A strain of *Streptomyces niger* for the control of clubroot disease in cruciferous plants, its screening method, application, and fermentation broth of derived strains.

Abstract

This invention relates to the field of Streptomyces technology, and discloses a *Streptomyces nigricans* strain for controlling clubroot disease in cruciferous plants in the field, its screening method, and the application of fermentation broth from derived strains. The *Streptomyces nigricans* strain, named X216, was isolated and screened from soil in cruciferous vegetable fields affected by clubroot disease and deposited at the China Center for Type Culture Collection (CCTCC) on May 10, 2023, with accession number CCTCC No: M2023723. This invention utilizes *Streptomyces nigricans*, an antagonistic strain against indicator fungi, screened from rapeseed fields affected by clubroot disease. Compared to existing chemical pesticides, it effectively increases the lethality of dormant *Plasmodiophora brassicae* spores and reduces the root hair infection rate of dormant *Plasmodiophora brassicae* spores, demonstrating a significant effect on clubroot disease control. Furthermore, its direct application in natural field environments shows significant efficacy in controlling clubroot disease in cruciferous plants.

Description

Technical Field

[0001] This invention relates to the field of Streptomyces technology, and in particular to a Streptomyces niger for the prevention and control of clubroot disease in cruciferous plants in the field, a screening method thereof, and the application of fermentation broth of derived strains. Background Technology

[0002] Clubroot, caused by the obligate parasite *Platycodon brassicola*, is one of the most serious soil-borne diseases affecting cruciferous plants worldwide. It is estimated that global crop losses due to clubroot are 10-15%, with severely infected rapeseed crops potentially suffering yield losses of 30-100%. The main symptom associated with clubroot is the formation of root sulci on the roots of cruciferous plants, restricting the plant's absorption of water and nutrients needed for above-ground growth. This leads to stunted plant development, accelerated flowering, and premature senescence. In rapeseed, it reduces yield and oil quality. Ultimately, the root system of infected plants disintegrates, releasing dormant spores into the soil, where they can remain infectious for up to 20 years, making clubroot difficult to control.

[0003] Traditional methods of breeding disease-resistant varieties to control clubroot have limited effectiveness, and the control of clubroot still mainly relies on chemical pesticides. However, large-scale use of pesticides cannot eradicate clubroot. To reduce the environmental pollution caused by the use of chemical pesticides for clubroot control, researchers are continuously exploring biological resources for its control. Soil contains abundant actinomycetes, which are important microbial resources for the biological control of plant diseases. Antagonistic soil actinomycetes can be screened and identified using methods such as soil gradient dilution spreading, plate confrontation, morphological identification, and molecular biological identification.

[0004] Chinese Patent Application No. 202211726509.5 discloses a *Streptomyces chrysogenum* CD1-1 that can control clubroot disease and its application. The *Streptomyces chrysogenum* CD1-1 exhibits strong inhibitory effects on various plant pathogens on agar plates, and its fermentation broth metabolites show good control effects on clubroot disease. The germination inhibition rate of dormant spores of *Streptomyces chrysogenum* is 32.64%, and the root hair infection inhibition rate of Chinese cabbage is 10.93%. The control effect on potted Chinese cabbage treated with CD1-1 fermentation broth is 49.33%, and when treated 7 days in advance, the control effect on potted Chinese cabbage is 66.10%. However, this patent's research on the antagonistic actinomycetes for clubroot disease control stops at pot experiments and does not further determine the control effect of the target actinomycetes on clubroot disease in natural field environments. This is not conducive to the application and promotion of actinomycetes in the biological control of clubroot disease. Furthermore, the antagonistic actinomycetes may not have ideal colonization capabilities in the field environment, which would lead to unstable control effects on plant diseases. Summary of the Invention

[0005] To address the current lack of biocontrol techniques using Streptomyces for clubroot disease in cruciferous plants in field environments, this invention provides a *Streptomyces niger* strain for controlling clubroot disease in cruciferous plants in fields. This strain exhibits a high corrected lethality rate against dormant spores of *Platycodon brassicae*, demonstrating significant effectiveness in controlling clubroot disease in cruciferous plants. Furthermore, this invention provides a screening method for *Streptomyces niger* strains for controlling clubroot disease in cruciferous plants in fields, enabling the selection of strains suitable for use in controlling clubroot disease in field vegetable gardens. This invention relates to *Streptomyces niger*, a fungus used to control clubroot disease in cruciferous plants. It also provides an application of *Streptomyces niger* for controlling clubroot disease in field cruciferous plants, demonstrating significant effectiveness in this treatment. Furthermore, this invention provides a fermentation broth prepared from a strain of *Streptomyces niger* for controlling clubroot disease in field cruciferous plants, which exhibits a high corrected lethality rate against dormant spores of *Platycodon brassicae* and reduces the infection rate of dormant spores on the root hairs of cruciferous plants.

[0006] This invention is achieved through the following technical solution:

[0007] A strain of *Streptomyces melanosporofaciens*, used for the control of clubroot disease in cruciferous plants in the field, is described. This strain, named X216, was isolated and screened from soil in cruciferous vegetable fields where clubroot disease had occurred and was deposited at the China Center for Type Culture Collection (CCTCC) on May 10, 2023, with accession number CCTCC No.: M2023723. The control efficacy of strain X216 against clubroot disease in cruciferous plants under field conditions reached 30.84±3.91%–43.16±1.40%.

[0008] Preferably, the X216 strain has the morphological characteristics of Streptomyces, and its physiological and biochemical characteristics show that the X216 strain has the ability to produce amylase, cellulase and β-1,3-glucanase.

[0009] Preferably, the corrected lethality of *Plasmodiophora brassicae* dormant spores after treatment with the fermentation broth prepared using the X216 strain for several days was significantly higher than the lethality of spores treated with 75% chlorothalonil, 50% carbendazim, and 20% fluazinam during the same period.

[0010] Preferably, after treating cruciferous plants infected with dormant spores of *Plasmodiophora brassicae* using the fermentation broth prepared from the X216 strain for several days, the root hair infection rate of *Plasmodiophora brassicae* dormant spores on cruciferous plants was significantly lower than the root hair infection rate of spores treated with 75% chlorothalonil and 50% carbendazim during the same period.

[0011] A method for screening *Streptomyces nigricans* for controlling clubroot disease in cruciferous plants in the field, comprising the following steps:

[0012] 1) Add the collected soil to sterile water and mix well to a concentration of 10. -1 A suspension of g / mL was prepared and incubated with shaking; the suspension was then diluted with sterile water to a concentration of 10 g / mL. -2 g / mL, 10 -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 10 -6 A g / mL dilution solution was prepared; the dilution solution was evenly spread onto Gao's No. 1 medium, and several equal numbers of replicate samples were spread for each concentration. The samples were incubated at room temperature for several days to obtain colony 1.

[0013] 2) Count the colonies cultured in step 1), and separate each colony after counting on Gao's No. 1 medium, and purify them by streak plate method to obtain colony 2.

[0014] 3) The colony 2 obtained from step 2) was screened using pathogens with cell wall structures similar to those of *Plasmodiophora brassicae* as indicator bacteria, and strain X216 was selected.

[0015] The cotton wilt pathogen, *Fusarium oxysporum*, among the indicator bacteria, has a similar cell wall composition to *Plasmodiophora brassicae*. Based on the principle that similar cell wall structures influence interactions between strains, it can be deduced that the screened strains can effectively antagonize *Plasmodiophora brassicae* cells. In the antagonistic experiment between the test strains extracted from soil and the indicator bacteria on agar plates, the strain showing significant antagonistic effects was the X216 strain to be screened in this invention. It was found that *Phytophthora blight*, *Pseudomonas aeruginosa*, *Pseudomonas aeruginosa*, *Fusarium wilt*, *Fusarium oxysporum*, *Citrus sclerotium*, and *Citrus aurantium* all served as indicator bacteria.

[0016] Preferably, the screening in step 5) includes the following steps:

[0017] (1) Initial screening: The indicator bacteria are divided into initial screening indicator bacteria and secondary screening indicator bacteria. The initial screening indicator bacteria are activated, and the initial screening indicator bacteria cake is inoculated in the center of Gao's No. 1 culture medium plate. At the same time, the test bacteria isolated from the soil are inoculated around the initial screening indicator bacteria. Each test bacteria is repeated several times and cultured at a constant temperature. After several days, observe whether an inhibition zone is formed.

[0018] (2) Secondary screening: The antagonistic effect of the strains initially screened in step (1) on the secondary screening indicator bacteria was determined by the plate confrontation method; the secondary screening indicator bacteria cake was inoculated in the center of Gao's No. 1 medium, and the test strain was streaked on both sides of the secondary screening indicator bacteria cake. The experimental group was obtained by equidistant from the secondary screening indicator bacteria cake and the edge of the plate. The blank plate inoculated only with the secondary screening indicator bacteria cake was used as the control group; multiple experimental groups were set up.

[0019] (3) Replace the secondary screening indicator bacteria in step (2) with other secondary screening indicator bacteria, and then screen again. After all experimental groups are incubated at constant temperature for several days, calculate the inhibition rate.

[0020] Preferably, the indicator fungus is one or more of the following: cotton wilt fungus, strawberry blight fungus, strawberry root rot fungus, eggplant brown spot fungus, pepper wilt fungus, water chestnut Fusarium, citrus scab fungus, and corn leaf blight fungus.

[0021] The application of *Streptomyces niger* prepared by the above-mentioned screening method for controlling clubroot disease in field-grown cruciferous plants, or *Streptomyces niger* prepared by the above-mentioned screening method for controlling clubroot disease in field-grown cruciferous plants, in the control of clubroot disease in field-grown cruciferous plants.

[0022] A fermentation broth prepared from a strain of *Streptomyces nigricans* prepared using the above-described screening method for *Streptomyces nigricans* used to control clubroot disease in field-grown cruciferous plants, or using the above-described screening method for *Streptomyces nigricans* used to control clubroot disease in field-grown cruciferous plants.

[0023] The beneficial effects of this invention are:

[0024] (1) This invention uses Streptomyces nigricans, which is antagonistic to the indicator fungus, screened from rapeseed fields where clubroot disease has occurred. Compared with existing chemical pesticides, it effectively increases the mortality rate of dormant spores of Clubroot and reduces the infection rate of dormant spores of Clubroot on the root hairs of cruciferous plants, thus playing a significant role in the prevention and control of clubroot disease. Furthermore, it can be directly applied to the natural environment in the field to play a significant role in the prevention and control of clubroot disease in cruciferous plants.

[0025] (2) The *Streptomyces niger* used in this invention is more environmentally friendly than existing chemical pesticides, thus reducing the environmental pollution caused by chemical pesticides penetrating into the soil.

[0026] (3) The *Streptomyces niger* strain of the present invention is derived from rapeseed field soil, which is easy to obtain, easy to replicate and proliferate, low in cost, and simple in process, which is conducive to large-scale industrial promotion. Attached Figure Description

[0027] Figure 1The colony characteristics and microstructure of strain X216 are shown below: A, B: front and back of the colony; C: colony morphology on Gao's No. 1 medium; F: colony morphology on PDA medium; D: mycelial morphology; E: spore hyphae.

[0028] Figure 2 Results of detection of amylase (A), cellulase (B), and β-1,3-glucanase (C) produced by strain X216.

[0029] Figure 3 The lethality of strain X216 and different agents on clubroot dormant spores was investigated.

[0030] Figure 4 The greenhouse control effects of strain X216 and different agents on clubroot disease in rapeseed were investigated. A: CK; B: Original fermentation broth of strain X216; C: Fermentation broth of strain X216 diluted 2 times; D: Fermentation broth of strain X216 diluted 5 times; E: 100g / L cyazofamid SC diluted 1000 times; F: 75% chlorothalonil WP diluted 500 times; G: 50% carbendazim WP diluted 600 times; H: Quicklime as a percentage of soil 1.2g / kg.

[0031] Figure 5 The results of field trials of strain X216 and different agents for the control of clubroot disease in rapeseed are shown. A: CK; B: Original fermentation broth of strain X216; C: Fermentation broth of strain X216 diluted 2 times; D: Fermentation broth of strain X216 diluted 5 times; E: 100g / L cyazofamid SC diluted 1000 times; F: 75% chlorothalonil WP diluted 500 times; G: 50% carbendazim WP diluted 600 times; H: Quicklime content in soil is 1.2g / kg. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention. The terms "comprising," "including," or any other variations thereof used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not explicitly listed or elements inherent to such a composition, step, method, article, or apparatus. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. The experimental materials used in the embodiments and comparative examples of the present invention are all commercially available products.

[0033] Example 1

[0034] A method for screening *Streptomyces nigricans* for controlling clubroot disease in cruciferous plants in the field, comprising the following steps:

[0035] Initial screening: Using a hole punch (d=5mm), holes were made in the activated cotton wilt pathogen agar plates. The mycelial cake of cotton wilt pathogen was inoculated in the center of the Gao Shi No. 1 agar medium plate, and different microorganisms isolated from the soil were symmetrically inoculated around the mycelial cake. Each strain was replicated 3 times. The plates were inverted and incubated in a constant temperature incubator at 28℃. After 5 days, the presence of an inhibition zone was observed.

[0036] Secondary screening: The antagonistic effects of the initially screened strains against *Fusarium wilt*, *Phytophthora blight*, *Fungiella root rot*, *Brachystomata variegata*, *Fusarium wilt*, *Fusarium oxysporum*, *Fusarium solani*, *Citrus medica*, and *Heliotropium indicum* were determined using the plate confrontation method. Pathogen fungal discs (d = 5 mm) were inoculated in the center of Gao's No. 1 agar medium. The test strains were streaked on both sides of the pathogen fungal discs, equidistant from the edge of the plate. Plates inoculated only with pathogen fungal discs served as controls. Each treatment was replicated in six places. After incubation at 28°C upside down for 7 days, the inhibition rate was calculated. The strain with strong antagonistic effect obtained by isolation and screening was named X216. Its inhibitory effects on cotton wilt fungus, strawberry blight fungus, strawberry root rot fungus, eggplant brown spot fungus, pepper wilt fungus, wild rice Fusarium, citrus scab fungus, and corn leaf blight fungus are shown in Table 1. The X216 strain was obtained.

[0037] like Figure 1 As shown, *Streptomyces nigricans* strain X216, isolated from soil infested with rapeseed clubroot disease in Xidu Town, Hengyang County, Hengyang City, Hunan Province, exhibited a distinct radial colony pattern when cultured on Gao's No. 1 solid medium at 28°C. Initially, the colonies were white and nearly circular, growing slowly. The hyphae grew radially on the solid medium, forming dense and compact colonies that were slightly raised and firm. Later, the center of the colony gradually turned black, producing a pale yellow soluble pigment over time. On PDA medium, the growth of the strain differed significantly from that on Gao's No. 1 medium. The colony growth rate was slightly faster than on Gao's No. 1 medium. Initially, the colonies were pale yellow and wrinkled, then dense white or pale yellow hyphae grew on the wrinkles, and the colonies were clearly raised. Later, the colonies turned black and produced yellow pigment, staining the medium pale yellow. When the coverslips after the culture were used for microscopic observation, the mycelia of this strain grew intertwined and densely connected, without septa, with a diameter of 0.5-1.1 μm, and the spore filaments were curved.

[0038] Table 1. Inhibitory effects of strain X216 against different tested pathogens

[0039] Table 1 Inhibition rate of X216 strain against different pathogens

[0040]

[0041] A strain with strong antagonistic effects, isolated and screened, was named X216. Its inhibitory effects on *Fusarium wilt* of cotton, *Phytophthora blight* of strawberry, *Pseudomonas aeruginosa* of strawberry root rot, *Fusarium wilt* of eggplant, *Fusarium wilt* of pepper, *Fusarium oxysporum* of wild rice, *Fusarium solani* of citrus, and *Leptochloa crus-galli* of corn are shown in Table 1. The inhibition rates of strain X216 against these eight pathogens ranged from 51.87% to 71.19%, and its inhibition rate against *Fusarium wilt* of cotton, which has a cell wall composition similar to that of *Plasmodiophora stenophylla* dormant spores, was (63.84±1.35)%.

[0042] Comparative Example 1

[0043] One chemical pesticide used to control clubroot disease in cruciferous plants in the field is 100 g / L cypermethrin.

[0044] Comparative Example 2

[0045] One chemical pesticide used to control clubroot disease in cruciferous plants in the field is 20% fluazinam.

[0046] Comparative Example 3

[0047] One chemical pesticide used to control clubroot disease in cruciferous plants in the field is 75% chlorothalonil.

[0048] Comparative Example 4

[0049] One chemical pesticide used to control clubroot disease in cruciferous plants in the field is 50% carbendazim.

[0050] Comparative Example 5

[0051] Quicklime is a chemical pesticide used to control clubroot disease in cruciferous plants in the field.

[0052] control group

[0053] Sterile water.

[0054] 1. The efficacy of X216 strain, cyazofamid, fluazinam, chlorothalonil, and carbendazim against *Plasmodiophora brassicae* dormant spores was tested using a suspension of *Plasmodiophora brassicae* dormant spores:

[0055] Preparation of dormant spore suspension of *Cladosporium brassicae*: The clubroots of rapeseed stored at -20℃ were thawed and placed in the dark at 25℃ for 5 days to rot. The rhizomatous roots were crushed using a grinder, mixed with sterile water, and filtered through 8 layers of gauze. The filtrate was centrifuged at 500 rpm for 15 min, the precipitate was discarded, and the supernatant was collected. The supernatant was then centrifuged at 2500 rpm for 5 min in a 50 mL centrifuge tube, and the precipitate was collected. This process was repeated 3-4 times to obtain more dormant spores of *Rhizomatous*. The precipitate was disinfected with 2% chloramine-T for 20 min, then centrifuged at 3000 rpm for 5 min to collect the precipitate. The precipitate was resuspended in sterile water and centrifuged and washed 3 times. The precipitate was then resuspended in sterile water, and colistin sulfate, vancomycin hydrochloride, and cefotaxime sodium were added to achieve concentrations of 1, 1, and 6 μg / mL, respectively. The precipitate was placed in the dark at 24°C for 24 h, then centrifuged at 3000 rpm for 5 min to collect the precipitate. The precipitate was resuspended in sterile water, centrifuged at 3000 rpm for 5 min, and washed 3 times. The concentration of dormant spores was calculated using a hemocytometer. Finally, the dormant spore suspension was adjusted to 1×10⁻⁶ with sterile water. 8 Quantity / mL, store at 4℃ for later use.

[0056] Preparation of X216 strain fermentation broth: 150 mL of Gao's No. 1 liquid culture medium was placed in a 300 mL Erlenmeyer flask, and 15 pieces of X216 strain mycelium were inoculated. The culture was maintained at 28℃ and a shaking speed of 180 r / min for 7 days. Five experimental concentrations of the X216 strain fermentation broth were set up, and the concentrations of the sterile X216 strain fermentation broth were diluted with sterile water to 1, 2, 5, 10, and 20 times. The dormant spore suspension of *Plasmodiophora* was diluted with root exudate to a final concentration of 1 × 10⁻⁶. 7 100 g / L of cyazofamid, 20% fluazinam, 75% chlorothalonil, and 50% carbendazim were added to the dormant spore suspension at the lowest dosage specified in the instructions, with each treatment repeated 3 times. The spores were shaken to ensure uniformity daily and incubated in the dark at 25°C.

[0057] The activity of dormant spores was observed under a microscope at 7, 10, and 14 days. After comparison of procedures, 2% Evans blue solution was selected as the staining agent. The dormant spore suspension and 2% Evans blue solution were mixed thoroughly at a 2:1 volume ratio and stained at room temperature for 24 hours. 10 μL of the solution was pipetted onto a temporary slide for observation under an optical microscope. The activity of dormant spores was determined by the degree of staining; inactive dormant spores were stained dark blue, while active dormant spores were not stained blue. 100 dormant spores were observed each time, with three replicates.

[0058] Root hairs were observed under a microscope 3, 7, and 10 days after inoculation. Four seedling roots were taken from each treatment at each stage. The seedlings were first rinsed with sterile water to remove some spores attached to the roots, then stained with aniline blue solution (125 mg / L) prepared in 50% acetic acid for 1-2 minutes. The surface stain was then washed away with sterile water. Temporary slides were prepared, and the number of infected and uninfected root hairs was observed and counted under a microscope to calculate the root hair infection rate. 100 root hairs were observed each time, and the observations were repeated three times.

[0059] like Figure 3 As shown, after treatment with sterile fermentation broth of strain X216 and several agents, the mortality rate of dormant spores of *Plasmodiophora* significantly increased, and the mortality rate of dormant spores was positively correlated with time. Both the original X216 strain and fermentation broth diluted 2, 5, 10, and 20 times were lethal to dormant spores. After 14 days of treatment, the corrected lethality rate of the original X216 strain reached (56.59±1.97)%, which was not significantly different from the lethality rate of 50% carbendazim WP at the same time point, but significantly higher (P<0.05) than the corrected lethality rate of 75% chlorothalonil WP (51.03±0.80)% and 20% fluazinam SC (37.62±0.72)%. The corrected lethality rates of fermentation broth diluted 2 and 5 times after 14 days of treatment were 40-45%, while the lethality rates of fermentation broth diluted 10 and 20 times were approximately 30%. Among the nine treatments, 100 g / L cypermethrin SC had the strongest lethal effect on dormant spores, with a corrected lethality rate of (63.08 ± 2.28)% after 14 days of treatment.

[0060] 2. Greenhouse potted plant control efficacy test

[0061] The *Plasmodiophora brassicae* fungus was inoculated using the soil-inoculation method, resulting in a dormant spore count of 1.0 × 10⁻⁶ per gram of soil. 8 The soil moisture content should be such that it can be formed into a clump when squeezed but crumbles easily when pinched. It should be sealed at 25℃ for 48 hours before use. Treatment groups: Actinomycete fermentation broth diluted 1, 2, and 5 times; 100g / L cyazofamid SC diluted 1000, 1500, and 2000 times; 75% chlorothalonil WP diluted 500, 1000, and 1500 times; 50% carbendazim WP diluted 600, 800, and 1000 times; quicklime 1.2g, 1.4g, and 1.6g per kilogram of soil; the control group was a water control; a total of 16 groups were formed, with 6 pots per group and 10 rapeseed seedlings per pot. At sowing, 7 days after sowing, and 14 days after sowing, each pot was drenched with 100mL of the solution from each treatment group (except the quicklime group). The greenhouse temperature was 25℃, with 12 hours of light per day. The occurrence of clubroot disease was investigated 45 days after rapeseed emergence. The experimental data are shown in Table 2.

[0062] Table 2. Greenhouse control efficacy of strain X216 and different agents against clubroot disease in rapeseed.

[0063] Table 2 Control effect of different treatments against oilseed rapeclubroot in pot test

[0064]

[0065] Table 2 (continued)

[0066]

[0067] like Figure 4 As shown, the control efficacy of X216 strain fermentation broth and different pesticides against rapeseed clubroot disease was positively correlated with the root irrigation concentration. The control efficacy of X216 strain fermentation broth against rapeseed clubroot disease was 62.14%, which was not significantly different from the control efficacy of 100g / L cyazofamid SC diluted 1000 times (67.37%), but significantly higher (P<0.05) than the control efficacy of 75% chlorothalonil and 50% carbendazim WP against rapeseed clubroot disease (26.18% and 27.23%, respectively).

[0068] 3. Determination of the control effect of clubroot disease in natural field

[0069] In fields where clubroot disease had occurred for several years, a randomized block design experiment was conducted. Holes measuring 30×30 cm were dug on the ridges, with 15 rapeseed plants planted in each hole. Four holes were planted per row, with two rows per treatment, and the experiment was repeated three times. All agricultural operations were consistent. For the quicklime group, soil mixed with different concentrations was placed into the holes beforehand. For the other treatment groups, 500 mL of the solution from each treatment group was applied to each hole for root irrigation at sowing time and 14 days after sowing. Forty-five days after rapeseed emergence, after the control group developed symptoms, the incidence of clubroot disease in each group was investigated. The experimental data are shown in Table 3.

[0070] Table 3 shows the control effect of different treatments against clubroot disease in rapeseed in field test.

[0071]

[0072] like Figure 5As shown, the use of X216 strain fermentation broth in rapeseed fields can significantly reduce the disease index of clubroot disease in rapeseed fields. The field control effect of X216 strain fermentation broth on clubroot disease in rapeseed is 43.16%, which is comparable to the control effect of 100g / L cyazofamid SC diluted 2000 times, 75% chlorothalonil WP diluted 1000 times, and 50% carbendazim WP diluted 1000 times.

[0073] The above embodiments are merely examples of several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.

[0074] For those skilled in the art, various modifications and improvements can be made without departing from the concept of this invention, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of *Streptomyces niger* in the production of antibacterial agents, characterized in that, The *Streptomyces nigricans* strain was isolated and screened from rapeseed field soil infested with clubroot disease, named strain X216, and deposited at the China Center for Type Culture Collection (CCTCC) on May 17, 2023, with accession number CCTCC No: M2023723; the antibacterial agent includes one or more antibacterial agents for inhibiting one or more of the following: *Brachystomata glomerata*, *Fusarium oxysporum*, *Fusarium oxysporum*, and *Citrus sclerotium*, and the antibacterial effect reached (56.77±1.71)c~(66.31±2.23)b.

2. The application of *Streptomyces niger* according to claim 1 in the preparation of antibacterial agents, characterized in that, The X216 strain has the ability to produce amylase, cellulase and β-1,3-glucanase.

3. A bacteriostatic agent prepared using *Streptomyces niger*, characterized in that, The *Streptomyces nigricans* strain was isolated and screened from rapeseed field soil infested with clubroot disease. It was named strain X216 and deposited at the China Center for Type Culture Collection (CCTCC) on May 17, 2023, with accession number CCTCC No: M2023723. The antibacterial agent is the fermentation broth of the strain. The preparation process includes: filling 150 mL of Gao's No. 1 liquid culture medium into a 300 mL Erlenmeyer flask, inoculating 15 pieces of mycelial cake of strain X216, and culturing at 28℃ and shaking speed of 180 r / min for 7 days.

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