Cold-tolerant paenibacillus ky5 and application thereof

Abstract

This invention provides a cold-resistant *Bacillus subtilis* KY5 and its applications, belonging to the field of agricultural microbiology technology. The cold-resistant *Bacillus subtilis* KY5 provided by this invention is classified and named *Bacillus subtilis*. Peribacillus frigoritolerans The preservation number is CGMCC No. 36309. The cold-resistant *Bacillus subtilis* KY5 provided by this invention was isolated from the epidermis of healthy potato tubers in Heilongjiang Province. It is a local indigenous flora, possessing a natural affinity for the potato host, strong ecological adaptability, and the ability to adapt to cold climates and soil environments. Furthermore, its cultivation conditions are simple, making it easy to produce and apply. It also exhibits significant inhibitory activity against various potato scab pathogens, demonstrating a significant control effect on potato scab. In pot experiment tests, the control effect against potato scab reached 53.69%.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a cold-resistant Bacillus subtilis KY5 and its applications. Background Technology

[0002] Potato scab is caused by Streptomyces ( ) Streptomyces scabiei Soil-borne diseases caused by (etc.) primarily infect potato tubers, leading to the formation of lignified, scab-like lesions on the tuber surface. This severely reduces the commercial value and causes significant economic losses to the global potato industry. The pathogen population exhibits high genetic diversity and complex distribution.

[0003] Currently, the control of potato scab mainly relies on chemical pesticides and the breeding of resistant varieties. However, chemical control has problems such as environmental pollution, pesticide residues, and pathogen resistance; the breeding cycle for resistant varieties is long, and due to the high genetic diversity of the pathogen population, it is difficult to obtain broad-spectrum and durable resistant varieties. Biological control utilizes beneficial microorganisms to inhibit pathogens, which has the advantages of being environmentally friendly and sustainable. However, there is currently no research on developing novel biocontrol strains derived from potato tuber epidermis that are environmentally friendly and have significant control effects against potato scab. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a cold-resistant Bacillus subtilis KY5, which has a significant control effect on potato scab, obtained from the epidermis of healthy potato tubers, and to solve the environmental pollution and residue problems caused by existing chemical pesticide control.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a cold-resistant *Bacillus repens* KY5, which is classified and named *Bacillus repens* KY5. Peribacillus frigoritolerans It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36309.

[0006] The present invention also provides a culture medium obtained by liquid culture of the above-mentioned cold-resistant Bacillus subtilis KY5.

[0007] Preferably, the viable count of *Bacillus KY5* induced frostbite in the culture medium is 1 × 10⁻⁶. 8 CFU / mL or higher.

[0008] Preferably, the culture medium for liquid culture includes LB liquid medium; the LB liquid medium includes peptone at a final concentration of 10 g / L, yeast extract at a final concentration of 5 g / L, and NaCl at a final concentration of 10 g / L; the pH of the LB liquid medium is 7.0~7.2.

[0009] Preferably, the liquid culture conditions are 25~30℃, 160~200rpm shaking culture for 24~50 hours.

[0010] The present invention also provides the application of the above-mentioned cold-resistant Bacillus subtilis KY5 or the above-mentioned culture medium in inhibiting the pathogen of potato scab.

[0011] Preferably, the pathogen causing potato scab includes Streptomyces scabiei , Streptomyces europaeiscabiei , Streptomyces luridiscabiei , Streptomyces griseus and / or Streptomyces thermocarboxydus .

[0012] This invention also provides the application of the above-mentioned cold-resistant Bacillus subtilis KY5 or the above-mentioned culture medium in the prevention and control of potato scab.

[0013] The present invention also provides a method for preventing potato scab disease by irrigating the soil in which potatoes are planted with the above-mentioned culture solution.

[0014] Preferably, irrigation should begin one month after potato seedlings emerge, once a week for a total of four times. The amount applied each time should be at least 50 mL per potato seedling.

[0015] The beneficial effects of this invention are: The cold-resistant *Bacillus KY5*, provided by this invention, was isolated from the epidermis of healthy potato tubers in Heilongjiang Province. As a native local flora, it possesses a natural affinity for the potato host and can adapt to cold climates and soil environments. *Bacillus KY5* exhibits strong environmental resilience, tolerating concentrations of 6% NaCl and 0.1% phenol. It also demonstrates significant inhibitory activity against various potato scab pathogens, showing a significant control effect on potato scab. In pot experiment tests, the control effect against potato scab reached 53.69%. The cold-resistant *Bacillus KY5* provided by this invention has simple culture conditions, grows well on LB medium, and a high-concentration fermentation broth can be obtained by culturing at 25-30℃ for 24-50 hours, making it easy to produce and apply. Attached Figure Description

[0016] Figure 1The inhibition zone diameters are for 24 antagonistic bacterial strains. CKA represents Oxford cups with LB medium added (also labeled CKLB), and CKB represents Oxford cups with sterile water added (also labeled CK sterile water). A to X represent different strains isolated and purified, specifically: A: KY1, B: KY4, C: KY5, D: KY6, E: KY14, F: KY19, G: SD6, H: SDF, I: SD14, J: WL1, K: WL2, L: WL3, M: WL4, N: WL5, O: WL6, P: WL8, Q: WL9, R: WL11, S: WL12, T: WL13, U: WL14, V: WL19, W: WL20, X: WLR; Figure 2 Disease survey diagrams for different groups of potted plants; Figure 3 The table shows the statistical results of the incidence and disease index in the pot experiment. A represents the incidence result, and B represents the disease index result. Different lowercase letters above the bars indicate statistically significant differences (P<0.05). Figure 4 Colony morphology of cold-resistant Bacillus KY5 (LB medium); Figure 5 Phylogenetic tree of cold-resistant Bacillus KY5; Figure 6 For cold-resistant Bacillus subtilis KY5 pairs S. scabiei The antagonistic effect; Figure 7 For cold-resistant Bacillus subtilis KY5 pairs S. europaeiscabiei The antagonistic effect; Figure 8 For cold-resistant Bacillus subtilis KY5 pairs S. luridiscabiei The antagonistic effect; Figure 9 For cold-resistant Bacillus subtilis KY5 pairs S. griseus The antagonistic effect; Figure 10 For cold-resistant Bacillus subtilis KY5 pairs S. thermocarboxydus The antagonistic effect.

[0017] Biological Preservation Information The cold-resistant *Ligusticum striatum* KY5 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on October 23, 2025, with accession number CGMCC No. 36309, and classified as *Ligusticum striatum*. Peribacillus frigoritolerans . Detailed Implementation

[0018] This invention provides a cold-resistant *Bacillus repens* KY5, which is classified and named *Bacillus repens* KY5. Peribacillus frigoritolerans It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36309.

[0019] The cold-resistant *Bacillus kymophilus* KY5 provided by this invention was isolated from the epidermis of healthy potato tubers. After culturing on LB medium at 28°C for 24 hours, the colonies were round with irregular, raised edges, smooth, moist surfaces, and a pale yellow color; Gram-positive, and rod-shaped. The cold-resistant *Bacillus kymophilus* KY5 provided by this invention can utilize L-histidine as a nitrogen source (strongly positive), and weakly utilizes D-mannitol, sucrose, and L-methionine; it cannot utilize D-glucose, D-fructose, α-lactose, sorbitol, inositol, or L-tyrosine; it can grow in a medium containing 0.1% phenol; it can tolerate 1% and 3% NaCl, and weakly tolerates 6% NaCl; it has nitrate reducing ability; the VP test is negative; and it is sensitive to streptomycin (20 μg / mL) and penicillin (10 μg / mL). The 16S rRNA gene sequence of the cold-resistant *Bacillus kymophilus* KY5 provided by this invention is similar to... Peribacillus frigoritolerans The similarity to the standard strain DSM 8801 (NR_117474.1) reached 99.93%. A phylogenetic tree constructed using the Neighbor-Joining method showed that KY5 clustered with this strain in the same branch, with a bootstrap support rate of 93%. The cold-resistant *Bacillus subtilis* KY5 provided by this invention exhibits strong ecological adaptability and precise site of action (the strain derived from the tuber epidermis occupies the same ecological niche as the pathogen (tuber periderm), exhibiting direct competitive and antagonistic advantages at key sites of pathogen infection, thus providing more effective protection). Potato scab is an extremely difficult soil-borne disease to control. The pathogen can survive for a long time in the soil, and it is difficult for antagonistic bacteria to completely reach the tubers when applied to the roots. The control efficacy of commonly used chemical agents in this field is often only about 40%. However, in the pot control efficacy test, the cold-resistant Bacillus KY5 provided by this invention achieved a control effect of 53.69% against potato scab, which far exceeded conventional expectations. This indicates that the cold-resistant Bacillus KY5 of this invention can be used as a biocontrol agent for potato scab.

[0020] The present invention also provides a culture medium obtained by liquid culture of the above-mentioned cold-resistant Bacillus subtilis KY5.

[0021] In this invention, the viable count of *Bacillus kyptilus* KY5 in the culture medium is preferably 1 × 10⁻⁶. 8 CFU / mL or higher, preferably 1×10⁻⁶ 8 CFU / mL ~ 1×10 9CFU / mL. In this invention, the culture medium for liquid culture preferably includes LB liquid medium; the LB liquid medium preferably includes peptone at a final concentration of 10 g / L, yeast extract at a final concentration of 5 g / L, and NaCl at a final concentration of 10 g / L, and the solvent in the LB liquid medium is preferably distilled water or deionized water; the peptone is preferably tryptone, and the pH of the LB liquid medium is preferably 7.0-7.2, more preferably 7.1. This invention does not have specific limitations on the specific sources of each raw material in the LB liquid medium; commercially available products in the art can be used. The liquid culture conditions are preferably 25-30°C, 160-200 rpm shaking culture for 24-50 hours, more preferably 26-29°C, 170-190 rpm shaking culture for 26-48 hours, and even more preferably 28°C, 180 rpm shaking culture for 28-48 hours.

[0022] The present invention also provides the application of the above-mentioned cold-resistant Bacillus subtilis KY5 or the above-mentioned culture medium in inhibiting the pathogen of potato scab.

[0023] In this invention, the pathogen of potato scab preferably includes Streptomyces scabiei , Streptomyces europaeiscabiei , Streptomyces luridiscabiei , Streptomyces griseus and / or Streptomyces thermocarboxydus .

[0024] This invention also provides the application of the above-mentioned cold-resistant Bacillus subtilis KY5 or the above-mentioned culture medium in the prevention and control of potato scab.

[0025] The present invention also provides a method for preventing potato scab disease by irrigating the soil in which potatoes are planted with the above-mentioned culture solution.

[0026] In the method described in this invention, the culture medium serves as a microbial inoculant and can be used directly or diluted appropriately according to soil conditions. Preferably, irrigation begins one month after potato emergence (seedling stage), with an optimal irrigation frequency of once a week for a total of four applications. The amount applied each time is based on the number of potato seedlings, preferably more than 50 mL per seedling.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the following embodiments are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1 Isolation and purification of cold-resistant Bacillus subtilis KY5 (1) Sample collection and processing: Healthy potato tubers grown in Heilongjiang Province were collected, and after rinsing with running water to remove surface soil, they were disinfected with 75% (v / v) ethanol. Under aseptic conditions, the periderm tissue of the tubers was cut with a sterile scalpel and transferred to a sterile mortar.

[0031] (2) Preparation of bacterial suspension: Add about 5 mL of sterile phosphate buffer (PBS) to the mortar, grind thoroughly, filter through three layers of sterile gauze to remove plant residue, and collect the filtrate.

[0032] (3) Dilution and coating: The filtrate is serially diluted 10 times (10 -2 10 -3 10 -4 ), take 100 μL of the dilution solution and spread it on LB, PDA, TWYE, TSB and M715 agar plates respectively, and incubate at 28℃ inverted for 24~48 hours.

[0033] (4) Purification culture: Select single colonies with different morphology, color and edge characteristics, and purify them 2 to 3 times on LB plates using the three-waystreak method until a pure culture is obtained (confirmed by microscopic examination).

[0034] (5) Preservation of strains: For short-term preservation, use LB slant at 4℃; for long-term preservation, use LB liquid medium containing 30% (v / v) glycerol at -80℃.

[0035] Results: A total of 64 microbial strains were obtained.

[0036] Example 2 Confrontation culture method for screening antagonistic activity The Oxford cup method was used to determine the activity of each isolated strain obtained in Example 1 against the pathogen causing potato scab. Streptomyces scabiei Antagonistic activity.

[0037] Methods: 100 μL of pathogenic bacterial spore suspension was evenly spread onto Gause's No. 1 agar plates. After surface drying, the plates were placed in sterile Oxford cups. 100 μL of the fermentation broth of the test strain (LB liquid medium, incubated at 28°C and 180 rpm for 48 hours with shaking) was added to each Oxford cup. Sterile water and pure LB medium were used as controls (CKB and CKA). After incubation at 28°C for 7 days, the diameter of the inhibition zone was measured using calipers. The experiment was repeated three times.

[0038] The results showed that 24 of the strains exhibited resistance to the potato scab pathogen on plates.Streptomyces scabiei Antagonistic activity (antibacterial inhibition rate >10%) (see) Figure 1 The statistical results of the inhibition zone diameters of 24 antagonistic bacterial strains are shown in Table 1, indicating that there are high frequencies of antagonistic microorganisms on the surface of healthy potato tubers.

[0039] Table 1. Diameter of inhibition zones for 24 antagonistic bacterial strains

[0040] Note: Different lowercase letters in the table indicate statistically significant differences (P<0.05).

[0041] As shown in Table 1, the inhibition zone diameters of strains KY5, SD6, WLR, and WL9 were 35.00±0.30 mm, 36.62±1.0 mm, 32.91±3.50 mm, 30.19±3 mm, and 30.60±2.18 mm, respectively. Pot culture efficacy verification of these five strains will be conducted subsequently.

[0042] Example 3 Potted plant efficacy test The tested potato variety was Helan 5.

[0043] Tested pathogens: Streptomyces scabiei The spore suspension concentration was 1×10⁻⁶. 7 CFU / mL.

[0044] Experimental Design: (1) Substrate preparation: Use sterilized mixed substrate (to avoid contamination by other microorganisms) to fill the planting pot (38 cm high and 34.5 cm in diameter).

[0045] (2) Sowing and management: Sow 3 surface-sterilized "Dutch No. 5" seed potatoes in each pot and cover them with soil to a depth of about 15 cm. Water and fertilize according to standard potato cultivation management.

[0046] (3) Pathogen inoculation: One month after emergence (seedling stage), water each pot with 100 mL of pathogen spore suspension (1×10⁻⁶). 7 (CFU / mL), inoculated via soil irrigation.

[0047] (4) Biocontrol treatment: One week after inoculation with pathogens, biocontrol treatment began. Culture media of KY5, SD6, WLR, KY6, and WL9 (each culture medium containing approximately 1×10⁻⁶ viable bacteria) were respectively... 8The CFU / mL concentration was applied to the roots of each pot using 150 mL of the corresponding bacterial strain. All groups underwent seed coating preparation at the time of sowing and were subsequently labeled as KY6 (KY-6), KY5, WL9 (WL-9), WLR (WL-R), and SD6 (SD-6). Additionally, a group was established for KY5 that did not undergo seed coating and only underwent root drenching (also at 150 mL / pot), subsequently labeled as KY-5 (root drenching with mycotoxin) and also KY5 (Rd). The KY5 culture medium was obtained by inoculating KY5 onto LB liquid medium and incubating at 28°C and 180 rpm for 48 hours with shaking to obtain the culture medium (fermentation broth, viable count approximately 1 × 10⁻⁶). 8 CFU / mL). A pathogen control group (CK) was also established, receiving an equal volume of LB liquid culture medium. Treatment was performed weekly for a total of four weeks. Barrel cultivation was employed, as illustrated in the diagram below. Figure 2 The image shows a photo of the seed potatoes grown in a pot. The seed coating preparation method used above is as follows: First, the seed potatoes were soaked in a 0.1% potassium permanganate solution for 5 minutes for surface disinfection, and then dried for later use. Second, sodium alginate was slowly added to warm water below 60°C and stirred continuously until completely dissolved to form a uniform gel solution. Then, the antagonistic bacteria fermentation broth was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded and the antagonistic bacteria cells were retained. The bacteria cells were added to the sodium alginate gel solution and stirred evenly. Finally, the dried seed potatoes were immersed in the prepared sodium alginate bacterial solution for 2 minutes, and then immediately immersed in calcium chloride solution for cross-linking and solidification for 2 minutes to form a uniform gel film on the surface of the seed potatoes, thus completing the seed coating preparation.

[0048] (5) Disease survey: Three months after sowing, the tubers were harvested and the severity of scab disease was investigated according to the following grading standards (1-9): Grade 1: No lesions; Grade 2: Lesion area accounts for 0.1-0.8% of the tuber surface area; Grade 3: Lesion area accounts for 0.9-2.8% of the tuber surface area; Grade 4: Lesion area accounts for 2.9-7.9% of the tuber surface area; Grade 5: Lesion area accounts for 8.0-18.0% of the tuber surface area; Grade 6: Lesion area accounts for 18.1-34.0% of the tuber surface area; Grade 7: Lesion area accounts for 34.1-55.0% of the tuber surface area; Grade 8: Lesion area accounts for 55.1-77.0% of the tuber surface area; Grade 9: Lesion area accounts for 77.1-100% of the tuber surface area.

[0049] The following formulas are used to calculate the incidence rate, disease index, and prevention and control effectiveness: Disease incidence (%) = (Number of diseased tubers / Total number of tubers surveyed) × 100%; Disease Index = [Σ(Grade Number × Number of Tubers in That Grade) / (9 × Total Number of Tubers)] × 100; Prevention and control efficacy (%) = [(Pathogen control disease index - Treatment disease index) / Pathogen control disease index] × 100%; The results are as follows Figure 2 and Figure 3 As shown, the incidence rate of the soil root irrigation treatment group (KY5(Rd) group) of strain KY5 was 71.08% and the disease index was 31.46, while the incidence rate of the pathogen control group was 95.71% and the disease index was 67.94. Therefore, the control effect of soil root irrigation treatment of strain KY5 on potato scab was 53.69%.

[0050] Except for the KY5 root irrigation treatment, all other treatments (including the KY6 treatment) underwent seed coating at the initial planting stage, and all treatments were uniformly irrigated with roots in the later stages. The irrigation methods and operations were completely consistent across groups, with the only difference being the type of bacterial strain coated in the seed at the initial planting stage. Because of the seed coating, the KY6 treatment introduced a greater number of bacteria in the early stages compared to the KY5 treatment which did not undergo seed coating. The results showed that although KY6 introduced more bacteria through seed coating in the early stages, KY5, treated only with root irrigation, had a 0.2 lower disease index and a 1% lower incidence rate than KY6, and produced more potato tubers per plant, resulting in superior overall disease control and yield increase. Although KY6 carried a higher bacterial load in the early stages, its overall performance was inferior to KY5. The KY5 treatment significantly reduced the incidence rate and disease index (P<0.05), demonstrating a significantly better control effect than the other groups.

[0051] Example 4 Polyphasic classification of strain KY5 (1) Morphological observation: Colony characteristics: KY5 was inoculated onto LB agar plates and incubated at 28°C for 24 hours. Results are shown in the table below. Figure 4 The colonies are round with irregular margins, convex, smooth and moist surfaces, and pale yellow pigmentation.

[0052] Cell morphology: Gram-positive, appearing rod-shaped under a microscope.

[0053] (2) Determination of physiological and biochemical characteristics: Physiological and biochemical tests were performed according to Bergey's Manual of Systematic Bacteriology, and the results showed that: Carbon source utilization: D-glucose (-), D-fructose (-), D-mannitol (w), α-lactose (-), sucrose (w), sorbitol (-), inositol (-); Nitrogen source utilization: L-histidine (+), L-methionine (w), L-tyrosine (-); Salt tolerance: Can grow in 1% NaCl(+), 3% NaCl(+) and 6% NaCl(w); Other characteristics: 0.1% phenol tolerance (+), nitrate reduction (+), VP test (-), streptomycin sensitivity (20 μg / mL) (-), penicillin sensitivity (10 μg / mL) (-).

[0054] Note: "+" indicates a positive result, "w" indicates a weak positive result, and "-" indicates a negative result.

[0055] (3) Molecular biological identification: DNA extraction: Genomic DNA of the strain was extracted using the Solarbio Bacterial Genomic DNA Extraction Kit (D1600).

[0056] PCR amplification and sequencing: The full-length 16S rRNA gene sequence was amplified using universal primers, and the PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0057] Sequence analysis: The obtained sequence (approximately 1420 bp) was aligned with the NCBI database using BLASTn. The results showed that KY5 and... Peribacillus frigoritolerans The 16S rRNA gene sequence of DSM 8801 (NR_117474.1) has 99.93% homology.

[0058] Phylogenetic analysis: A phylogenetic tree was constructed using MEGA 11.0 software and the Neighbor-Joining method, based on 16S rRNA gene sequences, using a GTR+G+I substitution model. Branch support was assessed using 1000 bootstrap replicates (Bootstrap test with 1000 replicates). The scale bar corresponds to 0.01 substitutions per locus. Bacillus subtilis (substituent PP968145.1) was used as an outgroup. Results are as follows: Figure 5 As shown, KY5 and Peribacillus frigoritolerans DSM 8801 clustering is in the same branch, with bootstrap support at 93%, and... Peribacillus simplex and Peribacillus psychrosaccharolyticus They are also closely related by blood.

[0059] Identification conclusion: Based on morphological characteristics, physiological and biochemical properties, and 16S rRNA gene sequence analysis, strain KY5 was identified as a cold-resistant *Ligusticum striatum*. Peribacillus frigoritolerans .

[0060] Example 5 Preparation of culture medium (biocontrol bacterial solution) Strain KY5 was inoculated into LB liquid medium, which consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, with distilled water as the solvent and a pH of 7.1. The medium was incubated at 28°C with shaking at 180 rpm for 48 hours to obtain the culture broth (also known as biocontrol broth or fermentation broth). Plate count analysis showed that the viable cell count in the fermentation broth reached 1 × 10⁻⁶. 9 CFU / mL, can be directly used for potato soil irrigation treatment.

[0061] Example 6 Preparation of culture medium (biocontrol bacterial solution) Strain KY5 was inoculated into LB liquid medium, which consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, with distilled water as the solvent and a pH of 7.0. The medium was incubated at 30°C with shaking at 160 rpm for 40 hours to obtain the culture broth (also known as biocontrol broth or fermentation broth). Plate count analysis showed that the viable cell count in the fermentation broth reached 1 × 10⁻⁶. 8 CFU / mL, can be directly used for potato soil irrigation treatment.

[0062] Example 7 A method for preventing potato scab involves irrigating the soil in which potatoes are planted with the culture solution obtained in Example 5. The application period begins one month after potato emergence (seedling stage), and the solution is applied once a week for four consecutive weeks, with each application amount being 50 mL per plant.

[0063] Example 8 A method for preventing potato scab involves irrigating the soil in which potatoes are planted with the culture solution obtained in Example 6. The application period begins one month after potato emergence (seedling stage), and the solution is applied once a week for four consecutive weeks, with each application amount being 150 mL per plant.

[0064] Example 9 Cold-resistant *Streptomyces KY5* exhibits varying degrees of inhibitory effects against five *Streptomyces* species that cause potato scab. The cold-resistant *Bacillus KY5* was designed with five different pathogens ( S. scabiei , S. europaeiscabiei , S. luridiscabiei , S. griseus ,S. thermocarboxydus , among them S. yes Streptomyces (abbreviation) for a confrontation experiment with a target: The antagonistic effect of antagonistic bacteria on pathogens was determined using the Oxford cup method. Melted and cooled culture medium to 45°C was poured into sterile petri dishes, 20 mL per dish, and allowed to solidify completely. Single colonies of pathogens cultured for 7 days were scraped using a sterile inoculation loop and streaked onto the surface of the plate, then allowed to air dry for 10 min. Each treatment was repeated in triplicate. Using sterile forceps, sterilized Oxford cups were gently placed on the plates. 200 μL of filtered and sterilized antagonistic bacterial fermentation broth (treatment group, labeled KY5 group; the concentration of antagonistic bacterial fermentation broth was the same in all groups), sterile water (blank control, labeled CK sterile water group), and sterile LB liquid medium without antagonistic bacteria inoculation (medium control, labeled CKLB group) were added to each Oxford cup to eliminate interference from the LB liquid medium. The plates were inverted and incubated at 28°C for 7 days. The diameter of the inhibition zone around the Oxford cup was observed and measured; a larger inhibition zone indicated stronger antibacterial activity of the antagonistic bacteria.

[0065] The results are as follows: Figures 6 - 10 As shown, it can be seen that the cold-resistant Bacillus KY5 of the present invention has different degrees of inhibitory effect on five Streptomyces pathogens that cause potato scab.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cold-resistant *Bacillus subtilis* KY5, characterized in that, The cold-resistant *Ligusticum striatum* KY5 is classified as *Ligusticum striatum*. Peribacillus frigoritolerans It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36309.

2. A culture medium, characterized in that, It was obtained by liquid culture of the cold-resistant Bacillus subtilis KY5 as described in claim 1.

3. The culture medium according to claim 2, characterized in that, The viable count of *Ligusticum striatum* KY5 in the culture medium was 1 × 10⁻⁶. 8 CFU / mL or higher.

4. The culture medium according to claim 2, characterized in that, The culture medium for liquid culture includes LB liquid medium; the LB liquid medium includes peptone at a final concentration of 10 g / L, yeast extract at a final concentration of 5 g / L, and NaCl at a final concentration of 10 g / L; the pH of the LB liquid medium is 7.0~7.

2.

5. The culture medium according to claim 2, characterized in that, The liquid culture conditions are 25-30℃, 160-200rpm shaking culture for 24-50 hours.

6. The use of the cold-resistant Bacillus KY5 of claim 1 or the culture medium of any one of claims 2 to 5 in inhibiting the pathogen of potato scab.

7. The application according to claim 6, characterized in that, The pathogen causing potato scab includes Streptomyces scabiei , Streptomyces europaeiscabiei , Streptomyces luridiscabiei , Streptomyces griseus and / or Streptomyces thermocarboxydus .

8. The application of the cold-resistant Bacillus KY5 of claim 1 or the culture medium of any one of claims 2 to 5 in the prevention and control of potato scab.

9. A method for preventing and controlling potato scab, characterized in that, The culture solution described in claim 2 is poured into the soil in which potatoes are grown.

10. The method according to claim 9, characterized in that, Begin watering one month after potato seedlings emerge, once a week for a total of four times. Each application should be at least 50 mL per seedling.

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