Microbial complex microbial inoculant, preparation method thereof and application of microbial complex microbial inoculant in prevention and treatment of potato scab

Abstract

The invention relates to the technical field of microbial agents, and discloses a composite microbial agent, a preparation method thereof and application of the composite microbial agent in prevention and treatment of potato scab. Wherein the microbial complex microbial inoculant is prepared from bacillus aglaia pairwise 6096, streptomyces sp. 6219 and bacillus atrophaeus 4618; the aglaia odorata pairwise bacillus 6096, the streptomyces 6219 and the bacillus atrophaeus 4618 are all preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation numbers of the aglaia odorata pairwise bacillus 6096, the streptomyces 6219 and the bacillus atrophaeus 4618 are respectively CGMCC NO.34691, CGMCC NO.34692 and CGMCC The composite microbial inoculant is good and stable in antibacterial effect, and the morbidity and disease index of potato scab can be remarkably reduced after the composite microbial inoculant is used.

Description

Technical Field

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

[0002] Potato Common Scab (PCS) is a soil-borne and seed-borne bacterial disease caused by pathogenic Streptomyces, and is one of the four major diseases in potato production. Pathogenic Streptomyces invades potato tubers through lenticels, stomata and other ways. At the initial stage of infection, light brown spots with a diameter of 5-8 mm will appear on the surface of the tubers; as the pathogen infection intensifies, the tissues around the infection points will necrosis, the texture will become suberized, and the surface of the tubers will become rough, generally showing sunken, raised or flat lesions in black, rust or brown. This disease is extremely likely to occur in high-temperature alkaline environments. The pathogen can overwinter in the soil and remaining tubers, becoming the source of infection in the following year. The produced spores will quickly transfer and reproduce along with the seed potatoes, soil, wind, rain and insects.

[0003] The pathogen of potato common scab can survive in the soil for up to 10 years with continuous reproduction. The accumulation of toxins will destroy the soil microbial community structure and pollute the environment. At the same time, the appearance of diseased potatoes deteriorates, they are not resistant to storage, and the quality is significantly reduced, thus affecting the edible value and processing value of potatoes. Santos-Cervantes et al. found that the pathogen of common scab can also infect taproot crops such as sugar beet, carrot, sweet potato and parsnip. The host range of this disease continues to expand, and the control technology remains to be broken through. It has become one of the major diseases in global agricultural planting. Therefore, there is an urgent need to explore environmentally friendly means for preventing and treating potato common scab. Summary of the Invention

[0004] In view of the above deficiencies of the prior art, the purpose of the present invention is to provide a microbial compound agent, a preparation method thereof, and an application thereof in preventing and treating potato common scab, aiming to solve the problem of unstable control effect of existing potato common scab.

[0005] The technical solution of the present invention is as follows: In the first aspect, a microbial compound agent is provided, and the microbial compound agent includes: Paenibacillus thiaminolyticus Dyadobacter milanwu 6096, Streptomyces Streptomyces sp. 6219, and Bacillus atrophaeus Bacillus atrophaeus 4618; The Paenibacillus milanensis 6096 was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was May 28, 2025, and the deposit number was CGMCC NO. 34691; The Streptomyces sp. 6219 was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was May 28, 2025, and the deposit number was CGMCC NO. 34692; The Bacillus atrophaeus 4618 was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was June 19, 2025, and the deposit number was CGMCC NO. 34952.

[0006] In a preferred technical solution, the viable cell number ratio of the Paenibacillus milanensis 6096, Streptomyces sp. 6219, and Bacillus atrophaeus 4618 is (1 - 3):(1 - 3):(1 - 3).

[0007] In a preferred technical solution, the viable cell number ratio of the Paenibacillus milanensis 6096, Streptomyces sp. 6219, and Bacillus atrophaeus 4618 is 2:3:3.

[0008] In a preferred technical solution, the microbial complex bactericide further includes agriculturally acceptable excipients.

[0009] In a preferred technical solution, the agriculturally acceptable excipients are selected from one or more of a dispersant, a stabilizer, a filler, and a solvent.

[0010] In a second aspect, there is provided a preparation method of the microbial complex bactericide as described in the first aspect, including the steps: Co - culture the Paenibacillus milanensis 6096, Streptomyces sp. 6219, and Bacillus atrophaeus 4618 to obtain the microbial complex bactericide.

[0011] In a preferred technical solution, the conditions for the co - culture include: a culture temperature of 18 - 30 °C, a culture rotation speed of 100 - 300 rpm, and a culture time of 24 - 72 h.

[0012] In a preferred technical solution, the co - culture is carried out in an NB medium, and the NB medium is prepared with water and includes: 3 g / L of beef extract, 10 g / L of tryptone, and 5 g / L of sodium chloride.

[0013] Third aspect, there is provided an application of the microbial complex bactericide as described in the first aspect or the microbial complex bactericide prepared by the preparation method as described in the second aspect in preventing and controlling potato common scab.

[0014] Beneficial effects: In the early stage of the present invention, Bacillus atrophaeus 4618 with excellent antagonistic performance against the pathogenic bacterium P139 of potato common scab was isolated from the soil. Through screening, Paenibacillus milians 6096 and Streptomyces 6219, which have a promoting effect on the antagonistic effect of Bacillus atrophaeus 4618, were obtained and compounded with Bacillus atrophaeus 4618 to obtain a microbial complex bactericide. The bacteriostatic effect of this microbial complex bactericide is good and stable. When it is applied to the prevention and control of potato common scab, the incidence rate and disease index are significantly lower than those of the positive control group and the treatment group that only applies Bacillus atrophaeus 4618. Description of the drawings

[0015] Figure 1 It is the antagonistic effect diagram of the combination of Bacillus atrophaeus 4618 and candidate functional strains on the pathogenic bacterium P139 of common scab in Example 1.

[0016] Figure 2 It is the experimental result diagram of radish seedlings when Bacillus atrophaeus 4618 and candidate functional strains are mixed at a viable cell number ratio of 1:1 and immediately applied in Example 2.

[0017] Figure 3 It is the experimental result diagram of radish seedlings when Bacillus atrophaeus 4618 and candidate functional strains are mixed at a viable cell number ratio of 1:1 and co-cultured for 48 h and then applied in Example 2.

[0018] Figure 4 It is the experimental result diagram of a double-layer plate of the microbial complex bactericide compounded in the optimal ratio in Example 3.

[0019] Figure 5 It is the experimental result diagram of radish seedlings of the microbial complex bactericide compounded in the optimal ratio in Example 3.

[0020] Figure 6 It is the experimental result diagram of a pot experiment of the microbial complex bactericide compounded in the optimal ratio in Example 3. Detailed implementation manners

[0021] The present invention provides a microbial complex bactericide, its preparation method and its application in preventing and controlling potato common scab. To make the purpose, technical solution and effects of the present invention clearer and more definite, the present invention will be further described below through specific examples.

[0022] Bacillus atrophaeus is a major biocontrol bacterium that can protect plants from pathogens through mechanisms such as bacteriolysis, antagonism, competition, and induced resistance. Its application in the control of potato common 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 research found that the stability of Bacillus in the rhizosphere of plants can be improved by adding prebiotics derived from rhizosphere secretions / sediments, synthetic microbial communities, etc., thereby enhancing the prevention and control of plant diseases.

[0023] Based on this, the embodiments of the present invention provide a microbial compound bactericide, and the microbial compound bactericide includes: Paenibacillus milanensis ( Dyadobacter milanwu ), 6096, Streptomyces ( Streptomyces sp. ), 6219, and Bacillus atrophaeus ( Bacillus atrophaeus ), 4618; The Paenibacillus milanensis 6096 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is May 28, 2025, and the preservation number is CGMCC NO. 34691; The Streptomyces 6219 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is May 28, 2025, and the preservation number is CGMCC NO. 34692; The Bacillus atrophaeus 4618 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is June 19, 2025, and the preservation number is CGMCC NO. 34952.

[0024] In one embodiment, the viable cell number ratio of the Paenibacillus milanensis 6096, Streptomyces 6219, and Bacillus atrophaeus 4618 is (1 - 3):(1 - 3):(1 - 3).

[0025] In one embodiment, the viable cell number ratio of the Paenibacillus milanensis 6096, Streptomyces 6219, and Bacillus atrophaeus 4618 is 2:3:3.

[0026] In one embodiment, the microbial compound bactericide further includes agriculturally acceptable excipients.

[0027] In one embodiment, the agriculturally acceptable adjuvants are selected from one or more of a dispersant, a stabilizer, a filler, and a solvent.

[0028] The embodiment of the present invention provides the preparation method of the microbial complex bactericide as described above, including the steps: Coculturing Paenibacillus myricae 6096, Streptomyces sp. 6219, and Bacillus atrophaeus 4618 to obtain the microbial complex bactericide.

[0029] In one embodiment, the conditions of the coculture include: a culture temperature of 18 - 30 °C, a culture rotation speed of 100 - 300 rpm, and a culture time of 24 - 72 h.

[0030] In one embodiment, the coculture is carried out in an NB medium, which is prepared with water and includes: 3 g / L of beef extract, 10 g / L of tryptone, and 5 g / L of sodium chloride.

[0031] The present invention provides the application of the microbial complex bactericide as described above or the microbial complex bactericide prepared by the preparation method as described above in preventing and treating potato common scab.

[0032] The present invention is further illustrated below by specific examples.

[0033] In the following examples, the materials and culture medium components involved are specifically as follows: (1) Strains and materials The potato common scab pathogen P139 was isolated from the potato common scab diseased tissue 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 and named Streptomyces scabiei .

[0034] Paenibacillus myricae 6096 was isolated from a potato field in Nanning, Guangxi Zhuang Autonomous Region in March 2023, and was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was May 28, 2025, and the deposit number was CGMCC NO. 34691, and was classified and named Dyadobacter milanwu .

[0035] Streptomyces sp. 6219 was isolated from a potato field in Nanning, Guangxi Zhuang Autonomous Region in March 2023, and was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was May 28, 2025, and the deposit number was CGMCC NO. 34692, and was classified and named Streptomyces sp. .

[0036] Bacillus atrophaeus 4618 was isolated from the rhizosphere soil of corn in the Inner Mongolia University Farm, Hohhot City, Inner Mongolia Autonomous Region in September 2022. It is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is June 19, 2025, and the preservation number is CGMCC NO. 34952. The taxonomic name is Bacillus atrophaeus .

[0037] The tested radish seedling seeds are YR Xinbaiyuchun, which are produced by Beijing Shinong Seedling Co., Ltd.

[0038] The tested potato seeds are Lucinda (V7), which are purchased from Gansu Dingxi Potato Research Institute.

[0039] (2) Tested culture media and reagents Nutrient Agar (NA) is prepared with water and includes: beef extract 3 g / L, tryptone 10 g / L, sodium chloride (NaCl) 0.5 g / L, and agar 15 g / L.

[0040] Nutrient Broth (NB) is prepared with water and includes: beef extract 3 g / L, tryptone 10 g / L, and sodium chloride (NaCl) 5 g / L.

[0041] R2A agar medium is prepared with water and includes: R2A agar 18.1 g / L.

[0042] 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, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.01 g / L, manganese chloride heptahydrate (MnCl2·7H2O) 0.01 g / L, soluble starch 10 g / L, and agar 20 g / L.

[0043] ISP4 liquid medium is based on Streptomyces medium No. 4 without adding agar.

[0044] Water agar is prepared with water and includes: agar 7 g / L.

[0045] Example 1 Isolation, identification and screening of soil microorganisms (1) Isolation of soil microorganisms A. Sample: Rhizosphere soil of potato plants in Nanning, Guangxi Zhuang Autonomous Region, China.

[0046] B. Sample treatment: Weigh 0.1 g of rhizosphere soil and mix it with 1 mL of sterile water to obtain the original sample solution.

[0047] C. Dilution: Dilute 30 μL of the original sample solution and 270 μL of sterile water in a volume ratio of 1:9 for 8 gradients. Take 10 μL of each dilution gradient and spot them on NA, R2A, and ISP4 culture media respectively, and culture them in an incubator at 28 °C for 2 - 3 days to obtain the optimal dilution gradient.

[0048] D. Coating: Take 100 μL of the optimal gradient dilution solution and coat it on NA, R2A, and ISP4 culture media respectively, and culture them in an incubator at 28 °C for 4 - 5 days.

[0049] E. Streak purification: Select all bacteria with different morphologies, streak them on NA, R2A, and ISP4 culture media, and culture them in an incubator at 28 °C for 2 - 5 days until purified to single colonies.

[0050] F. Preservation: A total of 97 strains of bacteria were isolated. Pick a single colony of each strain of bacteria and inoculate it into NB medium, culture it at 28 °C with 200 rpm for 2 - 3 days, mix the bacterial suspension with glycerol at a volume ratio of 60% at 1:1, and store it in an ultra - low temperature freezer at - 80 °C for long - term preservation.

[0051] (2) Identification of soil microorganisms In this example, universal bacterial primers 8F and 1492R were used for colony PCR amplification. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Galaxy∣Europe (https: / / usegalaxy.eu / ) was used for sequence splicing, and the spliced sequences were compared with the sequences in NCBI - BLAST (https: / / www.ncbi.nlm.nih.gov / ). The sequencing and comparison results showed that among the 97 strains of bacteria, there were 37 genera. One strain of bacteria from each genus was selected for subsequent experiments.

[0052] (3) Screening for bacteria with antagonistic effects against Bacillus atrophaeus 4618 A. Effects of bacteria on Bacillus atrophaeus 4618 Bacillus atrophaeus 4618 and the bacteria to be tested were respectively inoculated into NB medium and cultured at 28 °C with shaking at 200 rpm for 1 - 2 days. 200 μL of the Bacillus atrophaeus 4618 bacterial solution was taken and placed in a 15 mL centrifuge tube, and water agar at 40 - 45 °C was added and spread on NA medium. 10 μL of the bacterial solution to be tested was spotted on the NA medium spread with Bacillus atrophaeus 4618, and cultured at 28 °C for 1 - 2 days, and the formation of inhibition zones was observed. Strains without inhibition zones were selected for the next step.

[0053] Effect of B. atrophaeus 4618 on bacteria 150 μL of the bacteria to be tested without inhibition zones in step A were respectively taken and placed in a 15 mL centrifuge tube, and water agar at 40 - 45 °C was added and spread on NA medium. 10 μL of the Bacillus atrophaeus 4618 bacterial solution was spotted on the NA medium spread with the bacteria to be tested, and cultured at 28 °C for 1 - 2 days, and the formation of inhibition zones was observed. Strains without inhibition zones were selected for the next step.

[0054] C. Effect of bacteria and B. atrophaeus 4618 on the scab pathogen P139 The scab pathogen P139 was inoculated into ISP4 liquid medium and cultured at 28 °C with shaking at 200 rpm for 3 - 5 days; the bacteria to be tested without inhibition zones in step B and B. atrophaeus 4618 were respectively inoculated into NB medium and cultured at 28 °C with shaking at 200 rpm for 1 - 2 days. 100 μL of the P139 bacterial solution was evenly spread on ISP4 medium. The B. atrophaeus 4618 and the bacterial solution to be tested were mixed evenly at a volume ratio of 1:1, and 10 μL of the mixed solution was spotted into the ISP4 medium coated with P139. The ISP4 medium with only B. atrophaeus 4618 was used as a control, and cultured at 28 °C for 3 - 5 days. The formation of inhibition zones was observed, and the area of the inhibition zones was calculated using Image J.

[0055] D. Screening results Based on the experimental results of steps A and B in this example, a total of 14 strains of bacteria without mutual antagonism with B. atrophaeus 4618 were screened. Combining the experimental results of step C in this example, after mixing the two strains of bacteria, Paenibacillus milanensis 6096 and Streptomyces 6219, with B. atrophaeus 4618 respectively, both had significant antibacterial effects ( Figure 1 ), and could promote the antagonistic effect of B. atrophaeus 4618 against the scab pathogen P139. Therefore, they were selected as candidate functional strains for constructing the microbial composite agent.

[0056] Example 2 Construction of microbial composite agent (1) Antagonism test between candidate functional strains The whole combination droplet method was used to observe the antagonistic phenomenon among candidate functional strains. The bacteria screened for promoting the antagonism of Bacillus atrophaeus 4618 in Example 1 were inoculated into NB medium respectively, and cultured at 28 °C with 200 rpm for 1 - 2 days; 200 μL of the bacterial liquid of one of the bacteria was taken into a 15 mL centrifuge tube, and water agar at 40 - 45 °C was added and spread on NA medium as the indicator bacterium, and the remaining strains were used as the test bacteria, and 3 plates were spread for each indicator bacterium; the Petri dishes of the indicator bacteria were divided into 4 fan-shaped areas, 10 μL of the test bacteria liquid was taken at 4 points in the fan-shaped area as duplicates, and 10 μL of the indicator bacteria liquid was taken at 1 upper point in the fan-shaped area as the control, and the distance between each point was about 1 cm; cultured at 28 °C for 2 - 3 days, the antagonistic phenomenon was observed, and the test results are shown in Table 1. There was no mutual antagonistic effect between the two candidate functional strains.

[0057] Table 1 Antagonistic effect among candidate functional strains

[0058] Note: “+” indicates that there is an antagonistic effect between strains and the antagonistic effect is strong; “-” indicates that there is no antagonism or the antagonistic effect between strains.

[0059] (2)Screening of the compound form of microbial complex bactericide The radish stem height measurement method was used to screen the compound form of functional strains.

[0060] A. Radish seedling germination: The radish seedling seeds were soaked in 75% (v / v) ethanol for 5 min, rinsed 3 times with sterile water, and the seeds were dispersed and placed on a Petri dish lined with wet filter paper, and germinated overnight in an incubator at 28 °C.

[0061] B. Radish seedling planting: About 15 g of soil was loaded into a tissue culture tube, autoclaved at 121 °C for 20 min, and after natural cooling, 5 mL of sterile water was added. After the soil was completely wet, the germinated seeds were transplanted into the tissue culture tube with forceps, and 2 - 3 seeds were inoculated into each tube. Light:Dark = 16:8 h, and cultured for 2 days.

[0062] C. Screening of compound form: The scab pathogen P139, Bacillus atrophaeus 4618, Paenibacillus milanensis 6096 and Streptomyces 6219 were inoculated into ISP4 liquid and NB medium respectively, and cultured at 28 °C with 200 rpm; the treatment groups included: Blank control group: Add an equal volume of sterile water.

[0063] Scab pathogen P139 group (abbreviated as P139): Only add the scab pathogen P139.

[0064] Scab pathogen P139 + Bacillus atrophaeus 4618 group (abbreviated as P139 + 4618): Add scab pathogen P139 and Bacillus atrophaeus 4618.

[0065] Scab pathogen P139 + (Bacillus atrophaeus 4618 + candidate functional strain) group (abbreviated as P139 + 4618 + 6096 or P139 + 4618 + 6219): Add scab pathogen P139, Bacillus atrophaeus 4618 and a candidate functional strain (Paenibacillus milanensis 6096 or Streptomyces 6219).

[0066] Among them, there are two inoculation forms for the groups that add Bacillus atrophaeus 4618 and the candidate functional strain at the same time: one is to immediately apply the mixture of Bacillus atrophaeus 4618 and the candidate functional strain after mixing them at a viable cell number ratio of 1:1, and the other is to apply the mixture after co - culturing Bacillus atrophaeus 4618 and the candidate functional strain at a viable cell number ratio of 1:1 for 48 h. Take 600 μL of the bacterial liquid from each treatment group and add it to the tissue culture tubes with emerged seedlings. Light:Dark = 16:8 h, and measure the height of the radish seedlings in each treatment group after culturing for 4 - 5 days. The experimental results are as Figure 2 and Figure 3 shown. It can be seen from Figure 2 and Figure 3 that applying the mixture after co - culturing Bacillus atrophaeus 4618 and the candidate functional strain for 48 h has a better antagonistic effect against scab pathogen P139.

[0067] (3) Screening of the compounding ratio of the microbial complex bactericide Compound three candidate functional strains into a microbial complex bactericide. In order to screen the optimal ratio of each strain in the microbial complex bactericide, an L9(3 3 ) multi - factor and multi - level orthogonal experiment is adopted. Taking the inoculation volume ratio of each strain as the experimental factor, setting a volume ratio of 1:2:3, and using the inhibition zone area of the microbial complex bactericide against scab pathogen P139 as the evaluation index. Combining with the double - layer plate confrontation method, evenly coat 150 μL of the scab pathogen P139 bacterial liquid onto the ISP4 medium, and spot 20 μL of the microbial complex bactericide into the ISP4 medium coated with scab pathogen P139. Incubate at 28 °C for 3 - 5 days, observe the generation of the inhibition zone, and calculate the inhibition zone area using Image J; through range analysis, further determine the optimal ratio of each strain in the microbial complex bactericide. The results of the orthogonal experiment and range analysis are shown in Table 2.

[0068] Table 2 Results of the orthogonal experiment of the microbial complex bactericide

[0069] Note: The test factor A refers to Bacillus paracatenulatus 6096, the test factor B refers to Streptomyces 6219, and the test factor C refers to Bacillus atrophaeus 4618. K is the sum of the test data of each factor at each level; k is the average value of the K value, that is, the average value of the test data of each factor at each level; R is the range, that is, the difference between the maximum and minimum values of the k values at each level of the same factor. The numbers 1-3 in the test factors refer to the volume ratio.

[0070] As can be seen from Table 2, when the viable counts of Bacillus paracatenulatus 6096, Streptomyces 6219, and Bacillus atrophaeus 4618 are 2:3:3, the antibacterial effect against the scab pathogen P139 is the best.

[0071] Example 3 Antibacterial verification test of microbial complex bactericide (1) Double-layer plate test Verify the antibacterial ability of the microbial complex bactericide constructed according to the optimal ratio. The method is the same as step (1) in Example 1. The experimental results are as Figure 4 shown. As Figure 4 can be seen, the antibacterial effect of adding the microbial complex bactericide is significantly higher than that of adding only Bacillus atrophaeus 4618.

[0072] (2) Radish seedling test Conduct a radish seedling test on the microbial complex bactericide constructed according to the optimal ratio. The method is the same as step (2) in Example 2. The treatment groups include: Scab pathogen P139 group (abbreviated as P139): Only add the scab pathogen P139.

[0073] Scab pathogen P139 + Bacillus atrophaeus 4618 group (abbreviated as P139 + 4618): Add the scab pathogen P139 and Bacillus atrophaeus 4618.

[0074] Scab pathogen P139 + microbial complex bactericide group (abbreviated as P139 + microbial complex bactericide): Add the scab pathogen P139 and the microbial complex bactericide.

[0075] Blank control group: Add an equal volume of sterile water.

[0076] The experimental results are as Figure 5 shown. As Figure 5 can be seen, the plant height of the radish seedlings in the P139 + microbial complex bactericide group is significantly higher than that in the P139 group and the P139 + 4618 group, indicating that the antibacterial effect of adding the microbial complex bactericide is better than that of adding only Bacillus atrophaeus 4618.

[0077] (3) Pot experiment The cultivated soil was collected from the on-campus farm of Inner Mongolia University. The seed potatoes were cut and sprouted under dark conditions at 18 - 25°C, and then cultivated in flower pots with a diameter of 25 cm and grown in an artificial climate chamber. The conditions of the artificial climate chamber were: temperature 22 - 25°C, humidity 30 - 35%, light condition 12 h light and 12 h dark. 5 - 6 g of granular base fertilizer (N:P2O5:K2O = 12:19:16) was applied to each pot, and the soil water content was maintained at 60 - 70%. The treatment groups were the same as in step (2) of this example. Each treatment group had 3 replicates, with 5 plants in each replicate, and the whole experiment was repeated 2 times.

[0078] Specific experimental operation: The scab pathogen P139 was inoculated into ISP4 liquid medium and cultured at 200 rpm and 28°C. 200 μL was taken and spread on ISP4 solid medium, and cultured in an incubator at 28°C for about 15 days. Then it was further transferred to a blender, added with distilled water to break, and the concentration was adjusted to 10 8 cfu / mL. 200 mL of the scab pathogen P139 was added to the soil and mixed evenly. Each strain in the microbial complex agent was inoculated into NB medium and cultured at 200 rpm and 28°C for 48 h. After compounding according to the optimal compounding ratio, it was continued to be cultured for 48 h, and the concentration was adjusted to 10 8 cfu / mL. 24 h after adding the scab pathogen P139, 200 mL of the microbial complex agent was added to the soil and mixed evenly. Finally, the germinated potato tubers were sown in the flower pots of each treatment group.

[0079] After 120 days of planting, the tubers were harvested and the incidence and disease severity were evaluated. The severity of potato scab can be divided into six grades according to the scab coverage rate: grade 0 = asymptomatic, grade 1 < 1%, grade 2 = 1% - 10%, grade 3 = 11% - 20%, grade 4 = 21% - 50%, grade 5 > 51%.

[0080] The formula for calculating the incidence of potato scab is: .

[0081] The formula for calculating the disease index of potato scab is: .

[0082] The experimental results are as Figure 6 shown, where A is the result of the incidence and B is the result of the disease index. From Figure 6It can be seen that the incidence rate of the P139 group is 87.37%; the incidence rate of the P139 + 4618 group is 62.45%, which is significantly lower than that of the P139 group; the incidence rate of the P139 + microbial complex bacterium agent group is 34.86%, which is significantly lower than that of the P139 group and the P139 + 4618 group; this result is the same as the trend of the plate verification and radish seedling verification. The incidence rate of the P139 + microbial complex bacterium agent group is reduced by 52.51% compared with that of the P139 group, and is reduced by 27.59% compared with that of the P139 + 4618 group, indicating that it has better effects in inhibiting potato common scab. The disease index of the P139 + 4618 group and the P139 + microbial complex bacterium agent group are both significantly lower than that of the P139 group, and the disease index of the P139 + microbial complex bacterium agent group is lower. To sum up, the microbial complex bacterium agent shows good effects in preventing and controlling potato common scab and improves the control effect of Bacillus atrophaeus 4618 on this disease.

[0083] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. Microbial complex bactericide, characterized in that, The microbial composite bacterium agent includes: Paenibacillus milanensis ( Dyadobacter milanwu ), Streptomyces sp. ( Streptomyces sp. ), and Bacillus atrophaeus ( Bacillus atrophaeus ); The Dyadobacter milanwu 6096 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 28, 2025, and the deposit number is CGMCC NO. 34691; The Streptomyces 6219 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 28, 2025, and the deposit number is CGMCC NO. 34692; The Bacillus atrophaeus 4618 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. 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.

2. The microbial complex bacterial agent according to claim 1, wherein The viable count ratio of the Dyadobacter milanwu 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is (1 - 3):(1 - 3):(1 - 3).

3. The microbial complex bacterial agent according to claim 1, wherein The viable count ratio of the Dyadobacter milanwu 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 is 2:3:

3.

4. The microbial complex bacterium agent according to claim 1, wherein The microbial complex bactericide further includes agriculturally acceptable excipients.

5. The microbial complex bacterial agent according to claim 4, wherein The agriculturally acceptable excipients are selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

6. The preparation method of the microbial complex bacterial agent according to claim 1, wherein Including the steps: Co-culturing the Dyadobacter milanwu 6096, Streptomyces 6219 and Bacillus atrophaeus 4618 to obtain the microbial complex bactericide.

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

8. The preparation method according to claim 6, characterized in that, The co-culture is carried out in an NB medium, and the NB medium is prepared with water and includes: 3 g / L of beef extract, 10 g / L of tryptone, and 5 g / L of sodium chloride.

9. Use of the microbial complex bactericide according to any one of claims 1 - 5 or the microbial complex bactericide prepared by the preparation method according to any one of claims 6 - 8 in the prevention and control of potato common scab.

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