptoms; 1 = lesion area <5% of leaf area; 2 = lesion area 5 to 10% of leaf area; 3 = lesion area 10 to 25% of leaf area; 4 = lesion area 25 to 50% of leaf area; 5 = lesion area ≥50% of leaf area).

[0108] Disease index percentage = Σ(number of diseased leaves of honeysuckle under each grade × relative grade) / (total number of leaves surveyed × highest disease grade) × 100%.

[0109] Prevention and control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0110] Experimental results are shown in Tables 7-9.

[0111] Table 7. Control efficacy of each treatment in field trials after spraying of the control group.

[0112]

[0113] Table 8. Control efficacy of each treatment in field trials after spraying of treatment group 1.

[0114]

[0115] Table 9. Control efficacy of each treatment in field trials after spraying in treatment group 2.

[0116]

[0117] As shown in Tables 7-9, in the field trial, the control effect of the wettable powder group on leaf spot disease of honeysuckle was significantly higher than that of the group using commercially available microbial inoculants. The control effect of the wettable powder group reached 46.46%.

[0118] As demonstrated by the above embodiments, this invention utilizes Bacillus subtilis NEAU-L03 and Bacillus velezensis NEAU-L05 to prepare compound bacterial solutions and wettable powder compound bacterial agents. For honeysuckle leaf spot disease caused by Alternaria alternata, the compound bacterial solution showed a pot control efficacy exceeding 40%, the wettable powder compound bacterial agent showed a pot control efficacy exceeding 50%, and the wettable powder compound bacterial agent showed a field control efficacy exceeding 40%. This provides an effective biological control solution for honeysuckle leaf spot disease, possessing significant economic and environmental benefits.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex microbial agent, characterized in that, Including Bacillus subtilis ( Bacillus subtilis NEAU-L03 and Bacillus belesiensis ( Bacillus velezensis NEAU-L05; The Bacillus subtilis NEAU-L03 has the accession number CGMCC No.33129; The Bacillus belyssus NEAU-L05 has the accession number CGMCC No.33130.

2. A liquid preparation containing the complex bacterial agent of claim 1, characterized by, The preparation method of the liquid formulation includes the following steps: 1) Bacillus subtilis NEAU-L03 and Bacillus belye NEAU-L05 were inoculated into LB culture medium and cultured to obtain Bacillus subtilis NEAU-L03 and Bacillus belye NEAU-L05 bacterial suspensions, respectively. 2) Mix Bacillus subtilis NEAU-L03 bacterial solution and Bacillus belye NEAU-L05 bacterial solution to obtain a mixed bacterial solution. Inoculate the mixed bacterial solution into a fermentation medium and culture it to obtain the liquid preparation.

3. The liquid formulation according to claim 2, characterized in that, Step 1) The inoculation amount of Bacillus subtilis NEAU-L03 and Bacillus belyi NEAU-L05 is 0.5-1.5% of the LB culture medium volume; the culture temperature is 25-30℃, the culture rotation speed is 150-200 rpm, and the culture time is 18-30 h. Step 2) The volume ratio of Bacillus subtilis NEAU-L03 bacterial suspension and Bacillus belyi NEAU-L05 bacterial suspension is 2-6:4-8; the inoculation amount of the mixed bacterial suspension is 3-5% of the fermentation medium volume; the fermentation medium comprises the following components at the following concentrations: maltose 15-25 g / L, yeast extract 30-40 g / L, magnesium sulfate 5-8 g / L, and the balance being water; the culture temperature is 28-32℃, and the culture time is 42-54 h.

4. A wettable powder comprising the complex bacterial agent of claim 1, characterized by, The preparation method of the wettable powder includes the following steps: 1) The liquid formulation described in claim 2 or 3 is mixed with kaolin according to the saturated adsorption capacity, and then dried to obtain the mother powder; 2) Mix the masterbatch powder, dispersant, wetting agent and protective agent, crush and sieve to obtain the wettable powder.

5. The wettable powder according to claim 4, characterized in that, In step 2), the mass ratio of the masterbatch, dispersant, wetting agent, and protective agent is 85–90: 6–7: 2–3: 0.01–0.

02. The dispersant includes sodium lignosulfonate, the wetting agent includes sodium dodecylbenzenesulfonate, and the protective agent includes sodium alginate.

6. The application of the compound microbial agent according to claim 1, the liquid preparation according to claim 2 or 3, and the wettable powder according to claim 4 or 5 in the prevention and control of leaf spot disease of honeysuckle caused by Alternaria spp.

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