Yokenella regensburgei and application thereof to prevention and treatment of tobacco black shank

Abstract

The present application relates to a yokenella strain and its application for preventing and treating tobacco black shank disease, the strain number of the yokenella is 733, the species preservation number of the yokenella is CGMCC No.38056.The yokenella 733 is used for preventing and treating tobacco black shank disease.The yokenella 733 of the present application has good biocontrol potential for tobacco black shank disease, and provides a new strain resource for using yokenella to prevent and treat tobacco black shank disease in production; the biocontrol agent of the yokenella 733 has a greenhouse potting test prevention effect of 76.2% on tobacco black shank disease, and a field test prevention effect of 80.5%, the prevention effect is remarkable, can reduce the pesticide use amount, effectively alleviate the environmental pollution caused by the use of chemical pesticides, and meets the current environment-friendly green agricultural development direction.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to a strain of Yorkshire and its application in the control of tobacco black shank disease. Background Technology

[0002] Tobacco black shank is caused by Phytophthora indicum (Tobacco black shank). Phytophthora nicotianae Tobacco black shank is a devastating soil-borne root and stem disease that severely impacts tobacco yield and quality. This pathogen can survive in the soil for years, accumulating in its effects, and spreads through rainwater, irrigation water, and agricultural operations, causing infection and disease throughout the entire tobacco growth cycle. Currently, tobacco black shank is prevalent in major tobacco-producing areas such as Chongqing, Sichuan, Guizhou, Yunnan, Henan, and Shandong, with an average incidence rate of around 10% in some areas and exceeding 75% in severely affected fields, even leading to total crop failure. In recent years, with the extension of continuous cropping years in tobacco cultivation, the reduction of varietal genetic diversity, and the increasing intensification of planting areas, the prevalence of tobacco black shank in core production areas has become increasingly severe, with a significant upward trend in field infection rates and disease severity, making it one of the major biological disasters restricting the sustainable development of the tobacco industry.

[0003] In current tobacco production, agricultural, chemical, and biological control methods are mainly used to control tobacco black shank. Among these, chemical control remains the primary method. Chemical control refers to the application of chemical agents to control plant diseases, typically using agents such as metalaxyl, fluopyram, and cymoxanil hydrochloride to control tobacco black shank. Although metalaxyl and other agents show significant initial efficacy, long-term single application easily induces drug resistance in the pathogen, leading to a gradual decline in efficacy and causing problems such as soil microbial community imbalance and environmental pollution. With the urgent need for green control technologies in modern agriculture, biological control, due to its advantages such as long-lasting effectiveness, environmental friendliness, and no pesticide residues, has gradually become a research hotspot in tobacco disease control. Studies have shown that screening and creating highly effective biocontrol agents is one of the important ways to achieve green control of tobacco black shank. Some scholars have isolated Bacillus hygroscopicus (Bacillus altissima) from tobacco rhizosphere soil. Bacillus altituudinis CY1, after fermentation optimization, achieved an inhibition rate of 68.6% against Tobacco Black Shank Bacterium; an endophytic short-spore bacillus screened from healthy tobacco plants ( Bacillus punilus Y4 showed control efficacy of 76.16% and 54.70% against tobacco black shank in greenhouses and field, respectively; other studies have shown that Bacillus subtilis ( Bacillus subtilis ) and Pseudomonas putida ( Pseudomonas putida It also shows good biocontrol potential.

[0004] However, while biocontrol bacteria offer advantages such as environmental friendliness, the stability of their field efficacy remains a challenge due to differences in soil ecology and planting systems across different tobacco-growing regions. Therefore, systematically screening for regionally adaptable native functional microbial strains will provide core germplasm resources for optimizing the biological control system of tobacco black shank, and is of great significance for promoting the green and sustainable development of tobacco.

[0005] Therefore, it is necessary to improve one or more of the problems existing in the above-mentioned related technical solutions.

[0006] It should be noted that this section is intended to provide background or context for the technical solutions of the invention as set forth in the claims. The description herein does not imply acceptance as prior art simply because it is included in this section. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Yorkshire and its application in the prevention and control of tobacco black shank disease, thereby at least partially solving one or more problems caused by the limitations and defects of related technologies.

[0008] In a first aspect, the present invention provides a Yorkshire strain, wherein the strain number of the Yorkshire strain is 733 and the strain preservation number of the Yorkshire strain is CGMCC No. 38056.

[0009] Secondly, the present invention provides a biological agent, wherein the active ingredient of the biological agent is the aforementioned Yorkshire bacteria.

[0010] Thirdly, the present invention provides an application of Yorkshire bacteria, specifically Yorkshire 733, for the prevention and treatment of tobacco black shank disease.

[0011] Fourthly, the present invention provides a method for preventing and treating tobacco black shank disease, the method comprising the following steps: S1, the biological agent is diluted in water to obtain a biological solution; S2, in the early stage of tobacco transplanting, this biological bacterial solution is applied to control tobacco black shank disease.

[0012] In this invention, the concentration of the biological bacterial solution is 1.5 × 10⁻⁶. 7 ~1.5×10 8 CFU / mL, for example 1.5 × 10⁻⁶ 7 1×10 8 1.5×10 8 CFU / mL, etc.

[0013] In this invention, in step S1, after dilution, the mass of the biological agent is 0.01~0.1 g per 1 mL of water, for example, 0.05 g, 0.09 g, etc. The biological agent solution can be diluted with sterile water or clean water.

[0014] In this invention, in step S2, the roots of the tobacco plants are irrigated with a biological bacterial solution on the day of transplanting, with a dosage of 20-30 mL / plant, such as 22 mL / plant, 25 mL / plant, 28 mL / plant, etc.

[0015] In this invention, in S2, root irrigation is carried out 10 to 30 days after the tobacco seedlings are transplanted, with a dosage of 150 to 300 mL / plant. For example, on the 12th, 15th, 20th, and 25th days, the dosage can be 200 mL / plant, 250 mL / plant, etc.

[0016] The technical solution provided by this invention may include the following beneficial effects: Yorkshire 733 of this invention has good biocontrol potential against tobacco black shank, providing a new strain resource for the use of Yorkshire in production to control tobacco black shank. The Yorkshire 733 biological agent showed a control efficacy of 76.2% in greenhouse pot experiments and 80.5% in field experiments against tobacco black shank, demonstrating significant efficacy. It can reduce the amount of pesticides used and effectively alleviate the environmental pollution caused by the use of chemical pesticides, which is in line with the current development direction of environmentally friendly green agriculture. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It is obvious that the drawings described below are merely some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0018] Figure 1 This invention demonstrates the inhibitory effect of Yorkshire 733 on Phytophthora tobaccoii; where A is the Phytophthora tobaccoii control without Yorkshire 733 inoculation; and B is the inhibitory effect of Yorkshire 733 inoculation on Phytophthora tobaccoii. Figure 2 The colony morphology of Yorkshire 733 on LB medium is shown in this invention; Figure 3 This invention presents a phylogenetic tree of Yorkshire 733 based on 16S rDNA. Figure 4 The greenhouse control effect of Yorkshire 733 on tobacco black shank is shown in the present invention (A: Yorkshire 733 inoculum treatment, B: metalaxyl-mancozeb powder 600 times dilution treatment, C: sterile water treatment). Detailed Implementation

[0019] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, they are provided so that the invention will be more comprehensive and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0020] Furthermore, the accompanying drawings are merely illustrative diagrams of embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0021] This invention provides a strain of Yorkshire and its application in the prevention and control of tobacco black shank disease. The strain is Yorkshire (… Yokenella Strain 733 was deposited on April 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38056, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain grows on LB agar plates, producing round, smooth, raised, pale yellow colonies with regular edges and a moist texture.

[0022] In this application, the tested pathogen is: Phytophthora indicum (… Phytophthora nicotianae The following were provided by the Yunnan Provincial Academy of Tobacco Agricultural Sciences: Metalaxyl-Manganese Zinc Powder was purchased from Limin Chemical Co., Ltd., and the DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0023] Example 1: Isolation and purification of Yorkshire 733 1. Methods for strain isolation and purification (1) Source of separation Yorkshire B. 733 involved in this invention was isolated from mixed rhizosphere soil samples of tobacco plants suffering from black shank disease in various regions of Eryuan County, Dali Prefecture, Yunnan Province.

[0024] (2) Time and place of separation It was isolated in November 2024 at the Microbiology Laboratory of the Center for Traditional Chinese Medicine Resources, School of Pharmacy, Dali University.

[0025] (3) Separation and purification steps The sample was obtained by separation using a dilution coating method, the specific method of which is as follows: ① Sample Collection: Three samples of rhizosphere soil from healthy tobacco plants and tobacco plants suffering from black shank disease were collected from tobacco fields in 14 areas of Dali Prefecture, Yunnan Province: Binchuan County (Shangang Village of Zhoucheng Town and Xionglumo Village of Qiaodian Town), Eryuan County (Taiping Village of Niujie Township, Guanyindeng of Qiaohou Township, Tunhu Village of Fengyu Town, Xinongcun Village of Sanying Town and Yonglian Village of Cibihu Town), Heqing County (Jiyi Village of Xiyi Town and Boluo Village of Gui Town), and Nanjian County (Wenqi Village of Nanjian Town, Dapingdi Community of Nanjian Town, Fada Village of Wuliangshan Town, Xiaoboluo Village of Yongcui Township and Akdi Village of Leqiu Township). The samples were packaged in plastic bags and placed in foam boxes containing biological ice packs, and transported back to the laboratory. Each plastic bag was labeled with the collection time and location. ② Separation and Preservation: Weigh 5 g of soil sample and place it in 45 mL of sterile water. Incubate at 28℃ and 180 r / min for 24 h with shaking. Prepare 10 g samples using a serial dilution method. 2 Up to 10 5 Dilute the solution by 1:1 and spread 0.2 mL onto solid LB agar plates, incubating at 28°C for 2–4 days. Select single colonies with significantly different morphologies and streak them onto LB agar plates for purification. Repeat the purification experiment three times. Suspend the purified strains in 50% glycerol and store them at -80°C for later use.

[0026] 2. Results Analysis Through the above separation and purification processes, a total of 64 bacterial isolates were screened from the collected rhizosphere soil.

[0027] Example 2: Screening of Yorkshire 733 for highly effective inhibition of Phytophthora tobaccoii 1. Preparation of bacterial suspensions from different test strains Under aseptic conditions, 64 bacterial isolates to be tested were streaked on LB solid medium and activated at 28°C for 2 days. During this period, the presence or absence of contamination was observed to obtain different bacterial strains to be tested.

[0028] 2. Detection of the inhibitory effects of different bacterial strains on Phytophthora tobaccoii Under aseptic conditions, Phytophthora tobaccois was inoculated onto potato dextrose agar (PDA) medium and cultured at 28°C for 7 days. Circular mycelial blocks with a diameter of 5 mm were then taken using a punch and inoculated into the center of a new PDA medium. 50 μL of the test bacterial solution was then streaked parallel to each other at a distance of 30 mm from the mycelial block. After culturing at 28°C for 3–5 days, the radius of the inhibition zone was observed and recorded, and the inhibition rate was calculated. Each treatment was repeated three times, and the strain with the highest inhibition rate was finally selected for subsequent experiments.

[0029] Antibacterial rate = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100% 3. Results Analysis Using the plate confrontation method, it was found that all 31 bacterial isolates had inhibitory effects on *Phytophthora nicotineae*. Compared with the *Phytophthora nicotineae* control (…),… Figure 1 A), among which bacterial strain numbered 733 showed the most significant antibacterial effect ( Figure 1 B), with an antibacterial rate of 72.29%.

[0030] Example 3: Identification of Yorkshire 733 1. Colony morphological characteristics analysis: This strain grew on LB agar (10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 15 g agar powder, dissolved in 1 L water). Single colonies of strain 733 were round, smooth, raised, pale yellow, with regular edges and a moist texture. Figure 2 ).

[0031] 2. Physiological and Biochemical Characteristic Analysis: The physiological and biochemical characteristics of strain 733 were detected and analyzed, as shown in Table 1. The results showed that strain 733 was negative for Gram staining and amylase test, but positive for VP test, citrate test, catalase reaction, methyl red reaction and nitrate reaction. Strain 733 was identified as Yorkshire (…). Yokenella ).

[0032] Table 1. Physiological and biochemical characteristics of strain 733

[0033] 3. Molecular biological characteristics: (1) Test methods Genomic DNA was extracted from strain 733 using a bacterial genomic DNA extraction kit. The 16S rRNA gene of strain 733 was amplified by PCR. Universal primers for the bacterial 16S rRNA gene were used. 27F: 5'-AGAGTTTGATCATGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'; Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0034] The PCR reaction system for 16S rRNA (20 μL) consisted of: 10 μL of 2×Taq Plus PCR Master Mix, 2 μL of DNA template, 1 μL of 27F, 1 μL of 1492R, and ddH2O to a final volume of 20 μL.

[0035] The PCR reaction conditions for the 16S rRNA gene were: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 60 s, for 30 cycles; 72℃ for 5 min, and stored at 4℃.

[0036] PCR amplification products were detected by electrophoresis using a 1% agarose gel, and then sent to the Chinese Academy of Agricultural Sciences for sequencing analysis. The sequenced sequences were aligned using BLAST in GenBank, and 16S rDNA with high homology was selected. A phylogenetic tree was constructed using the maximum likelihood method in MEGA-7 software.

[0037] (2) Results Analysis The 16S rDNA gene sequence of this strain is SEQ ID NO.1, and the determination results are as follows: like Figure 3 As shown, the phylogenetic tree constructed from the 16S rDNA of strain 733 and closely related bacteria indicates that strain 733 is related to Yorkshire strain OR373617.1_ Yokenella Based on the clustering of sp. and combined with morphological, physiological, biochemical, and molecular biological characteristics, strain 733 was identified as Yorkshire (…). Yokenella This strain was deposited at the China General Microbiological Culture Collection Center on April 7, 2026, with accession number CGMCC No. 38056.

[0038] Example 4: Preparation process of Yorkshire 733 biological agent 1. Yorkshire seed culture medium formulation 14 g soybean flour, 3 g sucrose, 2 g peptone, 4 g corn flour, 6 g calcium carbonate, 2 g fish meal, 0.5 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g potassium dihydrogen phosphate, 0.3 g dipotassium hydrogen phosphate, 0.2 g manganese sulfate, 0.3 g sodium chloride, 1 g defoamer, and deionized water to a final volume of 1000 mL. Separately prepare an aqueous solution of 0.25 g sodium hydroxide and add it before sterilizing the culture medium to adjust the pH to 6.0–8.0.

[0039] 2. Preparation method of Yorkshire seed culture Under aseptic conditions, Yorkshire bacteria were streaked onto LB solid medium and activated at 30°C for 24 h. Single colonies were then picked and inoculated into seed culture medium and cultured at 37°C with shaking at 200 r / min for 48 h to obtain Yorkshire 733 seed culture.

[0040] 3. Seed tank expansion The above primary seed culture was inoculated into a 100 L seed tank containing the above culture medium at a volume ratio of 1:120. The fermentation conditions in the seed tank are as follows: (1) Sterilization: The air sterilization conditions are 0.15 MPa, 123℃, 1 h; the actual sterilization conditions are 0.15 MPa, 123℃, 35 min.

[0041] (2) Inoculation: Inoculate when the temperature of the culture medium drops to 37°C after sterilization.

[0042] (3) Rotation speed: The rotation speed is 80 r / min for the first 6 hours of fermentation. After that, the rotation speed is increased by 10 r / min every 2 hours until it reaches 120 r / min. Then, the rotation speed is maintained until the end of fermentation.

[0043] (4) Ventilation rate: Maintain ventilation rate of 10 m³ / h for the first 8 hours of fermentation, and then increase ventilation rate by 1 m³ / h every 1 hour until the ventilation rate reaches 15 m³ / h and maintain it until the end of fermentation; maintain tank pressure of 0.06 MPa throughout the process.

[0044] (5) Temperature: 37℃±0.5℃.

[0045] (6) Fermentation time: 25-30 h.

[0046] 4. Expanding production in fermentation tanks Transfer the culture medium from the seed tank to the production fermenter containing the same culture medium at the required inoculum volume (which can be adjusted according to the fermenter volume). The fermentation conditions in the fermenter are the same as in the seed tank, except for the aeration rate: (1) Rotation speed: Same as the segmented variable speed fermentation of the seed tank (80 r / min for the first 6 hours, then increase by 10 r / min every 2 hours until it reaches 120 r / min and is maintained).

[0047] (2) Ventilation: Maintain ventilation at 30 m³ / h for the first 8 hours of fermentation, and then increase ventilation by 5 m³ / h every 1 hour until the ventilation reaches 50 m³ / h and is maintained until the end of fermentation.

[0048] (3) Tank pressure: 0.06 MPa.

[0049] (4) Temperature: 37℃±0.5℃.

[0050] (5) Fermentation time: 25-30 h.

[0051] 5. Preparation of microbial agents After fermentation, Yorkshire Bacterium 733 biological agent was obtained through concentration and spray drying, with an effective viable count of 1.5 × 10⁻⁶ cells. 9 CFU / g.

[0052] Example 5: Efficacy test of Yorkshire B 733 biological agent and metalaxyl-manganese zinc powder against tobacco black shank 1. Greenhouse potted plant control efficacy test (1) Greenhouse potted plant efficacy test method Healthy tobacco plants of the main tobacco variety Yunyan 87, with uniform growth at the 4-6 leaf stage, were selected in Dali, Yunnan Province. On the day of transplanting, the plants were inoculated with a suspension of Phytophthora tobacco spores using the inoculum-grain method. The tobacco plants were then transplanted into nutrient bags containing inoculum-grain, and 10 holes were punched in the stem of each plant using a 5mL disposable syringe needle. 25mL of a 1.37×10⁻⁶ concentration of the inoculum-grain solution was then injected. 8Each tobacco plant was treated with a root drench of CFU / mL Yorkshire Bacterium 733 biological agent and a 600-fold dilution of metalaxyl-mancozeb powder. Greenhouse conditions were 30℃ (14 h light) / 30℃ (10 h darkness) and 85% humidity. Sterile water was used as a control (CK). Each treatment consisted of 6 tobacco plants, and each treatment was replicated 3 times.

[0053] The incidence of black shank disease was investigated according to the national standard GB / T23222-2008, "Classification of Tobacco Diseases and Pests." The formula for calculation is as follows: Incidence rate = (Number of infected plants / Total number of plants surveyed) × 100% Disease index = [∑(each level of diseased plants × disease level value) / (total number of plants surveyed × highest level value)] × 100 Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100% (2) Results Analysis As shown in Table 2, the results of the greenhouse pot control efficacy test indicate that Yorkshire 733 has a good effect on the greenhouse control of tobacco black shank disease. Figure 4 A), Figure 4 Group B is treated with a 600-fold dilution of metalaxyl-manganese zinc powder. Figure 4 C represents the sterile water treatment group (CK). As shown in Table 2, the results indicate that the incidence and disease index of tobacco black shank were significantly lower in the treatment group after applying Yorkshire 733 inoculum compared to the sterile water treatment group. The control efficacy of Yorkshire 733 inoculum against tobacco black shank in greenhouse pot experiments was 76.2%.

[0054] Table 2. Efficacy of Yorkshire B 733 biological agent against black shank disease in greenhouse potted tobacco.

[0055] Note: Different letters indicate significant differences (P<0.05).

[0056] 2. Field efficacy trials (1) Field efficacy test method A field trial was conducted in the tobacco experimental field of Wanqiao Town, Dali City, Yunnan Province, with Yunyan 87 as the main tobacco variety. Ten days after transplanting, 150 mL of 733 biological inoculum solution (concentration 1.37 × 10⁻⁶) was applied to the seedlings. 8 The roots were drenched with a 600-fold dilution of metalaxyl-mancozeb powder (CFU / mL) every 20 days for three consecutive treatments. Twenty days after the last treatment, the incidence of tobacco black shank was observed and recorded. Water was used as a control (CK). Each treatment consisted of 100 tobacco plants, and each treatment was replicated three times. In the field efficacy test, the biological agent was diluted with water to obtain the required concentration of biological solution.

[0057] The incidence of black shank disease was investigated according to the national standard GB / T23222-2008, "Classification of Tobacco Diseases and Pests." The formula for calculation is as follows: Incidence rate = (Number of infected plants / Total number of plants surveyed) × 100% Disease index = [∑(each level of diseased plants × disease level value) / (total number of plants surveyed × highest level value)] × 100 Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100% (2) Results Analysis As shown in Table 3, the field efficacy results indicate that both 733 biological fungicide and metalaxyl-mancozeb powder significantly inhibited tobacco black shank disease. When 733 biological fungicide was used alone, its field efficacy against black shank disease reached 80.5%; when metalaxyl-mancozeb powder was applied alone, its field efficacy against black shank disease reached 87.4%. This demonstrates that 733 biological fungicide can effectively inhibit the occurrence of black shank disease in tobacco fields, although its effect is slightly lower than that of metalaxyl-mancozeb powder, and it can effectively reduce the amount of pesticides used in the field.

[0058] Table 3. Results of the control efficacy of different formulations against black shank disease in field tobacco.

[0059] Note: Different letters indicate significant differences (P<0.05).

[0060] In summary, Yorkshire B. 733, screened in this invention, has good biocontrol potential against tobacco black shank, providing a new strain resource for the control of tobacco black shank using Yorkshire B. The Yorkshire B. 733 biological agent showed a control efficacy of 76.2% in greenhouse pot experiments and 80.5% in field experiments against tobacco black shank, demonstrating significant efficacy. This can reduce pesticide use and effectively alleviate environmental pollution caused by chemical pesticides, aligning with the current trend of environmentally friendly green agriculture.

[0061] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0062] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A strain of Yorkshire, characterized in that, The strain number of the Yorkshire bacteria is 733, and the strain preservation number of the Yorkshire bacteria is CGMCC No. 38056.

2. A biological agent, characterized in that, The active ingredient of the biological agent is Yorkshire as described in claim 1.

3. The method for preparing the biological agent as described in claim 2, characterized in that, The bio-initiator was fermented using a segmented variable-speed fermentation process for 25–30 hours. After the fermentation was completed, it was concentrated and spray-dried to obtain Yorkshire 733 bio-initiator.

4. The application of Yorkshire as described in claim 1, characterized in that, Yorkshire 733 was used to prevent and treat tobacco black shank disease.

5. A method for preventing and treating tobacco black shank disease, characterized in that, The method includes the following steps: S1, the biological agent described in claim 2 is added to water for dilution to obtain a biological solution; S2, in the early stage of tobacco transplanting, this biological bacterial solution is applied to control tobacco black shank disease.

6. The method according to claim 5, characterized in that, The concentration of the biological bacterial solution is 1.5 × 10⁻⁶. 7 ~1.5×10 8 CFU / mL.

7. The method according to claim 5, characterized in that, In S2, on the day of tobacco transplanting, the roots are irrigated with biological bacterial solution at a rate of 20-30 mL per plant.

8. The method according to claim 5, characterized in that, In S2, root irrigation should be carried out 10 to 30 days after the tobacco seedlings are transplanted, with a dosage of 150 to 300 mL per seedling.

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