Multi-strain microbial agents for the prevention and control of potato scab, their preparation methods and applications

Abstract

This invention relates to the field of microbial inoculants, and discloses a multi-strain microbial preparation for the prevention and control of potato scab, its preparation method, and its application. The multi-strain microbial preparation includes: *Bacillus atrophicus* 6052, *Bacillus compostii* 6069, *Microbacterium natriureticum* 6035, and *Bacillus atrophicus* 4618; all of these strains are deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC NO.34693, CGMCC NO.34694, CGMCC NO.34695, and CGMCC NO.34952, respectively. The multi-strain microbial preparation of this invention exhibits good and stable antibacterial effects, and its use can significantly reduce the incidence and disease index of potato scab.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to multi-strain microbial preparations for the prevention and control of potato scab, their preparation methods, and applications. Background Technology

[0002] Potato scab (PCS) is a soil-borne and seed-borne bacterial disease caused by pathogenic Streptomyces, and is one of the four major diseases affecting potato production. The pathogenic Streptomyces invades potato tubers through lenticels and stomata. In the early stages of infection, light brown spots with a diameter of 5-8 mm appear on the tuber surface. As the infection progresses, the tissue around the infection site necroses, becomes corky, and the tuber surface roughens, typically exhibiting black, rust-colored, or brown sunken, raised, or flat lesions. This disease thrives in hot, alkaline environments. The pathogen can overwinter in the soil and surviving tubers, becoming a source of infection for the following year. The spores produced are rapidly transported and multiplied via seed potatoes, soil, wind, rain, and insects.

[0003] The pathogen causing potato scab can multiply and survive in the soil for up to 10 years. The accumulation of toxins disrupts the soil microbial community and pollutes the environment. Simultaneously, diseased potatoes exhibit poor appearance, are less resistant to storage, and experience a significant reduction in quality, thus affecting their edible and processing value. Studies by Santos-Cervantes et al. have found that the scab pathogen can also infect staple root crops such as sugar beets, carrots, sweet potatoes, and parsnip. The host range of this disease continues to expand, and control technologies remain to be developed, making it one of the major diseases in global agriculture. Therefore, there is an urgent need to explore eco-friendly methods for controlling potato scab. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a multi-strain microbial preparation for the prevention and control of potato scab, its preparation method and application, in order to solve the problem of unstable control effect of existing potato scab control methods.

[0005] The technical solution of the present invention is as follows:

[0006] Firstly, a multi-strain microbial preparation for the prevention and control of potato scab is provided, said multi-strain microbial preparation comprising: Bacillus atrophus (… Bacillus atrophaeus )6052, Bacillus compostii ( Bacillus stercoris )6069, Microbacterium natans ( Microbacterium nayanwu )6035 and Bacillus atrophus ( Bacillus atrophaeus 4618;

[0007] The Bacillus atrophus 6052 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO.34693.

[0008] The Bacillus compostii 6069 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO.34694.

[0009] The aforementioned *Narcissus* 6035 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO. 34695.

[0010] The Bacillus atrophus 4618 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 19, 2025, with accession number CGMCC NO.34952.

[0011] In the preferred embodiment, the ratio of viable bacteria of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618 is (1-3):(1-3):(1-3):(1-3).

[0012] In a preferred embodiment, the ratio of viable bacteria of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035, and Bacillus atrophus 4618 is 1:3:3:2.

[0013] In a preferred embodiment, the multi-strain microbial preparation further includes agriculturally acceptable adjuvants.

[0014] In a preferred embodiment, the agriculturally acceptable adjuvant is selected from one or more of dispersants, stabilizers, fillers, and solvents.

[0015] Secondly, a method for preparing a multi-strain microbial preparation as described in the first aspect is provided, comprising the steps of:

[0016] The multi-strain microbial preparation was obtained by co-culturing Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618.

[0017] In a preferred embodiment, the co-cultivation conditions include: a cultivation temperature of 18-30℃, a cultivation rotation speed of 100-300 rpm, and a cultivation time of 24-72 h.

[0018] In a preferred embodiment, the co-culture 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.

[0019] Thirdly, the application of multi-strain microbial agents as described in the first aspect or multi-strain microbial agents prepared by the preparation method described in the second aspect in the prevention and control of potato scab disease.

[0020] Beneficial Effects: In the early stages of this invention, *Bacillus atrophicus* 4618, exhibiting excellent antagonistic properties against the potato scab pathogen P139, was isolated from the soil. Through screening, *Bacillus atrophicus* 6052, *Bacillus compostii* 6069, and *Microbacterium natriureticum* 6035, which promoted the antagonistic effect of *Bacillus atrophicus* 4618, were obtained. These were then combined with *Bacillus atrophicus* 4618 to obtain a multi-strain microbial preparation. This multi-strain microbial preparation showed good and stable antibacterial effects. When applied to the control of potato scab, the incidence and disease index were significantly lower than those of the positive control group and the treatment group treated with only *Bacillus atrophicus* 4618. Attached Figure Description

[0021] Figure 1 This is a diagram showing the antagonistic effect of the combined use of Bacillus atrophus 4618 and candidate functional strains in Example 1 against P139, the pathogen of scabies.

[0022] Figure 2 This is a diagram showing the experimental results of radish seedlings immediately after mixing Bacillus atrophus 4618 and candidate functional strains at a live count of 1:1 in Example 2.

[0023] Figure 3 This is a diagram showing the results of an experiment on radish seedlings after co-culturing Bacillus atrophus 4618 and candidate functional strains at a 1:1 ratio for 48 hours.

[0024] Figure 4 This is a graph showing the results of a double-layer plate test of the multi-strain microbial preparation formulated in the optimal ratio in Example 3.

[0025] Figure 5 This is a diagram showing the experimental results of radish seedlings prepared with a multi-strain microbial agent in the optimal ratio as described in Example 3.

[0026] Figure 6 This is a diagram showing the pot experiment results of the multi-strain microbial preparation formulated in the optimal ratio in Example 3. Detailed Implementation

[0027] This invention provides a multi-strain microbial preparation for the prevention and control of potato scab, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, specific embodiments are provided below to further illustrate this invention.

[0028] Bacillus atrophicus is a major biocontrol bacterium that protects plants from pathogens through mechanisms such as lysis, antagonism, competition, and induced resistance. Its application in controlling potato scab has been reported. However, the control efficacy of Bacillus atrophicus in the field is often unstable due to complex soil environments. Furthermore, the activity of Bacillus atrophicus is 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 atrophicus may be inhibited, thus reducing its disease control efficacy. Further research has found that adding prebiotics derived from rhizosphere exudates / sediments and synthesizing microbial communities can improve the stability of Bacillus atrophicus in the plant rhizosphere, thereby enhancing its control over plant diseases.

[0029] Based on this, embodiments of the present invention provide a multi-strain microbial preparation for the prevention and control of potato scab, the multi-strain microbial preparation comprising: Bacillus atrophus (… Bacillus atrophaeus )6052, Bacillus compostii ( Bacillus stercoris )6069, Microbacterium natans ( Microbacterium nayanwu )6035 and Bacillus atrophus ( Bacillus atrophaeus 4618;

[0030] The Bacillus atrophus 6052 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO.34693.

[0031] The Bacillus compostii 6069 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO.34694.

[0032] The aforementioned *Narcissus* 6035 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO. 34695.

[0033] The Bacillus atrophus 4618 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 19, 2025, with accession number CGMCC NO.34952.

[0034] In one embodiment, the ratio of viable bacteria of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618 is (1-3):(1-3):(1-3):(1-3).

[0035] In one embodiment, the ratio of viable bacteria of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618 is 1:3:3:2.

[0036] In one embodiment, the multi-strain microbial preparation further includes agriculturally acceptable adjuvants.

[0037] In one embodiment, the agriculturally acceptable adjuvant is selected from one or more of dispersants, stabilizers, fillers, and solvents.

[0038] This invention provides a method for preparing a multi-strain microbial preparation as described above, comprising the following steps:

[0039] The multi-strain microbial preparation was obtained by co-culturing Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618.

[0040] In one embodiment, the co-culture conditions include: a culture temperature of 18-30°C, a culture rotation speed of 100-300 rpm, and a culture time of 24-72 h.

[0041] In one embodiment, the co-culture 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.

[0042] This invention provides the application of multi-strain microbial agents prepared as described above or prepared by the method described above in the prevention and control of potato scab disease.

[0043] The present invention will be further described below through specific embodiments.

[0044] The materials and culture medium components involved in the following embodiments are specifically as follows:

[0045] (1) Strains and materials

[0046] The pathogen causing potato scab, P139, was isolated in May 2020 from potato scab lesions in an experimental field at the Potato Center Farm of Inner Mongolia University in Hohhot, Inner Mongolia Autonomous Region. It was classified and named... Streptomyces scabiei .

[0047] Bacillus atrophus 6052 was isolated from a potato field in Qingyuan City, Guangdong Province in March 2024. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO.34693. The classification name is... Bacillus atrophaeus .

[0048] Bacillus compostii 6069 was isolated from a potato field in Qingyuan City, Guangdong Province in March 2024. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO.34694. The classification name is... Bacillus stercoris .

[0049] *Narcospora nabinoides* 6035 was isolated from a potato field in Qingyuan City, Guangdong Province in March 2024. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO. 34694. Its classification is as follows: Microbacterium nayanwu .

[0050] Bacillus atrophus 4618 was isolated in September 2022 from the rhizosphere soil of maize at the Inner Mongolia University Farm in Hohhot, Inner Mongolia Autonomous Region. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 19, 2025, with accession number CGMCCNO. 34952. It is classified as follows: Bacillus atrophaeus .

[0051] The radish seedlings tested were YR New White Jade Spring, a variety produced by Beijing Shinong Seedling Co., Ltd.

[0052] The tested potato seed tuber was Lucinda (V7), purchased from the Dingxi Potato Research Institute in Gansu Province.

[0053] (2) Test culture medium and reagents

[0054] Nutrient agar (NA) medium was prepared with water and consisted of: 3 g / L beef extract, 10 g / L tryptone, 0.5 g / L sodium chloride (NaCl), and 15 g / L agar.

[0055] The nutrient broth (NB) medium was prepared with water and included: 3 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride (NaCl).

[0056] R2A agar medium is prepared with water and contains 18.1 g / L of R2A agar.

[0057] Streptomyces 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 10 g / L, and agar 20 g / L.

[0058] ISP4 liquid medium is based on Streptomyces medium No. 4, without the addition of agar.

[0059] Water agar is prepared with water and contains 7 g / L of agar.

[0060] Example 1: Isolation, identification, and screening of soil microorganisms

[0061] (1) Isolation of soil microorganisms

[0062] A. Sample: Rhizosphere soil of potato plants in Qingyuan City, Guangdong Province, China.

[0063] B. Sample preparation: Weigh 0.1g of rhizosphere soil and mix it with 1mL of sterile water to obtain the original sample solution.

[0064] C. Dilution: Dilute 30 μL of the original sample solution with 270 μL of sterile water at a volume ratio of 1:9 to obtain 8 dilution gradients. Take 10 μL of each dilution gradient and spot it onto NA, R2A and ISP4 medium respectively. Incubate in a 28℃ incubator for 2-3 days to obtain the optimal dilution gradient.

[0065] D. Plating purification: Take 100 μL of the optimal gradient dilution and plating it onto NA, R2A and ISP4 media respectively, and incubate in an incubator at 28℃ for 4-5 days.

[0066] E. Streaking: Select all bacteria with different morphologies and streak them on NA, R2A and ISP4 media. Incubate at 28°C for 2-5 days until purified to single colonies.

[0067] F. Preservation: A total of 76 bacterial strains were isolated. A single colony of each strain was picked and inoculated into NB medium. The culture was carried out at 200 rpm and 28°C for 2-3 days. The bacterial suspension was then mixed with 60% glycerol at a volume ratio of 1:1 and stored in an ultra-low temperature freezer at -80°C for long-term preservation.

[0068] (2) Identification of soil microorganisms

[0069] In this embodiment, universal bacterial primers 8F and 1492R were used for colony PCR amplification. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. Sequence assembly was performed using Galaxy|Europe (https: / / usegalaxy.eu / ), and the assembled sequences were compared with those in NCBI-BLAST (https: / / www.ncbi.nlm.nih.gov / ). The sequencing comparison results showed that the 76 bacterial strains belonged to 46 genera, and one strain from each genera was selected for subsequent experiments.

[0070] (3) Screening for bacteria that promote antagonism with Bacillus atrophus 4618

[0071] A. Effects of bacteria on Bacillus atrophus 4618

[0072] Bacillus atrophicus 4618 and the test bacteria were inoculated separately into NB medium and incubated at 200 rpm and 28°C for 1-2 days. 200 μL of Bacillus atrophicus 4618 culture was transferred to a 15 mL centrifuge tube, and water agar at 40-45°C was added. The tube was then plated onto NA medium. 10 μL of the test bacteria culture was spotted onto the NA medium plate containing Bacillus atrophicus 4618. The tubes were incubated at 28°C for 1-2 days, and the formation of inhibition zones was observed. Strains that did not produce inhibition zones were selected for the next step.

[0073] B. Effects of Bacillus atrophus 4618 on bacteria

[0074] Take 150 μL of each of the test bacteria that did not produce an inhibition zone in step A and place it in a 15 mL centrifuge tube. Add water agar (40-45℃) and spread it on NA medium. Spot 10 μL of Bacillus atrophicus 4618 bacterial suspension onto the NA medium containing the test bacteria. Incubate at 28℃ for 1-2 days and observe the formation of inhibition zones. Select the strains that did not produce inhibition zones for the next step.

[0075] C. Effects of bacteria and Bacillus atrophus 4618 on P139, the pathogen causing scabies.

[0076] Inoculate scab pathogen P139 into ISP4 liquid medium and incubate at 200 rpm and 28°C for 3-5 days. Inoculate the test bacteria that did not produce inhibition zones in step B and Bacillus atrophicus 4618 into NB medium and incubate at 200 rpm and 28°C for 1-2 days. Spread 100 μL of scab pathogen P139 evenly onto ISP4 medium. Mix Bacillus atrophicus 4618 and the test bacteria at a volume ratio of 1:1. Spot 10 μL of the mixture onto the ISP4 medium containing scab pathogen P139. Use the ISP4 medium containing only Bacillus atrophicus 4618 as a control. Incubate at 28°C for 3-5 days, observe the formation of inhibition zones, and calculate the area of ​​the inhibition zones using ImageJ.

[0077] D. Filtering Results

[0078] Based on the experimental results of steps A and B in this embodiment, a total of 13 bacteria strains without antagonistic interaction with Bacillus atrophicus 4618 were screened. Combined with the experimental results of step C in this embodiment, Bacillus atrophicus 6052, Bacillus compostii 6069, and Microbacterium natriureticum 6035, when mixed with Bacillus atrophicus 4618, all showed significant antibacterial effects. Figure 1 These strains were selected as candidate functional strains for constructing multi-strain microbial preparations because they could enhance the antagonistic effect of Bacillus atrophus 4618 on the scab pathogen P139.

[0079] Example 2 Construction of multi-strain microbial preparations

[0080] (1) Antagonism test among candidate functional strains

[0081] The antagonistic phenomenon among candidate functional strains was observed using the combined droplet method. The bacteria that promoted the antagonism of Bacillus atrophicus 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 one bacterial culture was placed in a 15 mL centrifuge tube, and water agar at 40-45°C was added. This was then spread on NA medium as an indicator bacterium. The remaining strains were used as test bacteria, with each indicator bacterium spread on three plates. The indicator bacterial culture dish was divided into four fan-shaped regions. 10 μL of test bacterial culture was placed at four points in each fan-shaped region as replicates, and 10 μL of indicator bacterial culture was placed at one point in each fan-shaped region as a control. The distance between each point was approximately 1 cm. The culture was incubated at 28°C for 2-3 days, and the antagonistic phenomenon was observed. The results are shown in Table 1. No antagonistic effect was observed among the three candidate functional strains.

[0082] Table 1. Antagonistic effects among candidate functional strains

[0083]

[0084] Note: "+" indicates that there is antagonistic effect between strains and the antagonistic effect is strong; "-" indicates that there is no antagonistic effect or no antagonistic effect between strains.

[0085] (2) Screening of multi-strain microbial preparation compound forms

[0086] The combination of functional strains was screened using the radish stem height determination method.

[0087] A. Radish seedling germination: Soak radish seedling seeds in 75% (v / v) ethanol for 5 minutes, rinse 3 times with sterile water, disperse the seeds in petri dishes lined with moistened filter paper, and incubate overnight at 28℃ for germination.

[0088] B. Radish seedling planting: Put about 15g of soil into the tissue culture tube, autoclave at 121℃ for 20min, and after natural cooling, add 5mL of sterile water. After the soil is completely moistened, use tweezers to transplant the germinated seeds into the tissue culture tube, with 2-3 seeds in each tube. Light:Dark = 16:8h, and culture for 2 days.

[0089] C. Screening of compound formulations: The scab pathogen P139, along with *Bacillus atrophus* 4618, *Bacillus atrophus* 6052, *Bacillus compostii* 6069, and *Microbacterium nalerum* 6035, were inoculated into ISP4 liquid and NB medium, respectively, and cultured at 200 rpm and 28°C. Treatment groups included:

[0090] Blank control group: Add an equal volume of sterile water.

[0091] Group P139 (abbreviated as P139): Only P139, the pathogen of scabies, was added.

[0092] Group P139 + Bacillus atrophus 4618 (abbreviated as P139+4618): Scab pathogen P139 and Bacillus atrophus 4618 are added.

[0093] Group P139+ (Bacillus atrophicus 4618+ candidate functional strain) (abbreviated as P139+4618+6052 or P139+4618+6069 or P139+4618+6035): This group includes P139, the pathogenic bacterium of scabies, Bacillus atrophicus 4618, and one candidate functional strain (one of Bacillus atrophicus 6052, Bacillus compostii 6069, and Microbacterium natriureticum 6035).

[0094] Among the groups where both Bacillus atrophicus 4618 and the candidate functional strain were added, two inoculation methods were used: one was to mix Bacillus atrophicus 4618 and the candidate functional strain at a 1:1 live count and apply immediately; the other was to mix Bacillus atrophicus 4618 and the candidate functional strain at a 1:1 live count and co-culture for 48 hours before application. 600 μL of bacterial solution from each treatment group was added to the germinated tissue culture tubes, with a light:dark ratio of 16:8 hours. After 4-5 days of incubation, 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. By Figure 2 and Figure 3 It can be seen that the antagonistic effect against the scab pathogen P139 is better when Bacillus atrophus 4618 is co-cultured with the candidate functional strain for 48 hours.

[0095] (3) Screening of the compound ratio of multi-strain microbial preparations

[0096] Three candidate functional strains were combined to form a multi-strain microbial preparation. To screen for the optimal ratio of each strain in the multi-strain microbial preparation, L9(3) was used. 4 A multi-factor, multi-level orthogonal experiment was conducted, using the inoculation volume ratio of each strain as the experimental factor, with a volume ratio of 1:2:3. The inhibition zone area of ​​the multi-strain microbial preparation against the scab pathogen P139 was used as the evaluation index. Using the double-layer plate confrontation method, 150 μL of the scab pathogen P139 bacterial suspension was evenly spread onto ISP4 medium, and 20 μL of the multi-strain microbial preparation was spotted into the ISP4 medium containing scab pathogen P139. The mixture was incubated at 28℃ for 3-5 days, and the formation of the inhibition zone was observed. The inhibition zone area was calculated using ImageJ. Range analysis was then used to determine the optimal ratio of each strain in the multi-strain microbial preparation. The results of the orthogonal experiment and range analysis are shown in Table 2.

[0097] Table 2. Results of orthogonal experiments on multi-strain microbial preparations

[0098]

[0099] Note: Experimental factor A refers to Bacillus atrophicus 6052, experimental factor B refers to Bacillus composting 6069, experimental factor C refers to Microbene natriureticus 6035, and experimental factor D refers to Bacillus atrophicus 4618. K is the sum of experimental data for each factor at all levels; k is the average value of K, i.e., the average value of experimental data for each factor at all levels; R is the range, i.e., the difference between the maximum and minimum values ​​of k for the same factor at each level. Numbers 1-3 in the experimental factors refer to volume ratios.

[0100] As shown in Table 2, the best antibacterial effect against the scab pathogen P139 was achieved when the viable counts of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriureticum 6035, and Bacillus atrophus 4618 were 1:3:3:2.

[0101] Example 3 Antimicrobial Validation Test of Multi-Strain Microbial Agent

[0102] (1) Double-layer plate test

[0103] The antibacterial ability of the multi-strain microbial preparation constructed in the optimal ratio was verified using the same method as step (1) in Example 1. The experimental results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the antibacterial effect of adding multi-strain microbial preparations is significantly higher than that of adding only Bacillus atrophus 4618.

[0104] (2) Radish seedling experiment

[0105] A radish seedling test was conducted on a multi-strain microbial preparation constructed in the optimal ratio, using the same method as step (2) in Example 2. The treatment groups included:

[0106] Group P139 (abbreviated as P139): Only P139, the pathogen of scabies, was added.

[0107] Group P139 + Bacillus atrophus 4618 (abbreviated as P139+4618): Scab pathogen P139 and Bacillus atrophus 4618 are added.

[0108] The P139+ multi-strain microbial preparation group (abbreviated as P139+ multi-strain microbial preparation): contains the P139 pathogen of scabies and a multi-strain microbial preparation.

[0109] Blank control group: Add an equal volume of sterile water.

[0110] Experimental results are as follows Figure 5 As shown. By Figure 5 It can be seen that the radish seedling height of the P139+ multi-strain microbial preparation group was significantly higher than that of the P139 group and the P139+ Bacillus atrophicus 4618 group, indicating that the antibacterial effect of adding multi-strain microbial preparation is better than that of adding only Bacillus atrophicus 4618.

[0111] (3) Pot experiment

[0112] The cultivation soil was collected from the farm on campus of Inner Mongolia University. Seed potatoes were cut into pieces and sprouted under dark conditions at 18-25℃. They were then planted in 25cm diameter pots and grown in an artificial climate chamber. The artificial climate chamber conditions were: temperature 22-25℃, humidity 30-35%, and light conditions of 12h light and 12h darkness. 5-6g of granular base fertilizer (N:P2O5:K2O=12:19:16) was applied to each pot, and the soil moisture content was maintained at 60-70%. The treatment groups followed the same steps as in step (2) of this embodiment. Each treatment group had 3 replicates, with 5 pots of plants per replicate. The entire experiment was repeated twice.

[0113] Specific experimental procedures: The scab causative agent P139 was inoculated into ISP4 liquid medium and incubated at 200 rpm and 28°C. 200 μL of the inoculum was then spread onto ISP4 solid medium and incubated at 28°C for approximately 15 days. The mixture was then transferred to a blender, and distilled water was added to disrupt the morphology, adjusting the concentration to 10. 8 Add 200 mL of the scab pathogen P139 to the soil at a concentration of cfu / mL and mix well. Inoculate each strain from the multi-strain microbial preparation into NB medium and incubate at 200 rpm and 28°C for 48 h. After recombining the mixture according to the optimal ratio, continue incubation for another 48 h, adjusting the concentration to 10. 8 After adding the scab pathogen P13924 h at cfu / mL, 200 mL of a multi-strain microbial preparation was added to the soil and mixed thoroughly. Finally, the sprouted potato tubers were sown in flowerpots in each treatment group.

[0114] 120 days after planting, the tubers were harvested and the incidence and severity of the disease were assessed. The severity of potato scab was classified into six levels based on 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%.

[0115] The formula for calculating the incidence rate of potato scab is:

[0116] .

[0117] The formula for calculating the potato scab disease severity index is as follows:

[0118] .

[0119] Experimental results are as follows Figure 6 As shown. By Figure 6The incidence rate was 87.37% in the P139 group; 62.45% in the P139+4618 group, significantly lower than that in the P139 group; and 37.99% in the P139+multi-strain microbial agent group, significantly lower than both the P139 and P139+4618 groups. This result is consistent with the trends observed in plate and radish seedling validation. The incidence rate in the P139+multi-strain microbial agent group was 49.38% lower than that in the P139 group and 24.46% lower than that in the P139+4618 group, indicating its superior efficacy in inhibiting potato scab. The disease index in both the P139+4618 and P139+multi-strain microbial agent groups was significantly lower than that in the P139 group, with the P139+multi-strain microbial agent group exhibiting an even lower disease index. In conclusion, the multi-strain microbial agent demonstrated good efficacy in controlling potato scab and enhanced the control effect of Bacillus atrophicus 4618 on this disease.

[0120] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A multi-strain microbial preparation for the prevention and control of potato scab, characterized in that, The multi-strain microbial preparation includes: Bacillus atrophus (… Bacillus atrophaeus )6052, Bacillus compostii ( Bacillus stercoris )6069, Microbacterium natans ( Microbacterium nayanwu )6035 and Bacillus atrophus ( Bacillus atrophaeus 4618; The Bacillus atrophus 6052 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO. 34693. The Bacillus compostii 6069 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO. 34694. The aforementioned *Narcissus* 6035 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 28, 2025, with accession number CGMCC NO. 34695. The Bacillus atrophus 4618 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 19, 2025, with accession number CGMCCNO.34952.

2. The multi-strain microbial preparation according to claim 1, characterized in that, The ratio of viable bacteria of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618 is (1-3):(1-3):(1-3):(1-3).

3. The multi-strain microbial preparation according to claim 1, characterized in that, The ratio of viable bacteria of Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618 is 1:3:3:

2.

4. The multi-strain microbial preparation according to claim 1, characterized in that, The multi-strain microbial preparation also includes agriculturally acceptable adjuvants.

5. The multi-strain microbial preparation according to claim 4, characterized in that, The agriculturally acceptable adjuvants are selected from one or more of dispersants, stabilizers, fillers, and solvents.

6. The method for preparing the multi-strain microbial preparation according to claim 1, characterized in that, Including the following steps: The multi-strain microbial preparation was obtained by co-culturing Bacillus atrophus 6052, Bacillus compostii 6069, Microbacterium natriuresis 6035 and Bacillus atrophus 4618.

7. The preparation method according to claim 6, characterized in that, The co-culture conditions include: a culture temperature of 18-30℃, 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 was carried out in NB medium, which was prepared with water and included: 3 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride.

9. The application of the multi-strain microbial preparation as described in any one of claims 1-5 or the multi-strain microbial preparation prepared by the preparation method as described in any one of claims 6-8 in the prevention and control of potato scab.

Images