Bacillus velezensis DSSR-2, biocontrol inoculant and application of biocontrol inoculant

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses bacillus velezensis DSSR-2, a biocontrol microbial agent and application of the bacillus velezensis DSSR-2, the bacillus velezensis DSSR-2 is preserved in the China General Microbiological Culture Collection Center on July 8, 2024, the preservation number is CGMCC No.31207, and the bacillus velezensis DSSR-2 is classified and named as bacillus velezensis. The bacillus velezensis DSSR-2 provided by the invention has broad-spectrum antibacterial activity and can be used for inhibiting pathogenic bacteria of plant diseases, so that the plant diseases are prevented and treated, and meanwhile, the growth and development of plants are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Bacillus velezensis DSSR-2, a biocontrol agent and applications thereof. Background Art

[0002] Plant diseases caused by plant pathogens are one of the important factors affecting the improvement of crop yield and quality. Among them, soil-borne diseases and foliar diseases are two common types of diseases in the process of crop cultivation. Soil-borne diseases are diseases caused by pathogenic organisms in the soil (such as fungi, bacteria, etc.) or diseased residues in the soil. Pathogens survive and accumulate in the soil, invade the plant body through the root system or the base of the stem, and cause various diseases such as root rot, damping-off, wilt, etc. This type of disease is characterized by strong concealment, long disease cycle, and difficulty in prevention and control. Because pathogens are difficult to detect and monitor in the soil, they are often not discovered until the disease occurs on a large scale, which increases the difficulty of prevention and control. Foliar diseases are diseases caused by pathogens that infect plant leaves. Common ones include leaf spot, downy mildew, powdery mildew, etc. Pathogens typically spread through air currents, rain, or insects, invading the leaves through the stomata or epidermis. These pathogens cause leaf spotting, discoloration, and necrosis, impacting photosynthesis and transpiration, and ultimately, the growth and development of the plant. Leaf diseases are characterized by rapid spread, explosiveness, and a direct impact on plant growth. Once established, they can cause extensive leaf damage within a short period of time, reducing plant productivity.

[0003] Chemical pesticides have long played an irreplaceable role in preventing the occurrence and spread of crop diseases and stabilizing crop yields. However, the long-term, large-scale, high-dose, and irrational application of chemical pesticides has led to increasingly serious environmental pollution, food safety issues, and pathogen resistance. Growing awareness of environmental protection and a greater emphasis on personal health are prompting a search for effective crop disease control measures that are both environmentally and food-safe. Research and practice both domestically and internationally have demonstrated that the use of living microorganisms or their metabolites is an effective method for controlling plant diseases.

[0004] Using antagonistic microorganisms and other biological factors to inhibit pathogens is safe, environmentally friendly, and can reduce the use of chemical agents, which is friendly to the ecological environment. Therefore, it is necessary to explore biocontrol microorganisms in order to provide a green and efficient new means for plant disease control. Summary of the Invention

[0005] To explore a microorganism for plant disease control, the present invention provides a Bacillus Velez subtilis DSSR-2, a biocontrol agent, and its use. The Bacillus Velez subtilis DSSR-2 provided by the present invention has broad-spectrum antibacterial activity and can be used to inhibit plant disease pathogens, thereby controlling plant diseases while also promoting plant growth and development.

[0006] The present invention provides a Bacillus velezensis DSSR-2. The Bacillus velezensis DSSR-2 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on July 8, 2024, with a deposit number of CGMCC No. 31207 and a classification name of Bacillus velezensis.

[0007] The Bacillus Velezii DSSR-2 provided by the present invention has a broad-spectrum antibacterial property and is used to inhibit plant disease pathogens, thereby preventing and controlling plant diseases and promoting plant growth and development.

[0008] The present invention also provides a biocontrol agent, which contains the Bacillus Velezii DSSR-2 as the only effective ingredient.

[0009] Furthermore, the concentration of live bacteria of Bacillus velezensis DSSR-2 in the biocontrol agent is 1×10 6 CFU / mL~1×10 13 CFU / mL.

[0010] Furthermore, the concentration of live bacteria of Bacillus velezensis DSSR-2 in the biocontrol agent is 1×10 7 CFU / mL.

[0011] The present invention also provides a use of the Bacillus Velez subtilis DSSR-2 or the biocontrol agent in antibacterial treatment, wherein the antibacterial treatment is to inhibit at least one of the following pathogens: Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, and Botrytis cinerea.

[0012] The present invention also provides a use of the Bacillus Velezii DSSR-2 or the biocontrol agent in the prevention and treatment of diseases of traditional Chinese medicines, wherein the plant disease is caused by any one of the following pathogens:

[0013] Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, and Botrytis cinerea.

[0014] Furthermore, the plant disease is at least one of root rot, phytophthora, leaf spot, powdery mildew and gray mold; wherein, root rot and phytophthora are soil-borne diseases, and leaf spot, powdery mildew and gray mold are leaf diseases.

[0015] Furthermore, the root rot pathogen is any one of Fusarium oxysporum, Fusarium solani and Fusarium solani; the blight pathogen is Phytophthora; the leaf spot pathogen is at least one of Phytophthora multilocus, Alternaria alternata and Alternaria tenuis; the powdery mildew pathogen is Powdery mildew; and the gray mold pathogen is Botrytis cinerea.

[0016] Furthermore, the plant is a Chinese medicinal plant, a fruit plant or a vegetable plant.

[0017] Furthermore, the Chinese medicinal plant is at least one of Salvia miltiorrhiza, Scutellaria baicalensis, Schisandra chinensis and Panax notoginseng.

[0018] Furthermore, the fruit plant is at least one of strawberry, grape, apple, pear and watermelon.

[0019] Furthermore, the vegetable plant is at least one of water spinach, cucumber, pumpkin, pepper, eggplant, tomato and potato.

[0020] The present invention also provides an application of the Bacillus Velezii DSSR-2 or the biocontrol agent in promoting the growth and development of Chinese medicinal plants.

[0021] Furthermore, the Chinese medicinal plant is Salvia miltiorrhiza or Scutellaria baicalensis;

[0022] The Bacillus Velez DSSR-2 or biocontrol agent is used for increasing the fresh weight of the above-ground part and the fresh weight of the underground part of the Chinese medicinal plant.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The Bacillus Velezii DSSR-2 provided by the present invention has a broad spectrum of activity against eight pathogens, including Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, and Botrytis cinerea. It can also control soil-borne diseases such as root rot and phytophthora of Salvia miltiorrhiza, Scutellaria baicalensis, Schisandra chinensis, and Panax notoginseng. It can also control leaf diseases such as leaf spot, powdery mildew, and gray mold of these plants. It can also promote the growth and development of traditional Chinese medicines. This provides high-quality strain data for agricultural disease prevention and control.

[0025] Information on the deposit of biological materials

[0026] DSSR-2, referred to as Bacillus velezensis DSSR-2 in this application, was deposited in the General Microbiology Center of the China Culture Collection Administration on July 8, 2024, with the deposit number CGMCC No. 31207. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the classification name is Bacillus velezensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 The results are as follows: CK is the blank control; DSSR-2 is the color reaction result of DSSR-2 strain with Salkowski colorimetric solution.

[0029] Figure 2 This is the result of the determination of the siderophore production ability of strain DSSR-2.

[0030] Figure 3The results of enzyme production of strain DSSR-2 are shown in FIG. 1 . From left to right, the results of amylase production, protease production, cellulase production, chitinase production, and β-1,3 glucanase production are shown in FIG. 1 .

[0031] Figure 4 These are the test results of the phosphate solubility ability of strain DSSR-2; the left figure is the test results of the phosphate solubility of strain DSSR-2 for insoluble organic phosphorus; the right figure is the test results of the phosphate solubility of strain DSSR-2 for insoluble inorganic phosphorus.

[0032] Figure 5 To identify the morphological characteristics of strain DSSR-2;

[0033] In the figure, A is the colony plate image of strain DSSR-2;

[0034] B is the Gram staining result of strain DSSR-2.

[0035] Figure 6 is the phylogenetic tree of strain DSSR-2;

[0036] In the figure, A is the phylogenetic tree of strain DSSR-2 16S rDNA;

[0037] B is the phylogenetic tree of strain DSSR-2gyrB.

[0038] Figure 7 This is the potted plant efficacy test result of strain DSSR-2 against root rot of Salvia miltiorrhiza;

[0039] In the figure, A shows the appearance of a plant infected with root rot of Salvia miltiorrhiza;

[0040] B is the longitudinal section of the root of Salvia miltiorrhiza with root rot.

[0041] Figure 8 These are the results of potted plant efficacy tests using strain DSSR-2 to control Scutellaria baicalensis root rot.

[0042] Figure 9 These are the results of potted plant efficacy tests using strain DSSR-2 to control Schisandra root rot.

[0043] Figure 10 These are the results of potted plant efficacy tests using strain DSSR-2 to control Salvia miltiorrhiza leaf spot.

[0044] Figure 11 These are the results of potted plant efficacy tests of strain DSSR-2 against Schisandra leaf spot.

[0045] Figure 12 These are the results of a potted experiment on the effect of strain DSSR-2 on promoting the growth of Salvia miltiorrhiza.

[0046] Figure 13These are the results of a potted experiment on the growth promotion of Scutellaria baicalensis by strain DSSR-2.

[0047] Figure 14 This is the plate confrontation result of strain DSSR-2 against different pathogens. DETAILED DESCRIPTION

[0048] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0049] Example 1: Isolation and identification of Bacillus velez DSSR-2.

[0050] 1. Isolation of strain DSSR-2

[0051] The rhizosphere soil of healthy Chinese medicinal materials was collected. The root samples of the collected plants were taken out. The large pieces of soil attached to the roots were gently shaken off. The root tissue was placed in a triangular flask filled with an appropriate amount of sterile water and fully shaken. After standing and settling, 1 mL of supernatant was drawn out. Subsequently, 10, 10, and 20 dilutions were made in 10 mL centrifuge tubes with sterile water. 2 , 10 3 and 10 4 times of soil suspension. 3 and 10 4 100 μL of the soil dilution was applied to LB medium (the final concentration was 5×10 -6 g / L potassium dichromate) and replicated three times for each treatment. Incubate at 28°C until a moderate number of clearly visible single colonies grow. Single colonies are then picked and purified until the colony morphology is stable and consistent.

[0052] 2. Determination of biological characteristics of strain DSSR-2

[0053] 1. Determination of the ability of strains to secrete indoleacetic acid (IAA)

[0054] Methods: The DSSR-2 strain was inoculated into LB liquid medium containing 100 mg / L L-tryptophan and incubated in a shaker at 28°C and 200 rpm for 24 hours. 500 μL of the bacterial suspension was then dripped into a blank test tube and an equal amount of Salkowski colorimetric solution was added for color development (DSSR-2). A blank control (CK) was created by replacing the bacterial suspension with an equal amount of LB liquid medium. The test tube was placed at room temperature, protected from light, and observed after 30 minutes. A red color indicated IAA production.

[0055] Results: The supernatant of strain DSSR-2 mixed with Salkowski reagent turned redder than that of the control group ( Figure 1 The qualitative test result was positive, indicating that the strain DSSR-2 has the ability to secrete IAA, which is one of the reasons why the strain has a promoting effect on the growth of Chinese medicinal materials.

[0056] 2. Determination of siderophore production capacity

[0057] Method: The strain DSSR-2 was inoculated onto CAS solid medium and cultured at 28℃ for 7 days. If an orange halo appeared around the colony, it indicated that the strain had the ability to produce siderophores.

[0058] Results: After 7 days of culture on CAS plates, the strain DSSR-2 produced a yellow halo ( Figure 2 ), the qualitative test result was positive, indicating that the strain had the ability to produce siderophore.

[0059] 3. Determination of enzyme production capacity

[0060] Methods: The strain DSSR-2 was spotted on the specific detection medium of amylase, protease, cellulase, chitinase and β-1,3 glucanase respectively, and the enzyme production was determined.

[0061] Results: The qualitative test results were all positive ( Figure 3 ), indicating that the strain has the ability to produce amylase, protease, cellulase, chitinase and β-1,3 glucanase.

[0062] 4. Determination of phosphate solubility

[0063] Method: The strain DSSR-2 was inoculated on the culture medium containing insoluble organic phosphorus and insoluble inorganic phosphorus respectively. The strain with the ability to solubilize phosphorus would grow normally on the culture medium.

[0064] Results: The strain DSSR-2 could grow normally after 7 days of culture in the insoluble organic phosphorus medium and the insoluble inorganic phosphorus medium, and the qualitative test results were positive ( Figure 4 ), indicating that the strain has the ability to dissolve organic and inorganic phosphorus.

[0065] 3. Identification of strain DSSR-2

[0066] 1. Morphological identification

[0067] Morphological observation results showed that strain DSSR-2 formed white to light yellow colonies on NA medium, which were opaque and had irregular colony edges ( Figure 5 A). DSSR-2 strain is Gram-positive and the cells are rod-shaped ( Figure 5 B).

[0068] 2. Molecular Biology Identification

[0069] 16S rDNA of strain DSSR-2 ( Figure 6 A) and gyrB( Figure 6 B) The results of sequence phylogenetic analysis showed that the bacterium had a high homology with Bacillus velezensis.

[0070] Based on the above test results, combined with morphological and biological characteristics, strain DSSR-2 was identified as Bacillus Velez subsp. and named Bacillus Velez subsp. DSSR-2. The 16s rDNA sequence of Bacillus Velez subsp. DSSR-2 is shown in SEQ ID NO. 1, and the gyrB sequence is shown in SEQ ID NO. 2.

[0071] Its 16s rDNA sequence:

[0072] AAGTCGAGCGGACAGATGAGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAgGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACcTTGACGGTAcCTAACCAGAAAGcCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGgTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTtGGGTtAAGTCCCGCAaCGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCC。

[0073] Its gyrB sequence:

[0074] GCGGTCTTCACGGTGTAGGGGCATCTGTCGTAAACGCCTTGTCGACCACTCTTGACGTTACGGTTCATCGTGACGGAAAAATCCACTATCAGGCGTACGAGCGCGGTGTACCTGTGGCCGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTACGCACTTCGTT CCGGATCCGGAAATCTTCAAAGAAACAACCGTATACGACTATGATCTGCTTTCAAACCGTGTCCGGGAATTGGCCTTCCTGACAAAAGGCGTAAΑСATCACGATTGAAGACAAACGTGAAGGACAAGAACGGAAAAACGAGTAССΑСTACGAAGGCGGAATCAAAAGCTATGTT GAGTACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGATTTATATCGAAGGCGAGAAAGACGGCATAACGGTTGAAGTTGCATTGCAATACAACGACAGCTATACAAGCAATATTTATTCTTTCACGAATAATATCAACACATAC GAAGGCGGGACGCACGAAGCCGGATTTAAAACCGGTCTGACCCGTGTCATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAAATGATCCGAATTTAAGCGGGGATGATGTGAGAGAAGGGCTGACTGCCATTATTTCAATTAAGC ACCCTGATCCGCAATTCGAAGGGCAGACGAAAACCAAGCTCGGCAACTCCGAAGCGAGAACGATCACTGATACGCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCAGCCCGCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCGAAAAAAGCACGGGAATTGACCCGGCGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGCAA.

[0075] Example 2: Application of Bacillus velez DSSR-2 as a biocontrol bacterium in the prevention and treatment of root rot and phytophthora of traditional Chinese medicines.

[0076] 1. Preparation of pathogen spore mixed suspension and Bacillus Velez DSSR-2 fermentation broth

[0077] Preparation of pathogen spore suspension: Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora and other pathogens cultured at 25°C for 10 days were scraped from PDA plates and dispersed in sterile water to prepare spore suspensions. The spore concentration was adjusted to 1.0×10 7 CFU / mL.

[0078] Fusarium oxysporum is publicly described in the following documents:

[0079] Wang Li, Wang Yazhu, Zhang Ru, Wang Juanjuan, Han Lirong * .Screening and identification of biocontrol bacteria against Scutellaria baicalensis root rot and determination of their biocontrol effects. Journal of Shanxi Agricultural University (Natural Science Edition), 2024, 44(5):068.

[0080] Fusarium spp. is publicly described in the following documents:

[0081] Wu Hao, Yu Tingting, Yuan Zhichun, Miao Jianqiang, and Han Lirong * , Liu Xili. Identification of the pathogen causing Schisandra root rot in central China and screening of control agents. Acta Phytopathologica Sinica, 2023, 53(3): 491-497.

[0082] Phytophthora is publicly documented in the following documents:

[0083] Xie Ziyu, Guo Enhui, Sun Yubo, Han Lirong, Feng Juntao, Zhang Xing. Disease prevention and growth promotion of tomato and pepper by Bacillus subtilis B1409. Acta Phytophylacica Sinica, 2018, 45(3): 520-527.

[0084] Fusarium solani was purchased from China General Microorganism Culture Collection Center with the collection number CGMCC 3.17848.

[0085] Preparation of Bacillus Velez DSSR-2 fermentation broth: The DSSR-2 strain was cultured on LB medium plates at 28°C for 24 h. After colonies grew, 4 mm diameter bacterial cakes were taken and 5 bacterial cakes were inoculated into 200 mL of culture medium. The culture was shaken at 28°C and 200 rpm for 48 h. The concentration of the obtained bacterial solution was determined using a hemocytometer.

[0086] 2. Potted plant experiment design

[0087] Healthy seedlings of Salvia miltiorrhiza, Scutellaria baicalensis, Schisandra chinensis, and Panax notoginseng with consistent growth were selected. These seedlings were divided into a control group (inoculated with pathogens) and a test group (inoculated with pathogens and biocontrol bacteria), with 12 plants in each group. The seedlings were transplanted into small plastic pots filled with mixed nutrient soil (nutrient soil: vermiculite: perlite = 3g:1g:1g). The nutrient soil was purchased from Sanli Chemical Glass Station, Yangling, Xianyang City, Shaanxi Province, with the product number: SL-01055. Fourteen days after transplanting, the control and test groups were inoculated with a spore suspension of the aforementioned pathogen (1×10 7 CFU / mL, and the inoculation volume for each pot was 5 mL. Three days after the pathogen inoculation, each pot of the experimental group was inoculated with 5 mL of the fermentation liquid of Bacillus Velezii DSSR-2 (1×10 7 CFU / mL), and the control group was replaced by an equal amount of sterile water.

[0088] 3. Determination of control efficacy: After 28 days of cultivation, observe and count the incidence of disease, and calculate the control efficacy according to the following disease grading standards.

[0089] Root disease grading standards:

[0090] Level 0: There are no lesions on the stem base and main root of the plant;

[0091] Level 1: There are a few lesions on the stem base and main root;

[0092] Level 3: There are many lesions at the base of the stem or on the main root, and the area of lesions accounts for 1 / 4 to 1 / 2 of the total area of the stem and root;

[0093] Level 5: There are many large lesions on the stem base or main root, and the area of lesions accounts for 1 / 2 to 3 / 4 of the total area of the stem base and root;

[0094] Level 7: The lesions are continuous at the base of the stem or on the main root, forming a stem-circling phenomenon, but the root system is not dead;

[0095] Level 9: Root necrosis, above-ground part of the plant wilts or dies.

[0096] The calculation formula is as follows:

[0097]

[0098] Table 1 Potted control effect of Bacillus velezensis DSSR-2 on root diseases of Chinese medicinal materials (%)

[0099]

[0100]

[0101] Note: The data in the table are mean ± standard error.

[0102] The test results are shown in Table 1. The results showed that Bacillus Velezii DSSR-2 had an effect on Salvia miltiorrhiza ( Figure 7), Scutellaria baicalensis ( Figure 8 ), Schisandra chinensis ( Figure 9 ) and Panax notoginseng and other Chinese medicinal materials have good prevention and control effects on root rot and blight, and the prevention efficiency is above 60%.

[0103] Example 3: Application of Bacillus velezensis DSSR-2 as a biocontrol bacterium against leaf spot and gray mold of traditional Chinese medicine.

[0104] Healthy, uniformly growing, one-year-old Salvia miltiorrhiza, Scutellaria baicalensis, and Schisandra chinensis were selected. Two weeks after transplantation, the leaves were evenly sprayed with a fermentation broth of Bacillus velezensis DSSR-2 containing 0.025% Tween 20. A control group was sprayed with an equal amount of sterile water. After 4 hours, the leaf surfaces were allowed to dry, and then wounded with a sterile inoculating needle on the underside. The leaves were then inoculated with a fungal cake containing either Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, or Botrytis cinerea. The cakes were covered with cotton to retain moisture. Each treatment was transferred to a greenhouse for further incubation. Six days later, the leaf lesions were observed and counted. The diameter of the lesions was measured using the cross-hatch method, and the inhibition rate was calculated as follows:

[0105]

[0106] Corynespora polyhomogeneum is publicly documented in the following articles:

[0107] Wang Li, Wang Yazhu, Duan Weiwei, Zhang Ru, Lin Weifeng, Guo Xin, and Han Lirong * .Screening, identification and biocontrol effect determination of antagonistic bacteria against Salvia miltiorrhiza leaf spot. Chinese Medicinal Materials, DOI: 10.13863 / j.issn1001-4454.2025.04.00.

[0108] Botrytis cinerea is publicly documented in the following articles:

[0109] Guo Enhui, Song Shuang, Han Lirong, Feng Juntao, Zhang Xing. Screening of antibacterial activity of acetone extracts from 85 plants. Journal of Northwest Agriculture and Forestry University (Natural Science Edition), 2017, 12(45): 77-83.

[0110] Alternaria alternata was purchased from China General Microorganism Culture Collection Center with the accession number CGMCC 3.15535. Alternaria tenuis was purchased from China General Microorganism Culture Collection Center with the accession number CGMCC 3.15531.

[0111] Table 2 Determination of the protective effect of DSSR-2 fermentation liquid on Chinese medicinal materials leaf blight

[0112]

[0113] Note: The data in the table are mean ± standard error.

[0114] The test results are shown in Table 2. The results showed that Bacillus Velezii DSSR-2 had an effect on Salvia miltiorrhiza ( Figure 10 ), Schisandra chinensis ( Figure 11 ) and other traditional Chinese medicines such as Scutellaria baicalensis have good prevention and control effects on leaf spot diseases, and their prevention rates are all above 65%.

[0115] Example 4: Application of Bacillus velezensis DSSR-2 as a biocontrol bacterium against powdery mildew of traditional Chinese medicine

[0116] At the early stage of powdery mildew on Salvia miltiorrhiza, Scutellaria baicalensis and Schisandra chinensis, the fermentation liquid of Bacillus velezensis DSSR-2 was sprayed, and the control group was sprayed with the same amount of sterile water. Each treatment plot had an area of 15m 2 Each treatment was replicated three times with a 7-day interval, using 1 L of liquid each time. Disease was assessed three weeks after the final application. Five-point leaf sampling was used, with 12 plants surveyed at each point and six leaves per plant surveyed from top to bottom. Disease index was calculated based on lesion area, and control efficacy was statistically analyzed.

[0117] The disease is graded as follows:

[0118] Level 0: no lesions on leaves;

[0119] Level 1: The lesion area accounts for less than 10% of the total leaf area;

[0120] Level 3: The lesion area accounts for 10-25% of the total leaf area;

[0121] Level 5: The lesion area accounts for 25-50% of the total leaf area;

[0122] Level 7: The lesion area accounts for 50-85% of the total leaf area;

[0123] Level 9: The area of lesions accounts for 85% or more of the total leaf area, or the leaves are severely wrinkled due to the disease.

[0124] The calculation formula is as follows:

[0125]

[0126] Table 3 Field control effect of Bacillus Velez DSSR-2 on powdery mildew of Chinese medicinal materials (%)

[0127]

[0128] Note: “ / ” means no such operation.

[0129] The test results are shown in Table 3. Bacillus Velezii DSSR-2 has a good control effect on powdery mildew of Chinese medicinal materials such as Salvia miltiorrhiza, Schisandra chinensis and Scutellaria baicalensis, and its control efficiency is above 65%.

[0130] Example 4: Application of Bacillus Velezii DSSR-2 in promoting the growth of Chinese medicinal materials.

[0131] 1. Test method

[0132] Salvia miltiorrhiza and Scutellaria baicalensis seeds were germinated on sterilized filter paper and then transferred to nutrient pots (containing nutrient soil: vermiculite: perlite = 3g:1g:1g) for further cultivation. The cultivation conditions were: 28°C, 75% humidity, 16 hours of light and 8 hours of darkness. The nutrient soil was purchased from Sanli Chemical Glass Station, Yangling, Xianyang City, Shaanxi Province, with the item number: SL-01055. After 30 days of cultivation, the biocontrol bacteria were inoculated by root irrigation. Before root irrigation, the soil was moistened with a small amount of clean water (10mL). A 10-fold dilution of the fermentation liquid of Bacillus velezensis DSSR-2 was inoculated once in the rhizosphere (the concentration of viable bacteria was 10 5 CFU / mL~10 6 CFU / mL), 10 mL for each strain.

[0133] The control group was inoculated with an equal volume of 10-fold dilution of NB liquid medium using the same method. Thirty days after inoculation, the seedlings were carefully excavated and the soil around the roots was washed away. Plant height, leaf width, leaf length, number of leaves, and above- and below-ground fresh weight of the Salvia miltiorrhiza and Scutellaria baicalensis seedlings from each treatment were measured and investigated. The results of Salvia miltiorrhiza growth promotion are shown in Table 4, and those of Scutellaria baicalensis growth promotion are shown in Table 5.

[0134] Table 4 Analysis of the growth promotion of Salvia miltiorrhiza by Bacillus velezensis DSSR-2

[0135]

[0136]

[0137] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences at the 0.05 level tested by Duncan's new multiple range method.

[0138] Table 5 Analysis of the growth promotion of Scutellaria baicalensis by Bacillus velezensis DSSR-2

[0139] deal with Plant height / cm Leaf width / cm Leaf length / cm Number of blades Fresh weight of aboveground parts / g Fresh weight of underground parts / g control group 8.65±0.64b 1.45±0.21b 2.15±0.18b 14.33±0.85b 147.43±3.88b 47.55±7.33b experimental group 12.13±0.63a 2.67±0.24a 4.47±0.39a 25.67±1.02a 289.76±4.22a 97.62±2.78a

[0140] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences at the 0.05 level tested by Duncan's new multiple range method.

[0141] According to the data in Table 4 and Table 5, Bacillus Velezii DSSR-2 can promote the increase of plant height, leaf width, leaf length and leaf number of Salvia miltiorrhiza and Scutellaria baicalensis, and increase the fresh weight of aboveground part and underground part ( Figure 12 and Figure 13 ), indicating that Bacillus Velezii DSSR-2 has a good growth-promoting effect on Salvia miltiorrhiza and Scutellaria baicalensis.

[0142] Comparative Example 1: Study on the broad-spectrum antibacterial activity of commercially available Bacillus Velezii.

[0143] 1. Strain Materials: Bacillus Velez subsp. was purchased from Shanghai Jiachu Bioengineering Co., Ltd. in March 2024 and designated as Bacillus Velez commercial strain No. 1, brand: SHMCC, catalog number: SHMCC D24818. Bacillus Velez subsp. was purchased from Henan Industrial Microbial Strain Engineering Technology Research Center and designated as Bacillus Velez commercial strain No. 2, brand: BNCC, catalog number: BNCC364390. Laboratory-preserved Bacillus Velez DSSR-18. Bacillus Velez DSSR-2 obtained in Example 1 of the present invention.

[0144] 2. Study the antibacterial effect and broad-spectrum analysis of the above-mentioned strain materials on different pathogens.

[0145] The pathogens are Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, and Botrytis cinerea described in the present invention.

[0146] The inhibitory effect of the above strains on the above pathogens was studied using the plate standoff method. The results are shown in Table 6.

[0147] Table 6 Antibacterial broad spectrum of different Bacillus velezensis

[0148]

[0149]

[0150] Note: "+" indicates antibacterial effect on pathogens. The number of "+" indicates the antibacterial strength. The antibacterial effect of "+++" is three times that of "+". "-" indicates no antibacterial effect.

[0151] As shown in Table 6, the strain isolated and preserved by the present application, Bacillus velezensis DSSR-2, has a significant inhibitory effect on eight pathogens, including Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, and Botrytis cinerea, with a high broad-spectrum inhibitory effect. Although the other three strains of Bacillus velezensis also have a certain inhibitory effect on some pathogens, their broad-spectrum inhibitory effect is significantly worse than that of Bacillus velezensis DSSR-2, and their inhibitory effects are also unsatisfactory. It can be seen that the present invention provides a broad-spectrum antibacterial Bacillus Velezii DSSR-2, which can not only broadly inhibit a variety of plant disease pathogens, but also promote the growth and development of Chinese medicinal materials, which is of great significance for the cultivation of Chinese medicinal materials.

[0152] The results of the plate confrontation of the Bacillus velezensis DSSR-2 of the present invention against the above pathogens are shown in FIG. Figure 14 As shown. Figure 14 It can be intuitively observed that the Bacillus Velezii DSSR-2 of the present invention has a significant inhibitory effect on different pathogens, and the inhibition rate is greater than 80%.

[0153] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0154] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A Bacillus velezensis DSSR-2, characterized in that The Bacillus velezensis DSSR-2 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 8, 2024, with a deposit number of CGMCC No. 31207 and a classification name of Bacillus velezensis.

2. A biocontrol agent, characterized in that The biocontrol agent contains the Bacillus Velezii DSSR-2 described in claim 1 as the only active ingredient.

3. The biocontrol agent according to claim 2, characterized in that The concentration of live bacteria of Bacillus velezensis DSSR-2 in the biocontrol agent is 1×10 6 CFU / mL~1×10 13 CFU / mL.

4. The biocontrol agent according to claim 3, characterized in that The concentration of live bacteria of Bacillus velezensis DSSR-2 in the biocontrol agent is 1×10 7 CFU / mL.

5. Use of the Bacillus Velezii DSSR-2 according to claim 1 or the biocontrol agent according to any one of claims 2 to 4 in antibacterial treatment, characterized in that: The antibacterial activity is to inhibit at least one of the following pathogens: Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima and Botrytis cinerea.

6. Use of the Bacillus Velez DSSR-2 according to claim 1 or the biocontrol agent according to any one of claims 2 to 4 in controlling plant diseases, characterized in that: The plant disease is caused by any of the following pathogens: Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Phytophthora, Corynespora cassiicola, Alternaria alternata, Alternaria tenuissima, and Botrytis cinerea.

7. The use according to claim 6, characterized in that The plant disease is at least one of root rot, phytophthora, leaf spot, powdery mildew and gray mold.

8. The use according to claim 7, characterized in that The plants are Chinese medicinal plants, fruit plants or vegetable plants.

9. Use of the Bacillus Velezii DSSR-2 according to claim 1 or the biocontrol agent according to any one of claims 2 to 4 in promoting the growth and development of Chinese medicinal plants.

10. The use according to claim 9, characterized in that The Chinese medicinal plant is Salvia miltiorrhiza or Scutellaria baicalensis; The Bacillus Velez DSSR-2 or biocontrol agent is used for increasing the fresh weight of the above-ground part and the fresh weight of the underground part of the Chinese medicinal plant.

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