Microbial composite agent, preparation method thereof and application in preventing and controlling potato scab

Abstract

The present invention relates to the technical field of microbial agents, and discloses a microbial composite agent, a preparation method thereof, and its use in preventing and treating potato scab. The microbial composite agent comprises: P. milanoxanus 6096, Streptomyces 6219, and Bacillus atrophaeus 4618; P. milanoxanus 6096, Streptomyces 6219, and Bacillus atrophaeus 4618 are all deposited in the General Microbiology Center of the China National Center for Microbiological Culture Collection, with deposit numbers CGMCC No. 34691, CGMCC No. 34692, and CGMCC No. 34952, respectively. The microbial composite agent of the present invention has a good and stable antibacterial effect and can significantly reduce the incidence and disease index of potato scab after use.

Description

Technical Field

[0001] The invention relates to the technical field of microbial agents, in particular to a composite microbial agent and a preparation method thereof, and application thereof in preventing and treating potato scab. Background Art

[0002] Potato common scab (PCS) is a soil-borne and seed-transmitted bacterial disease caused by pathogenic Streptomyces species and is one of the four major diseases in potato production. The pathogen invades potato tubers through lenticels and stomata. Initially, light brown spots 5-8 mm in diameter appear on the tuber surface. As the pathogen infects the tuber, the surrounding tissue necrotizes, becoming corky and roughened. These spots typically appear as sunken, raised, or flat black, rusty, or brown lesions. The disease is particularly susceptible to high temperatures and alkaline conditions. The pathogen can overwinter in the soil and remaining tubers, becoming a source of infection the following year. The spores produced are rapidly transferred and multiplied by seed potatoes, soil, wind, rain, and insects.

[0003] The pathogen causing potato scab can survive in the soil for up to 10 years, multiplying continuously. The accumulation of toxins disrupts the soil microbial community structure and pollutes the environment. Furthermore, the diseased potatoes deteriorate in appearance, become less storable, and significantly reduce their quality, which in turn affects their edible and processing value. Research by Santos-Cervantes et al. found that the scab pathogen can also infect staple root crops such as beets, carrots, sweet potatoes, and parsnips. The disease's host range continues to expand, and control technologies remain in need of breakthroughs. It has become a major global agricultural pest. Therefore, there is an urgent need to explore eco-friendly methods for controlling potato scab. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a microbial composite agent, a preparation method thereof, and an application thereof in preventing and treating potato scab, aiming to solve the problem of unstable effect of the existing potato scab prevention and treatment.

[0005] The technical solutions of the present invention are as follows:

[0006] In a first aspect, a microbial composite agent is provided, wherein the microbial composite agent comprises: Peptobacterium milanoxum ( Dyadobacter milanwu ) 6096, Streptomyces ( Streptomyces sp. )6219 and Bacillus atrophaeus ( Bacillus atrophaeus )4618;

[0007] The P. milan 6096 strain was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with the deposit number CGMCC NO.34691.

[0008] Streptomyces 6219 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with the deposit number CGMCC NO.34692.

[0009] The Bacillus atrophaeus 4618 is deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is June 19, 2025, and the deposit number is CGMCC NO.34952.

[0010] In a preferred technical solution, the ratio of the viable counts of P. milanoxum 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is (1-3): (1-3): (1-3).

[0011] In a preferred technical solution, the ratio of the live bacteria counts of P. milanoxum 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is 2:3:3.

[0012] According to a preferred technical solution, the microbial composite agent further includes agriculturally acceptable auxiliary materials.

[0013] According to a preferred technical solution, the agriculturally acceptable auxiliary material is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

[0014] In a second aspect, a method for preparing the microbial composite agent according to the first aspect is provided, comprising the steps of:

[0015] The microbial composite agent is obtained by co-culturing Pseudomonas milanoticus 6096, Streptomyces 6219 and Bacillus atrophaeus 4618.

[0016] In a preferred technical solution, the co-culture conditions include: culture temperature 18-30°C, culture speed 100-300 rpm, and culture time 24-72 h.

[0017] In a preferred technical solution, the co-cultivation is carried out in NB medium, which is prepared with water and includes: 3 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride.

[0018] In a third aspect, there is provided use of the microbial composite agent as described in the first aspect or the microbial composite agent prepared by the preparation method as described in the second aspect in preventing and treating potato scab.

[0019] Beneficial Effects: The present invention previously isolated Bacillus atrophaeus 4618 from soil, which exhibits excellent antagonistic properties against the pathogenic bacterium P139, a potato scab pathogen. Through screening, P. milanoxanus 6096 and Streptomyces 6219, which promote the antagonistic effects of Bacillus atrophaeus 4618, were obtained. These bacteria were then compounded with Bacillus atrophaeus 4618 to produce a microbial composite agent. This composite agent exhibits excellent and stable antibacterial effects. When used to control potato scab, the incidence rate and disease index were significantly lower than those in the positive control group and the group treated with Bacillus atrophaeus 4618 alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the antagonistic effect of the combined use of Bacillus atrophaeus 4618 and the candidate functional strain against the scab pathogen P139 in Example 1.

[0021] Figure 2 This is a graph showing the experimental results of Example 2 in which Bacillus atrophaeus 4618 and the candidate functional strain were mixed at a viable bacterial count of 1:1 and immediately applied to radish seedlings.

[0022] Figure 3 This is a graph showing the test results of Example 2 in which Bacillus atrophaeus 4618 and the candidate functional strain were co-cultured at a viable count of 1:1 for 48 hours and then applied to radish seedlings.

[0023] Figure 4 This is a graph showing the results of a double-layer plate test of the microbial composite agent prepared in the optimal ratio in Example 3.

[0024] Figure 5 This is a diagram showing the test results of radish seedlings using the microbial composite agent prepared in the optimal ratio in Example 3.

[0025] Figure 6 This is a diagram showing the potted plant test results of the microbial composite agent prepared in the optimal ratio in Example 3. DETAILED DESCRIPTION

[0026] The present invention provides a microbial composite agent, a preparation method thereof, and an application thereof in preventing and treating potato scab. To make the purpose, technical solution, and effects of the present invention clearer and more specific, the present invention is further described below with reference to specific examples.

[0027] Bacillus atrophaeus is a major biocontrol bacterium that can protect plants from pathogens through mechanisms such as lysis, antagonism, competition, and induced resistance. Its application in the prevention and control of potato scab has been reported. However, when Bacillus atrophaeus is applied in the field, its control effect is often unstable due to the complex soil environment. In addition, the activity of Bacillus atrophaeus is also affected by natural factors such as temperature, humidity, and light. For example, under high temperature or drought conditions, the germination and metabolic activity of Bacillus may be inhibited, thereby reducing the disease prevention effect. Further studies have found that the stability of Bacillus in the rhizosphere of plants can be improved by adding prebiotics derived from rhizosphere exudates / sediments, synthetic microbial communities, etc., thereby improving the prevention and control of plant diseases.

[0028] Based on this, an embodiment of the present invention provides a microbial composite agent, which includes:

[0029] Milanese Peneobacterium ( Dyadobacter milanwu ) 6096, Streptomyces ( Streptomyces sp. )6219 and Bacillus atrophaeus ( Bacillus atrophaeus )4618;

[0030] The P. milan 6096 strain was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with the deposit number CGMCC NO.34691.

[0031] Streptomyces 6219 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with the deposit number CGMCC NO.34692.

[0032] The Bacillus atrophaeus 4618 is deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is June 19, 2025, and the deposit number is CGMCC NO.34952.

[0033] In one embodiment, the ratio of the viable cell counts of P. milanoxum 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is (1-3):(1-3):(1-3).

[0034] In one embodiment, the ratio of the viable cell counts of P. milanoxum 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is 2:3:3.

[0035] In one embodiment, the microbial composite agent further includes agriculturally acceptable excipients.

[0036] In one embodiment, the agriculturally acceptable adjuvant is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

[0037] The present invention provides a method for preparing the microbial composite agent as described above, comprising the steps of:

[0038] The microbial composite agent is obtained by co-culturing Pseudomonas milanoticus 6096, Streptomyces 6219 and Bacillus atrophaeus 4618.

[0039] In one embodiment, the co-culture conditions include: culture temperature 18-30° C., culture speed 100-300 rpm, and culture time 24-72 h.

[0040] In one embodiment, the co-cultivation is carried out in NB medium, which is prepared with water and comprises: 3 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride.

[0041] The present invention provides use of the microbial composite agent as described above or the microbial composite agent prepared by the preparation method as described above in preventing and treating potato scab.

[0042] The present invention will be further described below with reference to specific examples.

[0043] In the following examples, the materials and culture medium components involved are as follows:

[0044] (1) Strains and materials

[0045] The pathogenic bacteria P139 of potato scab was isolated from potato scab lesions in the experimental field of the Potato Center Farm of Inner Mongolia University in Hohhot, Inner Mongolia Autonomous Region in May 2020 and was classified as Streptomyces scabiei .

[0046] P. milan 6096 was isolated from a potato field in Nanning, Guangxi Zhuang Autonomous Region in March 2023 and deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 28, 2025, with the deposit number CGMCC NO.34691 and the classification name Dyadobacter milanwu .

[0047] Streptomyces 6219 was isolated from a potato field in Nanning, Guangxi Zhuang Autonomous Region in March 2023 and deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 28, 2025, with the deposit number CGMCC NO.34692 and the classification name Streptomyces sp. .

[0048] Bacillus atrophaeus 4618 was isolated from the rhizosphere soil of corn at the Inner Mongolia University Farm in Hohhot, Inner Mongolia Autonomous Region in September 2022 and deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC). The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 19, 2025. The deposit number is CGMCC NO. 34952 and the classification name is Bacillus atrophaeus .

[0049] The radish seedlings used in the experiment were YR Xinbaiyuchun, a variety produced by Beijing Shinong Seedlings Co., Ltd.

[0050] The seed potato used in the experiment was Lucinda (V7), which was purchased from Dingxi Potato Research Institute in Gansu Province.

[0051] (2) Test culture medium and reagents

[0052] Nutrient agar (NA) medium was prepared with water and included 3 g / L beef extract, 10 g / L tryptone, 0.5 g / L sodium chloride (NaCl), and 15 g / L agar.

[0053] Nutrient broth (NB) was prepared with water and included 3 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride (NaCl).

[0054] R2A agar medium was prepared with water and included: R2A agar 18.1 g / L.

[0055] Streptomyces medium No. 4 (ISP MEDIUM NO.4, ISP4) is prepared with water and includes: dipotassium hydrogen phosphate (K2HPO4) 1 g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 1 g / L, sodium chloride (NaCl) 1 g / L, ammonium sulfate ((NH4)2SO4) 1 g / L, calcium carbonate (CaCO3) 2 g / L, ferric sulfate heptahydrate (FeSO4·7H2O) 0.01 g / L, manganese chloride heptahydrate (MnCl2·7H2O) 0.01 g / L, soluble starch (Soluble Starch) 10 g / L, and agar 20 g / L.

[0056] ISP4 liquid medium is based on Streptomyces medium No. 4, without agar.

[0057] Water agar is prepared with water and includes: agar 7g / L.

[0058] Example 1 Isolation, identification and screening of soil microorganisms

[0059] (1) Isolation of soil microorganisms

[0060] A. Sample: Rhizosphere soil of potato plants in Nanning, Guangxi Zhuang Autonomous Region, China.

[0061] B. Sample preparation: Weigh 0.1 g of rhizosphere soil and mix with 1 mL of sterile water to obtain the sample solution.

[0062] C. Dilution: Dilute 30 μL of the sample stock solution with 270 μL of sterile water at a volume ratio of 1:9 into eight dilution gradients. Spot 10 μL of each dilution gradient onto NA, R2A, and ISP4 culture media. Incubate in a 28°C incubator for 2-3 days to obtain the optimal dilution gradient.

[0063] D. Spreading: Take 100 μL of the optimal gradient dilution solution and spread it on NA, R2A and ISP4 culture media respectively, and culture in a 28°C incubator for 4-5 days.

[0064] E. Streak purification: Select all bacteria with different morphologies, streak them on NA, R2A, and ISP4 media, and culture them in a 28°C incubator for 2-5 days until single colonies are purified.

[0065] F. Storage: A total of 97 bacterial strains were isolated. A single colony of each strain was inoculated into NB medium and cultured at 200 rpm and 28°C for 2-3 days. The bacterial suspension was mixed with 60% glycerol at a 1:1 ratio by volume and stored in a -80°C freezer for long-term storage.

[0066] (2) Identification of soil microorganisms

[0067] In this example, colony PCR amplification was performed using universal bacterial primers 8F and 1492R. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. Sequences were assembled using Galaxy|Europe (https: / / usegalaxy.eu / ) and compared with sequences from NCBI-BLAST (https: / / www.ncbi.nlm.nih.gov / ). Sequencing results revealed that the 97 bacterial strains belonged to 37 genera, and one strain from each genus was selected for subsequent experiments.

[0068] (3) Screening of bacteria that promote antagonism against Bacillus atrophaeus 4618

[0069] A. Effects of bacteria on Bacillus atrophaeus 4618

[0070] Inoculate Bacillus atrophaeus 4618 and the test bacteria into NB medium, incubate at 200 rpm and 28°C for 1-2 days. Place 200 μL of Bacillus atrophaeus 4618 culture in a 15 mL centrifuge tube, add 40-45°C water agar, and plate on NA medium. Spot 10 μL of the test bacteria culture onto the NA medium with Bacillus atrophaeus 4618 and incubate at 28°C for 1-2 days. Observe for the formation of inhibition zones. Select strains that do not produce inhibition zones for the next step.

[0071] B. Effects of Bacillus atrophaeus 4618 on bacteria

[0072] Take 150 μL of each test bacteria that did not produce an inhibition zone in step A and place it in a 15 mL centrifuge tube. Add 40-45°C water agar and plate on NA medium. Then, spot 10 μL of Bacillus atrophaeus 4618 culture on the NA medium with the test bacteria. Incubate at 28°C for 1-2 days and observe for the formation of an inhibition zone. Select strains that did not produce an inhibition zone for the next step.

[0073] Effects of C. bacteria and Bacillus atrophaeus 4618 on the scab pathogen P139

[0074] Inoculate scab pathogen P139 into ISP4 liquid medium and incubate at 200 rpm and 28°C for 3-5 days. Inoculate the test bacteria and Bacillus atrophaeus 4618, which did not produce an inhibition zone in step B, into NB medium and incubate at 200 rpm and 28°C for 1-2 days. Evenly spread 100 μL of the scab pathogen P139 liquid onto the ISP4 medium. Mix Bacillus atrophaeus 4618 and the test bacteria liquid at a 1:1 volume ratio. Spot 10 μL of the mixture onto the ISP4 medium coated with scab pathogen P139. Use ISP4 medium coated with only Bacillus atrophaeus 4618 as a control. Incubate at 28°C for 3-5 days. Observe for the formation of inhibition zones, and calculate the area of ​​the inhibition zones using Image J.

[0075] D. Screening results

[0076] Based on the experimental results of steps A and B of this example, a total of 14 bacterial strains were screened out that had no mutual antagonistic effect with Bacillus atrophaeus 4618. Combined with the experimental results of step C of this example, the two bacterial strains, P. milanoxanus 6096 and Streptomyces 6219, had significant antibacterial effects after being mixed with Bacillus atrophaeus 4618 ( Figure 1), which can promote the antagonistic effect of Bacillus atrophaeus 4618 against the scab pathogen P139, so they were selected as candidate functional strains for constructing microbial composite agents.

[0077] Example 2 Construction of a microbial composite agent

[0078] (1) Antagonism test between candidate functional strains

[0079] The fully combined droplet method was used to observe antagonism between candidate functional strains. The bacteria that promoted antagonism against Bacillus atrophaeus 4618, screened in Example 1, were inoculated into NB medium and cultured at 200 rpm and 28°C for 1-2 days. 200 μL of the bacterial solution from one of the strains was placed in a 15 mL centrifuge tube, added to 40-45°C water agar, and plated on NA medium as an indicator bacterium. The remaining strains served as test bacteria, with three plates of each indicator bacterium plated. The indicator culture dish was divided into four sector-shaped areas, and 10 μL of the test bacterial solution was applied to four points in the sector as replicates. 10 μL of the indicator bacterial solution was applied to one point in the sector as a control, with each point approximately 1 cm apart. The culture was then cultured at 28°C for 2-3 days, and antagonism was observed. The test results are shown in Table 1, indicating no mutual antagonism between the two candidate functional strains.

[0080] Table 1 Antagonism between candidate functional strains

[0081]

[0082] Note: “+” indicates that there is antagonism between strains and the antagonism is strong; “-” indicates that there is no antagonism or antagonism between strains.

[0083] (2) Screening of compound forms of microbial compound agents

[0084] The radish stem height measurement method was used to screen the compound forms of functional strains.

[0085] A. Radish seedling germination: Soak radish seeds in 75% (v / v) ethanol for 5 min, rinse three times with sterile water, and place the seeds in a Petri dish covered with moistened filter paper. Germination was allowed to proceed overnight in a 28°C incubator.

[0086] B. Planting radish seedlings: Place approximately 15 g of soil in a tissue culture tube and sterilize with high-pressure steam at 121°C for 20 minutes. After natural cooling, add 5 mL of sterile water. Once the soil is completely soaked, use tweezers to transplant the germinated seeds into the tissue culture tube, seeding 2-3 seeds per tube. Incubate under a light:dark ratio of 16:8 hours for 2 days.

[0087] C. Screening of complex forms: The scab pathogen P139, Bacillus atrophaeus 4618, P. milanoxanus 6096, and Streptomyces 6219 were inoculated into ISP4 liquid and NB medium, respectively, and cultured at 200 rpm and 28°C. Treatment groups included:

[0088] Blank control group: add an equal volume of sterile water.

[0089] Scab pathogen P139 group (abbreviated as P139): only scab pathogen P139 was added.

[0090] Scab pathogen P139 + Bacillus atrophaeus 4618 group (abbreviated as P139+4618): Scab pathogen P139 and Bacillus atrophaeus 4618 were added.

[0091] Scab pathogen P139+(Bacillus atrophaeus 4618+candidate functional strain) group (abbreviated as P139+4618+6096 or P139+4618+6219): scab pathogen P139, Bacillus atrophaeus 4618 and a candidate functional strain (Penaebaeus milanese 6096 or Streptomyces 6219) were added.

[0092] Among them, there are two forms of inoculation for the group that added Bacillus atrophaeus 4618 and candidate functional strains at the same time: one is to mix Bacillus atrophaeus 4618 and candidate functional strains at a viable bacterial count of 1:1 and apply immediately; the other is to mix Bacillus atrophaeus 4618 and candidate functional strains at a viable bacterial count of 1:1 and co-culture for 48 hours before application. 600μL of bacterial solution was added to the tissue culture tube of the seedlings in each treatment group, with light:darkness = 16:8h. After culturing for 4-5 days, the height of the radish seedlings in each treatment group was measured. The experimental results are as follows. Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the antagonistic effect of Bacillus atrophaeus 4618 on the scab pathogen P139 was better when it was co-cultured with the candidate functional strain for 48 hours.

[0093] (3) Screening of compound ratio of microbial compound agents

[0094] The three candidate functional strains were compounded into a microbial composite agent. In order to screen and obtain the optimal ratio of each strain in the microbial composite agent, L9(3 3A multi-factor, multi-level orthogonal experiment was conducted, using the inoculum volume ratio of each strain as the experimental factor, with a volume ratio of 1:2:3. The inhibition zone area of ​​the microbial composite against P139, the causative agent of scab disease, was used as the evaluation indicator. Using the double-layer plate standoff method, 150 μL of the P139 bacterial suspension was evenly spread onto ISP4 medium. Then, 20 μL of the microbial composite was applied to the ISP4 medium coated with P139. The cells were incubated at 28°C for 3-5 days. The formation of inhibition zones was observed, and the inhibition zone area was calculated using Image J. The optimal ratio of the strains in the microbial composite was determined using range analysis. The results of the orthogonal experiment and range analysis are shown in Table 2.

[0095] Table 2 Results of orthogonal test of microbial composite agents

[0096]

[0097] Note: Experimental factor A refers to P. milanoxan 6096, experimental factor B refers to Streptomyces 6219, and experimental factor C refers to Bacillus atrophaeus 4618. K is the sum of the experimental data for each factor at all levels; k is the mean K value, i.e., the mean of the experimental data for each factor at all levels; R is the range, i.e., the difference between the maximum and minimum k values ​​for each factor at all levels. The numbers 1-3 in the experimental factors refer to volume ratios.

[0098] As shown in Table 2, when the viable cell counts of P. milanoxicola 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 were 2:3:3, the best antibacterial effect was achieved against the pathogenic bacteria P139 of scab disease.

[0099] Example 3 Antibacterial Verification Test of Microbial Composite Agent

[0100] (1) Double-layer plate test

[0101] The antibacterial ability of the microbial composite agent constructed according to the optimal ratio was verified by the same method as step (1) in Example 1. The experimental results are as follows: Figure 4 As shown. Figure 4 It can be seen that the antibacterial effect of adding the microbial composite agent is significantly higher than the antibacterial effect of adding only Bacillus atrophaeus 4618.

[0102] (2) Radish seedling experiment

[0103] The radish seedling test was conducted on the microbial composite agent constructed according to the optimal ratio, using the same method as step (2) in Example 2. The treatment group included:

[0104] Scab pathogen P139 group (abbreviated as P139): only scab pathogen P139 was added.

[0105] Scab pathogen P139 + Bacillus atrophaeus 4618 group (abbreviated as P139+4618): Scab pathogen P139 and Bacillus atrophaeus 4618 were added.

[0106] Scab pathogen P139 + microbial compound bacterial agent group (abbreviated as P139 + microbial compound bacterial agent): scab pathogen P139 and microbial compound bacterial agent were added.

[0107] Blank control group: add an equal volume of sterile water.

[0108] The experimental results are as follows Figure 5 As shown. Figure 5 It can be seen that the height of radish seedlings in the P139+microbial composite agent group was significantly higher than that in the P139 group and the P139+4618 group, indicating that the addition of the microbial composite agent had a better antibacterial effect than adding only Bacillus atrophaeus 4618.

[0109] (3) Potted plant experiment

[0110] Cultivation soil was collected from the farm on the campus of Inner Mongolia University. Seed potatoes were cut into pieces and germinated in the dark at 18-25°C. They were then cultivated in 25 cm diameter pots and grown in an artificial climate chamber. The artificial climate chamber conditions were: temperature 22-25°C, humidity 30-35%, and a light intensity of 12 h light and 12 h dark. 5-6 g of granular fertilizer (N:P2O5:K2O = 12:19:16) was applied to each pot to maintain soil moisture content at 60-70%. The treatment groups were the same as step (2) of this example. Each treatment group was set up with three replicates, each replicate had five pots of plants, and the entire experiment was repeated twice.

[0111] Specific experimental operation: The pathogenic bacteria P139 of scab disease were inoculated into ISP4 liquid culture medium, cultured at 200 rpm and 28 °C, 200 μL was spread on ISP4 solid culture medium, cultured in a 28 °C incubator for about 15 days, and then transferred to a wall breaking machine, added distilled water to break it, and adjusted the concentration to 10 8 cfu / mL, add 200mL of scab pathogen P139 to the soil and mix well. Inoculate each strain in the microbial composite agent into NB medium, culture at 200rpm and 28℃ for 48h, and continue to culture for 48h after compounding according to the optimal compound ratio. Adjust the concentration to 10 8 cfu / mL. 24 hours after adding the scab pathogen P139, 200 mL of the microbial compound was added to the soil and mixed thoroughly. Finally, germinated potato tubers were sown in pots in each treatment group.

[0112] The tubers were harvested 120 days after planting and the incidence and disease severity of the tubers were evaluated. The severity of potato scab can be divided into six levels according to the scab coverage: level 0 = no symptoms, level 1 <1%, level 2 = 1%-10%, level 3 = 11%-20%, level 4 = 21%-50%, and level 5 >51%.

[0113] The formula for calculating the incidence of potato scab is:

[0114] .

[0115] The calculation formula for potato scab disease index is:

[0116] .

[0117] The experimental results are as follows Figure 6 As shown, A is the incidence result and B is the disease index result. Figure 6 The P139 group had an 87.37% disease incidence rate; the P139+4618 group had a significantly lower disease incidence rate of 62.45% than the P139 group; and the P139+microbial complex group had a significantly lower disease incidence rate of 34.86% than both the P139 and P139+4618 groups. This trend mirrored the plate validation and radish seedling validation results. The P139+microbial complex group had a 52.51% lower disease incidence rate than the P139 group and a 27.59% lower disease incidence rate than the P139+4618 group, demonstrating its superior efficacy in suppressing potato scab. The disease indexes of the P139+4618 and P139+microbial complex groups were significantly lower than those of the P139 group, with the P139+microbial complex group having a lower disease index. In summary, the microbial complex demonstrated excellent efficacy in controlling potato scab and enhanced the efficacy of Bacillus atrophaeus 4618 against the disease.

[0118] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A microbial composite agent, characterized in that: The microbial composite agent includes: Pseudomonas milanensis ( Dyadobacter milanwu ) 6096, Streptomyces ( Streptomyces sp.) 6219 and Bacillus atrophaeus ( Bacillus atrophaeus )4618; The P. milan 6096 strain was deposited in the General Microbiology Center of the China Culture Collection Administration Committee, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with the deposit number CGMCC NO.34691. The Streptomyces 6219 was deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with the deposit number CGMCC NO.34692; The Bacillus atrophaeus 4618 was deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on June 19, 2025, with the deposit number CGMCC NO.34952; The ratio of the viable bacterial counts of Pseudomonas milanese 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is (1-3): (1-3): (1-3).

2. The microbial composite agent according to claim 1, characterized in that The ratio of the live bacteria counts of P. milanoxum 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is 2:3:

3.

3. The microbial composite agent according to claim 1, characterized in that The microbial composite agent also includes agriculturally acceptable auxiliary materials.

4. The microbial composite agent according to claim 3, characterized in that The agriculturally acceptable auxiliary material is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

5. The method for preparing the microbial composite agent according to claim 1, wherein: Including steps: The microbial composite agent is obtained by co-culturing Pseudomonas milanoticus 6096, Streptomyces 6219 and Bacillus atrophaeus 4618.

6. The preparation method according to claim 5, characterized in that The co-culture conditions include: culture temperature of 18-30° C., culture speed of 100-300 rpm, and culture time of 24-72 h.

7. The preparation method according to claim 5, characterized in that The co-cultivation is carried out in NB culture medium, which is prepared with water and includes: 3 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride.

8. Use of the microbial composite agent according to any one of claims 1 to 4 or the microbial composite agent prepared by the preparation method according to any one of claims 5 to 7 in preventing and treating potato scab.

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