Pseudomonas sp. and its application in tobacco black shank disease prevention and treatment

Abstract

The application belongs to the technical field of microorganisms, and discloses a Pseudomonas donghuensis and application of the Pseudomonas donghuensis in tobacco black shank disease prevention and treatment. Pseudomonas donghuensis A Pseudomonas donghuensis (YM3-423) is isolated and screened from rhizosphere soil of healthy plants in a tobacco black shank disease heavy incidence plot, and the preservation number is CCTCC M 2025914. Phytophthora nicotianae The Pseudomonas donghuensis YM3-423 has significant contact inhibition and volatile fumigation inhibition dual bacteriostatic activities on tobacco black shank disease pathogen Phytophthora parasitica, and has high selective bacteriostasis selectivity on the Phytophthora parasitica; and has significant growth promotion effect on tobacco seedlings. The strain provided by the application can be developed into a safe, efficient, disease prevention and growth promotion function combined biocontrol agent, and is used for green prevention and treatment of tobacco black shank disease.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to the isolation, identification and application of a functional bacterium, specifically to a strain of *Pseudomonas aeruginosa* and its application in the prevention and control of tobacco black shank disease. Background Technology

[0002] Tobacco black shank is caused by Phytophthora indicum (Tobacco Black Shank). Phytophthora nicotianae This soil-borne disease, caused by metalaxyl, affects the entire tobacco growing season, causing root and stem rot, reducing tobacco yield and quality, and is one of the most devastating diseases in tobacco production. Currently, the control of this disease mainly relies on chemical pesticides such as metalaxyl and mancozeb. However, long-term and excessive use of chemical agents can easily lead to drug resistance in pathogens, and pesticide residues pose a threat to the ecological environment and human health.

[0003] With the rapid advancement of modern biotechnology, biological control has ushered in a development opportunity as an environmentally friendly method for plant disease management. Biological control, with its outstanding advantages such as safety, reliability, environmental compatibility, high efficiency, and the low likelihood of pathogens developing resistance, effectively maintains the balance of the ecosystem while ensuring safe crop production. Compared with traditional chemical pesticides, biological pesticides generally have extremely low or even zero residue levels. By replacing or reducing the use of chemical pesticides with biological pesticides, the adverse effects of chemical pesticides on the ecological environment and human health can be mitigated at the source, thereby promoting sustainable socio-economic development.

[0004] In existing biological control research, strains such as *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, and *Pseudomonas spp.* have been used in research on tobacco disease control. Related published literature and patents indicate that some *Pseudomonas* species have a certain inhibitory effect on *Phytophthora tobaccoii*, with potted plant control efficacy generally between 60% and 80%. However, they generally suffer from problems such as a broad antibacterial spectrum, insufficient specificity, and unstable field effects, making it difficult to meet the dual demands of green control and increased yield and quality.

[0005] In summary, the existing technologies for the prevention and control of tobacco black shank have the following defects: (1) chemical pesticides are prone to developing resistance, residual pollution, and ecological damage; (2) existing biocontrol bacteria have low efficacy, poor specificity, and unstable effects; (3) there is a lack of excellent strains that combine high antibacterial properties, specific antagonism, and growth promotion.

[0006] Therefore, screening for novel biocontrol strains with high inhibitory activity, strong specificity, and growth-promoting properties against *Phytophthora indica*, and developing safe and efficient microbial agents, is of great significance for the green control of tobacco black shank and the sustainable development of the industry. A search revealed no information on *Pseudomonas aeruginosa* (Donghu Pseudomonas). Pseudomonas donghuensisThis invention reports on the use of this strain for the control of tobacco black shank disease caused by Phytophthora cirrhosa. This invention is the first to apply this strain to the biological control of tobacco black shank disease, filling a technological gap in the field of tobacco disease control. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a strain of *Pseudomonas donghuensis* and its application in the control of tobacco black shank disease. The *Pseudomonas donghuensis* YM3-423 is environmentally friendly, poses no crop safety risks, and does not produce harmful residues.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: A purified strain of *Pseudomonas aeruginosa* (Donghu Pseudomonas) Pseudomonas donghuensis The strain YM3-423 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2025914 and deposit date of April 27, 2025.

[0009] Furthermore, the 16S rDNA sequence of the *Pseudomonas aeruginosa* strain YM3-423 contains the nucleotide sequence shown in SEQ ID NO.1.

[0010] The present invention also provides the application of Pseudomonas aeruginosa strain YM3-423 in the prevention and control of tobacco black shank disease, and its application in the preparation of biocontrol agents for the prevention and control of tobacco black shank disease.

[0011] The present invention also provides the application of Pseudomonas aeruginosa strain YM3-423 in promoting tobacco growth, and its application in the preparation of biocontrol agents for promoting tobacco growth.

[0012] Furthermore, the *Pseudomonas aeruginosa* strain YM3-423 selectively inhibits *Phytophthora indicum* (…). Phytophthora nicotianae (This is used to prevent and treat tobacco black shank disease caused by this pathogen.)

[0013] The present invention also provides a biocontrol agent for preventing and / or promoting tobacco growth of tobacco black shank disease, wherein the active ingredient of the biocontrol agent comprises at least one of the cells of the *Pseudomonas aeruginosa* strain YM3-423 and the fermentation broth.

[0014] The biocontrol agent of this invention may also contain at least one of agriculturally acceptable carriers and adjuvants. The agriculturally acceptable carrier is selected from one or more of kaolin, diatomaceous earth, light calcium carbonate, corn starch, biochar, wheat bran, and sodium alginate. The adjuvant is selected from one or more of wetting and dispersing agents, water-retaining agents, nutrient enhancers, UV protectants, and binders. The wetting and dispersing agent may be sodium lignosulfonate or sodium dodecylbenzenesulfonate; the water-retaining agent may be sodium polyacrylate or sodium carboxymethyl cellulose; the nutrient enhancer may be glucose, yeast powder, or amino acid powder. The above-mentioned carriers and adjuvants are merely preferred examples of this invention. Conventional agricultural carriers and adjuvants in the art can be adapted to the strains of this invention and are not limited to the types listed above. The carriers and adjuvants are only auxiliary excipients or synergistic agents and do not change the antibacterial activity or growth-promoting effect of strain YM3-423.

[0015] Furthermore, the formulation of the biocontrol agent is one or more of the following: wettable powder, water-dispersible granules, suspension concentrate, and granules.

[0016] A method for controlling tobacco black shank disease, the method comprising: applying the fermentation broth and / or bacterial cells of the *Pseudomonas aeruginosa* strain YM3-423 to tobacco plants or the soil in which tobacco is grown; or applying the biocontrol agent to tobacco plants or the soil in which tobacco is grown.

[0017] A method for promoting tobacco growth, the method comprising: applying the fermentation broth and / or bacterial cells of the *Pseudomonas aeruginosa* strain YM3-423 to tobacco plants or soil in which tobacco is grown; or applying the biocontrol agent to tobacco plants or soil in which tobacco is grown.

[0018] Furthermore, the application methods include one or more of the following: root irrigation, spraying, seed dressing, and fumigation.

[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention isolated and screened a strain of *Pseudomonas aeruginosa* YM3-423 from the rhizosphere soil of healthy plants in areas severely affected by tobacco black shank disease. The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.1. *Pseudomonas aeruginosa* YM3-423 is environmentally friendly, poses no crop safety risks, and does not produce harmful residues.

[0020] The *Pseudomonas donghuensis* YM3-423 provided by this invention exhibits significant and specific inhibitory effects against *Phytophthora tobaccotae*, and possesses volatile antibacterial properties. Simultaneously, it significantly promotes the growth of tobacco seedlings, making it an excellent biocontrol microorganism. This invention is the first to reveal that *Pseudomonas donghuensis* YM3-423 has a significant inhibitory effect on *Phytophthora tobaccotae*, the pathogen of tobacco black shank, providing a novel strain resource for the biological control of tobacco black shank.

[0021] Furthermore, greenhouse pot experiments showed that the *Pseudomonas aeruginosa* YM3-423 of this invention achieved a 95.70% control effect against tobacco black shank disease, comparable to or even slightly better than the control effect of conventional chemical pesticide 58% metalaxyl-mancozeb (94.62%). Moreover, the biological agent leaves no chemical residue, is environmentally friendly, and has the potential to replace or reduce the use of chemical pesticides. This strain also significantly promoted the growth of tobacco seedlings, increasing seedling height by 29.4% and total fresh weight by 96.4% after treatment, achieving a dual function of "disease prevention through bacteria" and "growth promotion through bacteria." Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of Pseudomonas aeruginosa YM3-423 on LB solid medium in an embodiment of the present invention; Figure 2 This is a diagram showing the confrontation effect of Pseudomonas aeruginosa YM3-423 against Phytophthora nicotineae in an embodiment of the present invention (left: blank control, right: YM3-423). Figure 3 This is a diagram showing the effect of volatile substances from *Pseudomonas aeruginosa* YM3-423 on the mycelial growth of *Phytophthora indicum* in this invention (left: blank control, upper plate inoculated with *Phytophthora indicum*, lower plate blank LB medium; right: YM3-423 treatment, upper plate inoculated with *Phytophthora indicum*, lower plate inoculated with YM3-423). Figure 4 This is a graph showing the antibacterial spectrum of Pseudomonas aeruginosa YM3-423 against eight different common plant pathogens in an embodiment of the present invention (top: blank control, bottom: YM3-423). Figure 5 This is a diagram illustrating the efficacy of *Pseudomonas aeruginosa* YM3-423 in greenhouse pots against black shank disease in tobacco seedlings (Yunyan 87) in this embodiment of the invention. From left to right: blank control (CK), susceptible control (T1), pesticide control (T2), experimental group (T3); Figure 6 This is a top-view diagram of the effect of Pseudomonas aeruginosa YM3-423 on the growth promotion of tobacco (Yunyan 87) in a greenhouse potted plant according to an embodiment of the present invention (left two columns: CK, right two columns: YM3-423). Figure 7 This is a side view of the effect of Pseudomonas aeruginosa YM3-423 on the growth promotion of tobacco (Yunyan 87) in a greenhouse pot in an embodiment of the present invention (top: CK, bottom: YM3-423). Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: A purified strain of *Pseudomonas aeruginosa* YM3423, its preservation information is as follows: Strain name: *Pseudomonas aeruginosa*; Latin name: Pseudomonas donghuensis ; Strain number: YM3-423; Preservation institution: China Center for Type Culture Collection; Abbreviation for depository institution: CCTCC; Address of the depository: Wuhan University, Wuhan, China; Deposit date: April 27, 2025; Accession number: CCTCC M 2025914.

[0025] Specifically, the *Pseudomonas donghuensis* YM3-423 was isolated from the rhizosphere soil of healthy plants from an area severely affected by tobacco black shank disease. Observation of the colony morphology of the cultured *Pseudomonas donghuensis* revealed that the colonies of *Pseudomonas donghuensis* YM3-423 on LB agar plates were round, with a raised, glossy surface, a pale yellow color, and a distinctive odor.

[0026] Molecular classification and identification of the *Pseudomonas aeruginosa* YM3-423 were performed; its 16S rDNA nucleotide sequence is shown in SEQ ID NO.1.

[0027] Plate confrontation test, volatile substance inhibition test and greenhouse efficacy test showed that the *Pseudomonas aeruginosa* YM3-423 is effective against *Phytophthora indica*, the pathogen of tobacco black shank disease. P. nicotianae It exhibits significant inhibitory activity. Currently, no information has been found regarding *Pseudomonas aeruginosa* (Donghu Pseudomonas). Pseudomonas donghuensis Reports indicate that it has potential for biocontrol of tobacco diseases or has an inhibitory effect on Phytophthora indica.

[0028] Specifically, in this embodiment, the *Pseudomonas aeruginosa* YM3-423 was collected on June 24, 2024, from the rhizosphere soil of healthy plants in a region with severe tobacco black shank disease in a certain province, and was stored in a plastic resealable bag after collection.

[0029] Weigh 5 g of rhizosphere soil from healthy plants and pour it into a centrifuge tube containing 45 mL of sterile water. Incubate at 28°C and 200 rpm for 30 min with shaking. After thorough shaking, let stand for 5 min. The supernatant should be 10 g / mL. -1 The soil dilution was diluted to a certain degree; then diluted with 10... -1 The diluted soil solution is used as the mother liquor to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 Diluted soil solution, take 100 μL of 10 -4 10 -5 10 -6 The diluted soil solution was spread evenly on NA solid medium plates, with each concentration replicated three times. The plates were then placed in a constant temperature incubator and incubated at 28°C for 48 hours.

[0030] When single colonies grow on the plate, select single colonies of different shapes, inoculate them onto LB solid medium and culture them again. Repeat this process until several mediums with single colonies are obtained.

[0031] Bacterial cells from a medium with single colonies were selected for a plate confrontation experiment. *Phytophthora tobaccois* was inoculated in the center of an OA solid medium, and randomly selected isolated strains were placed around the pathogen on LB plates for confrontation culture. The plates were incubated at 28°C for 7 days, and the growth of the pathogen was observed.

[0032] The strains with good inhibitory effects were selected for purification. After two streaking tests to obtain single colonies, further screening was performed. Single colonies were picked and inoculated into 3 mL of LB liquid medium, and cultured at 28℃ and 200 rpm for 24 h with shaking. 20 μL of bacterial suspension was added dropwise at a distance of 1.5 cm from the pathogen, and the culture was incubated at 28℃ for 7 days. The diameter of *Phytophthora indicum* was measured, and the inhibition rate was calculated. Inhibition rate (%) = (Control plate colony diameter - Treatment plate colony diameter) / Control plate colony diameter × 100, repeated 3 times.

[0033] A strain exhibiting significant inhibitory activity against *Phytophthora indicum*, the pathogen causing tobacco black shank disease, was selected and designated YM3-423. The YM3-423 cells were placed in cryovials containing 20% ​​glycerol and stored at -80°C.

[0034] The colony morphology of strain YM3-423 on LB solid medium is shown in the figure. Figure 1 ,like Figure 1 As shown, the colonies of the YM3-423 strain appear as round, raised, and glossy colonies on LB solid medium plates, with neat, smooth edges, no serrations or rough edges, a pale yellow color, and a distinctive odor.

[0035] This embodiment also provides the classification and identification of the *Pseudomonas aeruginosa* YM3-423, the specific method of which is as follows: Genomic DNA was extracted from strain YM3-423 using a high-temperature lysis method. Its 16S rDNA was amplified by PCR and sequenced. The nucleotide sequence of the 16S rDNA of strain YM3-423 is shown in SEQ ID NO.1. BLAST alignment confirmed that this sequence was highly similar to that of *Pseudomonas donghuensis*.

[0036] Simultaneously, whole-genome sequencing was performed on strain YM3-423, and the obtained whole-genome sequence was compared with the genomes of known type strains in the NCBI / EzBioCloud database using average nucleotide identity (ANI) analysis. Strain YM3-423 and... Pseudomonas donghuensis strain The ANI value of P482 was 99.3196%, which is greater than the 95% species level threshold. Based on this, the strain of this invention was identified as *Pseudomonas donghuensis*. Pseudomonas donghuensis ).

[0037] Example 2: This example provides the application of Pseudomonas aeruginosa YM3-423 described in Example 1 in the prevention and control of tobacco black shank, as well as its application in the preparation of biocontrol agents for the prevention and control of tobacco black shank.

[0038] Specifically, the *Pseudomonas aeruginosa* YM3-423 is used to prevent and control tobacco black shank disease caused by *Phytophthora indicum*.

[0039] This embodiment uses the plate confrontation method to test the effect of *Pseudomonas aeruginosa* YM3-423 on the pathogen *Phytophthora indicum*, which causes black shank in tobacco. P. nicotianae The in-plate inhibition effect test of ) is as follows: A single colony of YM3-423 was picked and inoculated into 3 mL of LB liquid medium, and cultured at 28°C with shaking at 200 rpm for 24 h until OD reached. 600 The value was approximately 1.0. Using the plate confrontation method, mycelial cakes with a diameter of about 5 mm were taken from the periphery of a vigorous single colony of *Pseudomonas aeruginosa* and inoculated onto OA solid medium, incubated at 28℃ for 1 day. After mycelia emerged around the mycelial cake, 20 μL of bacterial solution was added at a distance of 1.5 cm from the pathogen, and incubated at 28℃ for 3-4 days. This was repeated 3 times. The diameter of *Pseudomonas aeruginosa* was measured using the cross-sectional method, and the average value of the measurements was taken. Inhibition rate (%) = (Coronary colony diameter of control plate - Coronary colony diameter of treatment plate) / Coronary colony diameter of control plate × 100. The average inhibition rate, after measurement and calculation, was 80.06% ± 4.63%. Therefore, *Pseudomonas aeruginosa* YM3-423 has a significant inhibitory effect on the growth of *Pseudomonas aeruginosa*. Figure 2As shown, the left image is the control group, and the right image is the experimental group.

[0040] This embodiment also provides an experiment on the inhibitory effect of the volatile substances of *Pseudomonas aeruginosa* YM3-423 on the mycelial growth of *Phytophthora indicum*, specifically including: A 5 mm diameter mycelial cake of the pathogen causing tobacco black shank was inoculated into the center of an OA medium and incubated at 28°C for 1 day to fix it onto the medium. A single colony of the biocontrol bacteria activated on an LB plate was inoculated into liquid LB medium and cultured at 28°C with shaking at 200 rpm for 24 h. 100 μL of the bacterial suspension was then plated onto a solid LB plate. The OA solid medium inoculated with *Phytophthora indicum* and the LB solid medium were then placed on top of the biocontrol plates, caps removed, and sealed together with airtight plastic wrap. The control group consisted of an OA plate inoculated with *Phytophthora indicum* and sterile LB solid medium, with the LB medium at the bottom and the OA medium on top. These were incubated at 28°C for 5 days. The growth of the pathogen was observed and recorded, and the experiment was repeated three times.

[0041] The results are as follows Figure 3 As shown in the figure, the left figure is the control group and the right figure is the experimental group. The volatile substances of Pseudomonas aeruginosa YM3-423 have a significant inhibitory effect on the mycelial growth of Phytophthora tobaccois, with an average inhibition rate of 77.72%±2.88%.

[0042] This embodiment also provides the *Pseudomonas aeruginosa* YM3-423. Inhibitory effect against different pathogens (antibacterial spectrum).

[0043] In this embodiment, the tested pathogens include: Fusarium wilt pathogen (… Fusarium oxysporum Tobacco black rot fungus ( Thielaviopsis basicola ), tobacco red spot bacterium ( Alternaria alternat a) Penicillium expansum ( Penicillium expansum ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), Staphylococcus aureus ( Botryosphaeria dothidea Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ).

[0044] The experimental method employed was the plate confrontation method: a single colony of YM3-423 was picked and inoculated into 3 mL of LB liquid medium, and incubated at 28°C with shaking at 200 rpm for 24 h until OD reached. 600 The value was around 1.0. Different pathogens were inoculated into the center of PDA solid medium, and 20 μL of YM3-423 fermentation broth was added 1.5 cm away from the pathogens. The medium was incubated at 28℃ for 3-7 days. When the control colonies grew to cover the entire surface of the plate, the process was repeated 3 times.

[0045] The results are as follows Figure 4As shown, from left to right, they are: Phytophthora indicum, Fusarium wilt of tobacco, Phytophthora indicum, Aureobasidium indicum, Penicillium expansum, Colletotrichum gloeosporioides, Staphylococcus aureus, and Rhizoctonia solani. Figure 4 As can be seen, the *Pseudomonas aeruginosa* YM3-423 has a significant inhibitory effect on *Phytophthora indicum*, but shows no inhibitory activity or poor activity against the other 7 common pathogenic fungi tested, indicating that strain YM3-423 has strong specificity or selectivity against *Phytophthora indicum*.

[0046] This embodiment also provides the inhibitory effect of the aforementioned *Pseudomonas aeruginosa* YM3-423 on *Phytophthora nicotineae* in tobacco seedlings under greenhouse pot conditions.

[0047] This embodiment used healthy Yunyan 87 seedlings at the 4-6 leaf stage. The following treatments were established: CK (equal volume of water irrigation), T1 (susceptible control, seedling neck punctured with a needle tip and inoculated with a fungal cake), T2 (pesticide control, fungal cake punctured, root drenching with 58% metalaxyl-mancozeb 750-fold diluted solution, 20 mL per plant), and T3 (experimental group, fungal cake punctured, root drenching with compound antagonistic bacterial solution 20 mL / plant). Seven days after transplanting and allowing the seedlings to recover, each treatment was drenched twice, three days before and three days after inoculation with the pathogen. Each treatment consisted of 8 plants, replicated three times. Black shank disease incidence was recorded every 7 days, per plant, with photographs taken and the incidence recorded for each group. The disease severity grading was based on the national standard GB / T 23222-2008; specific grading standards are shown in Table 1.

[0048] Table 1. Grading criteria for tobacco black shank disease The incidence rate, disease index, and control effect were calculated using the following formulas: Incidence rate = (Number of diseased plants / Total number of plants surveyed) × 100%; Disease index = [∑(Number of diseased plants or leaves at each level × Disease level) / (Total number of plants surveyed × Highest level value)] × 100; Control effect = [1 - (Disease index after treatment / Disease index of susceptible control)] × 100%.

[0049] See results Figure 5 (From left to right: blank control (CK), diseased control (T1), pesticide control (T2), experimental group YM3-423 (T3)). The incidence rate, disease index and control effect of each treatment group are shown in Table 2.

[0050] Table 2. Control efficacy of *Pseudomonas aeruginosa* YM3-423 against tobacco black shank in greenhouse potted plants. Depend on Figure 5As shown in Table 2, under greenhouse pot cultivation conditions, *Pseudomonas aeruginosa* YM3-423 significantly inhibited the infection of *Phytophthora nicotineae* on tobacco seedlings, with a control effect of 95.70%, which is at the same level as the control effect of conventional chemical pesticide 58% metalaxyl-mancozeb (94.62%). Moreover, it has no chemical residues and is environmentally friendly, showing excellent potential for alternative application.

[0051] Example 3: Preparation and application of Pseudomonas aeruginosa YM3-423 bacterial agent. This example provides a practical scheme for liquid bacterial agent preparation and also provides an exemplary approach to the conventional preparation of solid bacterial agent preparation.

[0052] The *Pseudomonas aeruginosa* YM3-423 described in Example 1 was inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 48 h with shaking. The resulting fermentation broth contained approximately 1 × 10⁻⁶ bacteria. 8 ~1×10 10 The above liquid bacterial agent (CFU / mL) can be used directly as a liquid bacterial agent. Dilute the above liquid bacterial agent to 1×10^8 CFU / mL and treat tobacco seedlings with the root irrigation method (20 mL / plant) as described in Example 2. Its control effect on tobacco black shank disease is shown in Example 2 and will not be repeated here.

[0053] In addition to the liquid bacterial agent described above, the *Pseudomonas aeruginosa* strain YM3-423 of this invention can also be prepared as a solid bacterial agent using conventional methods in the field of microbial agents. As an example, the fermentation broth can be centrifuged to collect the bacterial precipitate, which is then mixed with a sterilized carrier (such as diatomaceous earth, corn starch, or light calcium carbonate), dried at low temperature, pulverized, and sieved to obtain a powdered bacterial agent. This solid bacterial agent can be reconstituted with sterile water and diluted to the required concentration before application. The specific preparation process parameters of the above-mentioned solid bacterial agent (such as carrier type and ratio, drying temperature, and moisture content) can be routinely optimized and adjusted by those skilled in the art based on actual production conditions, without requiring any creative effort.

[0054] Example 4: This example demonstrates the growth-promoting effect of the *Pseudomonas aeruginosa* YM3-423 on tobacco seedlings under greenhouse conditions.

[0055] The healthy seedlings of Yunyan 87 at the 4-6 leaf stage used in this embodiment.

[0056] Experimental groups and treatments: CK: Irrigated with equal volume of water; T1: Root drenching with YM3-423 bacterial solution (20 mL / plant, 1×10⁻⁶). 8 CFU / ml). The roots were irrigated once every 7 days for a total of 2 times. Seven days after the last treatment, the growth parameters of the tobacco seedlings were measured, including plant height, root length, stem diameter, maximum leaf length, maximum leaf width, fresh weight and dry weight of the aboveground and underground parts.

[0057] The growth of each experimental group was recorded by taking photos, and the results are shown below. Figure 6 The growth parameters for each treatment group are shown in Table 3.

[0058] Table 3. Growth-promoting effect of *Pseudomonas aeruginosa* YM3-423 on tobacco seedlings. Depend on Figures 6-7 As shown in Table 3, under greenhouse pot cultivation conditions, the growth of treatment T1 was significantly better than that of the control group CK. This indicates that *Pseudomonas aeruginosa* YM3-423 has a significant growth-promoting effect on tobacco seedlings.

[0059] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, modifications or variations can still be made to the technical solutions described above, and these modifications and variations all fall within the protection scope of the present invention.

Claims

1. A purified strain of *Pseudomonas aeruginosa* (Donghu Pseudomonas) Pseudomonas donghuensis strain YM3-423, characterized in that, The strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2025914 and deposit date of April 27, 2025.

2. The *Pseudomonas aeruginosa* strain YM3-423 according to claim 1, characterized in that, The 16S rDNA sequence of the *Pseudomonas aeruginosa* strain YM3-423 contains the nucleotide sequence shown in SEQ ID NO.

1.

3. The application of the *Pseudomonas aeruginosa* strain YM3-423 as described in claim 1 or 2 in the prevention and control of tobacco black shank disease, and its application in the preparation of a biocontrol agent for the prevention and control of tobacco black shank disease.

4. The use of the *Pseudomonas aeruginosa* strain YM3-423 as described in claim 1 or 2 in promoting tobacco growth, and its use in preparing a biocontrol agent for promoting tobacco growth.

5. The application according to claim 3, characterized in that, The *Pseudomonas aeruginosa* strain YM3-423 selectively inhibits *Phytophthora indica* (…). Phytophthora nicotianae (This is used to prevent and treat tobacco black shank disease caused by this pathogen.) 6. A biocontrol agent for preventing and / or promoting tobacco growth in the form of black shank disease, characterized in that, The active ingredient of the biocontrol agent includes at least one of the bacterial cells and fermentation broth of the *Pseudomonas aeruginosa* strain YM3-423 as described in claim 1 or 2; at least one of the agriculturally acceptable carriers and adjuvants may be selectively added.

7. The biocontrol agent according to claim 6, characterized in that, The biocontrol agent is formulated as one or more of the following: wettable powder, water-dispersible granules, suspension concentrate, and granules.

8. A method for preventing and controlling tobacco black shank disease, characterized in that, The method includes: applying the fermentation broth and / or bacterial cells of the *Pseudomonas aeruginosa* strain YM3-423 of claim 1 or 2 to tobacco plants or the soil in which tobacco is grown; or applying the biocontrol agent of claim 6 or 7 to tobacco plants or the soil in which tobacco is grown.

9. A method for promoting tobacco growth, characterized in that, The method includes: applying the fermentation broth and / or bacterial cells of the *Pseudomonas aeruginosa* strain YM3-423 of claim 1 or 2 to tobacco plants or the soil in which tobacco is grown; or applying the biocontrol agent of claim 6 or 7 to tobacco plants or the soil in which tobacco is grown.

10. The method according to claim 8 or 9, characterized in that, The application methods include one or more of the following: root irrigation, spraying, seed dressing, and fumigation.

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