Pseudomonas donghuensis probiotics for preventing and controlling tobacco black shank and application thereof

Abstract

This invention belongs to the field of microbiology and relates to a *Pseudomonas donghuensis* synbiotic for the control of tobacco black shank disease and its application. This invention constructs a synbiotic by combining *Pseudomonas donghuensis* with glycolic acid. Experimental results show that glycolic acid not only inhibits the growth of *Phytophthora xanthoides* but also promotes the proliferation of *Pseudomonas donghuensis* and further enhances its antagonistic ability against *Phytophthora xanthoides*. The *Pseudomonas donghuensis* synbiotic provided by this invention can significantly reduce the disease index of tobacco black shank disease and reduce the amount of *Phytophthora xanthoides* in the rhizosphere soil, providing a new technical solution for the green control of tobacco black shank disease.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to a *Pseudomonas donghuensis* synbiotic for the prevention and control of tobacco black shank disease and its application. Background Technology

[0002] Tobacco black shank is an important soil-borne oomycete disease caused by *Phytophthora nicotianae*. It primarily infects the roots and stem base of tobacco plants, leading to blackening of the stem base, wilting, and even death. It is one of the most damaging and difficult-to-control diseases in tobacco production. *Phytophthora nicotianae* can survive and spread in soil, diseased plant debris, and irrigation water, exhibiting a wide host range. Once the disease occurs, it spreads rapidly and is difficult to control. Existing control measures mainly include resistant varieties, crop rotation, cultivation management, and chemical treatment. However, under conditions of continuous planting and high pathogen pressure, single measures are often insufficient for long-term stable control of the disease. Furthermore, long-term reliance on chemical agents may lead to decreased efficacy and increased environmental stress. Therefore, developing safe, green, and stable biological control technologies for tobacco black shank is of great significance.

[0003] Beneficial rhizosphere Pseudomonas spp. are functional microorganisms that have attracted widespread attention in the biocontrol of soil-borne diseases due to their role in promoting plant growth, competing for nutrients and ecological niches, producing siderophores, secreting antimicrobial substances, and inducing plant resistance. Previous studies have shown that some Pseudomonas strains can inhibit *Phytophthora indicum* and reduce the incidence of tobacco black shank under pot cultivation conditions. However, there are significant differences in the genetic background, metabolic characteristics, rhizosphere adaptability, and antagonistic activity among different strains within the genus *Pseudomonas*. Even within the same genus, different strains may exhibit significantly different inhibitory abilities against *Phytophthora indicum* and different field control effects. Therefore, it is not possible to simply determine whether a particular *Pseudomonas* strain has a stable ability to control tobacco black shank based on its genus name; resource adaptation and efficacy verification for specific strains are still necessary.

[0004] Furthermore, the efficacy of beneficial bacteria applied alone in actual soil environments is often unstable. On the one hand, nutrient resources in the rhizosphere soil are limited, and exogenously applied beneficial bacteria need to compete with native microorganisms for space and nutrients. On the other hand, soil physicochemical properties, the composition of crop root exudates, pathogen pressure, and environmental conditions all affect the colonization, growth, and functional expression of beneficial bacteria. Therefore, even if a certain beneficial bacteria shows good antagonistic effects under indoor conditions, its efficacy may decrease after being applied to the soil due to a lack of suitable resources, insufficient colonization, or limited expression of antagonistic activity.

[0005] Root exudates and small-molecule metabolites available to microorganisms can serve as nutritional resources for the growth of beneficial bacteria, but they can also affect the colonization ability and antagonistic function of these bacteria. By screening for resources that can be effectively utilized by target beneficial bacteria while not promoting or inhibiting the growth of pathogens, suitable resource conditions can be provided for beneficial bacteria to function in the rhizosphere. Combining beneficial bacteria with suitable resources to construct rhizosphere synbiotics is beneficial for improving the growth and functional stability of beneficial bacteria in the crop rhizosphere, thereby enhancing the control effect against soil-borne diseases.

[0006] In summary, existing control technologies have the following problems: 1) Tobacco black shank is a soil-borne disease, and the pathogen can accumulate continuously in the rhizosphere soil environment, making it difficult for conventional control measures to stably suppress the disease in the long term; 2) After beneficial Pseudomonas bacteria are applied to the soil, their growth, colonization, and expression of antagonistic substances are affected by the rhizosphere resource environment, and the control efficacy may be limited when beneficial bacteria are applied alone; 3) Different crops have different rhizosphere nutrient environments, and it is necessary to screen suitable combinations of beneficial bacteria and resource substances for the tobacco-Tobacco Phytophthora system. Summary of the Invention

[0007] To address the control of tobacco black shank disease, this invention provides a *Pseudomonas donghuensis* synbiotic and its application. This invention constructs a synbiotic by combining *Pseudomonas donghuensis* with glycolic acid. Experimental results show that glycolic acid not only inhibits the growth of *Phytophthora nicotine* but also promotes the proliferation of *Pseudomonas donghuensis*, further enhancing its antagonistic ability against *Phytophthora nicotine*. The *Pseudomonas donghuensis* synbiotic provided by this invention can significantly reduce the disease index of tobacco black shank and decrease the amount of *Phytophthora nicotine* in the rhizosphere soil, providing a new technical solution for the green control of tobacco black shank disease.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: A *Pseudomonas donghuensis* synbiotic for the prevention and control of tobacco black shank disease, wherein the *Pseudomonas donghuensis* synbiotic comprises *Pseudomonas donghuensis* (… Pseudomonas donghuensis ) strain YM3-423 and glycolic acid; The Donghu Pseudomonas ( Pseudomonas donghuensis Strain YM3-423 was deposited at the China Center for Type Culture Collection on April 27, 2025, with accession number CCTCC NO:M 2025914, originating from Northwest A&F University.

[0009] Glycolic acid, with CAS number 79-14-1, is classified as an organic acid and has a molecular weight of 76.05 g / mol.

[0010] Furthermore, the *Pseudomonas aeruginosa* synbiotic is a formulation prepared using glycolic acid, *Pseudomonas aeruginosa* YM3-423 bacterial suspension, and sterile water. In the formulation of *Pseudomonas aeruginosa* synbiotic, the final concentration of glycolic acid was 0.05–0.2 mM, and the final concentration of *Pseudomonas aeruginosa* YM3-423 was 1 × 10⁻⁶ mM. 7 ~1×10 9 CFU / mL.

[0011] Preferably, in the preparation of the *Pseudomonas aeruginosa* synbiotic, glycolic acid is applied in the form of a glycolic acid solution; the concentration of the glycolic acid stock solution is 2 mM. The glycolic acid solution is prepared by dissolving glycolic acid in deionized water, drawing it up with a sterile syringe, and filtering it through a 0.22 μm filter membrane to obtain the glycolic acid solution.

[0012] Furthermore, in the formulation of the *Pseudomonas aeruginosa* synbiotic, the final concentration of glycolic acid was 0.1 mM, and the final concentration of *Pseudomonas aeruginosa* YM3-423 was 1 × 10⁻⁶ mM. 8 CFU / mL.

[0013] Further, the *Pseudomonas aeruginosa* YM3-423 bacterial suspension was prepared by the following method: *Pseudomonas aeruginosa* YM3-423 strain was activated using LB solid medium; single colonies were picked and cultured in LB liquid medium at 28-30℃ with shaking for 14-18 hours, centrifuged, the supernatant was discarded, the cells were washed with sterile water, the bacterial cells were collected, and sterile water or sterile physiological saline was added to obtain the *Pseudomonas aeruginosa* YM3-423 bacterial suspension.

[0014] Preferably, the shaking rotation speed is 170 rpm. Preferably, the centrifugation conditions are 6000 rpm for 5-8 min. Preferably, the method for washing away residual culture medium with sterile water is as follows: wash the precipitate with sterile water, centrifuge at 6000 rpm for 5-8 min, wash the precipitate again with sterile water, centrifuge at 6000 rpm for 5-8 min, and repeat the sterile water washing and centrifugation three times in total.

[0015] The present invention also provides the application of *Pseudomonas donghuensis* synbiotic, which is used to control tobacco black shank disease.

[0016] Furthermore, the aforementioned tobacco black shank disease is caused by *Phytophthora indica*. Phytophthora nicotianae This can cause tobacco black shank disease or tobacco root rot-related black shank disease.

[0017] Furthermore, the application involves using a root irrigation method to combine glycolic acid with Pseudomonas aeruginosa YM3-423 to control tobacco black shank disease.

[0018] A method for controlling tobacco black shank disease involves applying the *Pseudomonas donghuensis* synbiotic to the tobacco rhizosphere soil via root irrigation. Preferably, the *Pseudomonas donghuensis* synbiotic is applied to the tobacco rhizosphere soil via root irrigation 5–10 days after tobacco transplanting. The volume of the *Pseudomonas donghuensis* synbiotic applied to each tobacco seedling is 10–30 mL.

[0019] Furthermore, the volume of the *Pseudomonas donghuensis* synbiotic applied to each tobacco seedling is 20 mL.

[0020] The beneficial effects of this invention are: This invention combines glycolic acid with Pseudomonas aeruginosa YM3-423 to form a Pseudomonas aeruginosa synbiotic for the control of tobacco black shank disease. This synbiotic can significantly reduce the incidence of tobacco black shank disease and decrease the number of soil pathogens, making it suitable for green control of tobacco black shank disease.

[0021] This invention is the first to discover that glycolic acid (GlyA) can inhibit the growth of Phytophthora tobaccois and promote the growth of Pseudomonas donghuensis YM3-423; the combined use of glycolic acid and Pseudomonas donghuensis YM3-423 can improve the plate antagonistic inhibition rate of Pseudomonas donghuensis YM3-423 against Phytophthora tobaccois; in the tobacco potted plant system, the synbiotic formulation composed of Pseudomonas donghuensis YM3-423 and glycolic acid can significantly reduce the disease index of tobacco black shank and reduce the number of Phytophthora tobaccois in the rhizosphere soil. Attached Figure Description

[0022] Figure 1 This illustrates the effect of glycolic acid on the growth of Phytophthora in tobacco in this embodiment of the invention. Figure 2 This invention illustrates the effect of glycolic acid on the growth of *Pseudomonas aeruginosa* YM3-423 in this embodiment. Figure 3 The effect of glycolic acid on the antagonistic ability of Pseudomonas aeruginosa YM3-423 against Phytophthora nicotine; Figure 4 The effect of Pseudomonas aeruginosa YM3-423-glycolic acid synbiotic on the disease index of black shank disease in potted tobacco; Figure 5 The effect of *Pseudomonas aeruginosa* YM3-423-glycolic acid synbiotic on the abundance of *Phytophthora nicotine* in soil. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0024] Example 1: A *Pseudomonas donghuensis* synbiotic for the prevention and control of tobacco black shank disease, wherein the *Pseudomonas donghuensis* synbiotic includes *Pseudomonas donghuensis* (… Pseudomonas donghuensis ) strain YM3-423 and glycolic acid; The Donghu Pseudomonas ( Pseudomonas donghuensis Strain YM3-423 was deposited at the China Center for Type Culture Collection on April 27, 2025, with accession number CCTCC NO:M 2025914.

[0025] The glycolic acid (GlyA) used in this invention, CAS number 79-14-1, is an organic acid with a molecular weight of 76.05 g / mol.

[0026] The *Pseudomonas aeruginosa* synbiotic is a formulation prepared using glycolic acid, *Pseudomonas aeruginosa* YM3-423 bacterial suspension, and sterile water. In the formulation, the final concentration of glycolic acid is 0.05–0.2 mM, and the final concentration of *Pseudomonas aeruginosa* YM3-423 is 1 × 10⁻⁶ mM. 7 ~1×10 9 CFU / mL.

[0027] Preferably, in the formulation of *Pseudomonas aeruginosa* synbiotic, the final concentration of glycolic acid is 0.1 mM, and the final concentration of *Pseudomonas aeruginosa* YM3-423 is 1 × 10⁻⁶ mM. 8 CFU / mL.

[0028] In this embodiment, the *Pseudomonas aeruginosa* YM3-423 bacterial suspension was prepared by the following method: the *Pseudomonas aeruginosa* YM3-423 strain was activated using LB solid medium; single colonies were picked and cultured in LB liquid medium at 28–30°C with shaking for 14–18 h; centrifuged, the supernatant was discarded, the bacterial cells were collected, and sterile water or sterile physiological saline was added to obtain the *Pseudomonas aeruginosa* YM3-423 bacterial suspension.

[0029] In this invention, *Phytophthora nicotianae* is the model pathogen that can cause black shank disease in tobacco.

[0030] LB solid medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, 15.0-20.0g agar, distilled water to a final volume of 1L, pH 7.0-7.2, sterilized at 121℃ for 20min.

[0031] LB liquid medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, distilled water to a final volume of 1L, pH 7.0-7.2, sterilized at 121℃ for 20min.

[0032] Glycolic acid (GlyA): Chinese name is ethanol acid, English name is Glycolic acid, CAS number is 79-14-1, molecular weight is 76.05 g / mol.

[0033] This invention investigated the indoor effects of glycolic acid on the growth of Phytophthora tobaccoii and Pseudomonas donghuensis YM3-423: Preparation of glycolic acid solution: Glycolic acid was dissolved in deionized water to prepare a 2mM stock solution, which was then filtered through a 0.22μm filter membrane for sterilization and set aside for use.

[0034] Effect of glycolic acid on the growth of Phytophthora xanthoides: The experiment was set up with two treatments: Phytophthora xanthoides alone (CK) and a group treated with glycolic acid plus Phytophthora xanthoides (GlyA). 50% PDB medium, Phytophthora xanthoides spore suspension, and glycolic acid stock solution were added to 96-well plates to achieve a Phytophthora xanthoides spore suspension concentration of 1×10^8 CFU / mL and an addition volume of 10 μL. The final glycolic acid concentration was 0.1 mM. The CK group was treated with an equal volume of sterile water instead of glycolic acid. OD630 was measured after 72 h of incubation, with six replicates for each treatment.

[0035] Effect of glycolic acid on the growth of *Pseudomonas aeruginosa* YM3-423: The experiment was set up with two treatments: YM3-423 alone (CK) and glycolic acid plus YM3-423 (GlyA). 50% PDB medium, 20 μL of YM3-423 bacterial suspension, and glycolic acid stock solution were added to 96-well plates to achieve a final glycolic acid concentration of 0.1 mM and a final YM3-423 bacterial concentration of 1 × 10⁻⁶ mM. 8 CFU / mL; the CK group was treated with an equal volume of sterile water instead of glycolic acid. OD600 was measured after 48 h of incubation, with 6 replicates per treatment.

[0036] See results Figures 1-2 See Table 1. Compared with the CK group, the growth of *Phytophthora indicum* in the GlyA group was significantly decreased, while the growth of *Pseudomonas donghuensis* YM3-423 was significantly increased, indicating that glycolic acid can significantly inhibit the growth of *Phytophthora indicum* and promote the proliferation of *Pseudomonas donghuensis* YM3-423 (P < 0.05). These results suggest that glycolic acid can serve as a resource for *Pseudomonas donghuensis* YM3-423, and can be used to construct a *Pseudomonas donghuensis* YM3-423-glycolic acid synbiotic, providing a foundation for subsequent control of tobacco black shank disease.

[0037] Table 1. Effects of glycolic acid on the growth of Phytophthora nicotinica and Pseudomonas donghuensis YM3-423 Note: Data are expressed as Mean ± SD (n = 6). One-way ANOVA was used, and Tukey's HSD multiple comparisons were employed. Different letters indicate significant differences at the P < 0.05 level. Tables 2 and 3-4 were performed using the same method.

[0038] This invention also investigated the effect of glycolic acid on promoting the antagonistic effect of *Pseudomonas aeruginosa* YM3-423 against *Phytophthora indicum*: Experimental setup: 1) CK group: YM3-423 alone; 2) GlyA group: glycolic acid plus YM3-423. The antagonistic effect of YM3-423 on *Phytophthora indicum* was evaluated using the plate confrontation method. 20 μL of YM3-423 bacterial suspension (final bacterial concentration 1 × 10⁻⁶) was used. 8 A mixed bacterial suspension for confrontation was prepared in 96-well plates at a ratio of CFU / mL and a final glycolic acid concentration of 0.1 mM (10 μL of 2 mM stock solution was added). The remaining volume was made up with 50% PDB. For confrontation, 10 μL of the mixed suspension was spot-inoculated onto OA medium plates. Each treatment was repeated in 6 replicates, and the inhibition rate was measured after incubation. The 50% PDB medium was prepared by diluting PDB medium with sterile water at a 1:1 volume ratio. The OA medium was oat medium, prepared and sterilized according to standard methods.

[0039] See results Figure 3 See Table 2. Compared with YM3-423 alone, the combined use of glycolic acid and Pseudomonas aeruginosa YM3-423 further enhanced the inhibitory effect on Phytophthora nicotine, increasing the inhibition rate by approximately 2.2%.

[0040] Table 2. Effect of glycolic acid on the antagonistic ability of *Pseudomonas aeruginosa* YM3-423 against *Phytophthora indicum*. Example 2: Application of a *Pseudomonas donghuensis* synbiotic, using the *Pseudomonas donghuensis* synbiotic described in Example 1 for the control of tobacco black shank disease. The tobacco black shank disease is caused by *Phytophthora nicotineae*. Phytophthora nicotianae This causes tobacco black shank disease or tobacco root rot-related black shank diseases. The application involves applying the *Pseudomonas aeruginosa* synbiotic to the tobacco rhizosphere soil via root irrigation.

[0041] This example studies the potted plant effect of *Pseudomonas aeruginosa* YM3-423-glycolic acid synbiotic on the control of tobacco black shank disease: Based on the effects of glycolic acid on the growth of *Phytophthora indicum* and YM3-423, and the effect of glycolic acid on promoting the antagonistic effect of YM3-423 against *Phytophthora indicum*, a pot experiment was conducted to further investigate the effects of YM3-423-glycolic acid synbiotic on the disease index of tobacco black shank and the number of *Phytophthora indicum* in the rhizosphere soil. The concentration of *Phytophthora indicum* spore solution was 1×10⁻⁶. 8 CFU / mL.

[0042] Experimental setup and treatments: 1) 9+GlyA group: One week after tobacco transplanting, each pot was inoculated with 20mL of a synbiotic formulation solution. The formulation solution consisted of 2mL of YM3-423 bacterial suspension, 1mL of 2mM glycolic acid solution, and the remainder sterile water. The final concentration of glycolic acid was 0.1mM, and the final concentration of YM3-423 bacteria was 1×10⁻⁶. 8CFU / mL; 2) P. nicotianae group: Inoculated with an equal volume of sterile water. One week after treatment, each pot was inoculated with 20 mL of Phytophthora nicotianae spore solution. After inoculation with Phytophthora nicotianae, disease incidence was recorded as soon as symptoms appeared in the tobacco plants.

[0043] The severity level of tobacco black shank disease is determined by referring to the relevant disease survey methods of the national tobacco industry, and is graded on a per-plant basis. The specific grading standards are as follows: Incidence rate, disease index, and biocontrol rate are calculated using the following formula: Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100% Disease index (%) = [∑(Number of diseased plants at each level × Corresponding level) / (Total number of plants surveyed × Highest level value)] × 100% Biological control rate (%) = [1 - (disease index after treatment / disease index of CK)] × 100% Among them, CK is the group P. nicotianae, with a superlative value of 9.

[0044] Simultaneously, DNA was extracted from rhizosphere soil, and the number of *Phytophthora indicum* was detected by quantitative PCR. The results were expressed as log10 copies·g. -1 Earth represents soil.

[0045] See results Figures 4-5 Tables 3 and 4. Compared with the *P. nicotianae* group, the 9+GlyA group showed a significant decrease in the tobacco black shank disease index, with a biocontrol rate of approximately 59%, indicating that the combined use of glycolic acid and *Pseudomonas aeruginosa* YM3-423 can effectively reduce the severity of tobacco black shank disease (P < 0.05). Simultaneously, the number of *Phytophthora nicotianae* in the rhizosphere soil of the 9+GlyA group was significantly lower than that of the *P. nicotianae* group, with a reduction rate of approximately 34.7%, indicating that the *P. aeruginosa* YM3-423-glycolic acid synbiotic can not only reduce the tobacco black shank disease index but also reduce the accumulation of *Phytophthora nicotianae* in the rhizosphere soil, thereby improving the biocontrol effect on tobacco black shank disease.

[0046] Table 3. Effects of *Pseudomonas donghuensis* YM3-423-glycolic acid synbiotic on the disease index and biocontrol efficacy of tobacco black shank. Table 4. Effects of *Pseudomonas donghuensis* YM3-423-glycolic acid synbiotic on the abundance of *Phytophthora nicotine* in tobacco rhizosphere soil. 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 *Pseudomonas aeruginosa* synbiotic for controlling tobacco black shank disease, characterized in that, The *Pseudomonas donghuensis* synbiotic includes *Pseudomonas donghuensis* (… Pseudomonas donghuensis ) strain YM3-423 and glycolic acid; The Donghu Pseudomonas ( Pseudomonas donghuensis Strain YM3-423 was deposited at the China Center for Type Culture Collection on April 27, 2025, with accession number CCTCC NO:M 2025914.

2. The *Pseudomonas aeruginosa* synbiotic for controlling tobacco black shank according to claim 1, characterized in that, The Donghu Pseudomonas synbiotic is a formulation prepared using glycolic acid, Donghu Pseudomonas YM3-423 bacterial suspension, and sterile water. In the formulation of *Pseudomonas aeruginosa* synbiotic, the final concentration of glycolic acid was 0.05–0.2 mM, and the final concentration of *Pseudomonas aeruginosa* YM3-423 was 1 × 10⁻⁶ mM. 7 ~1×10 9 CFU / mL.

3. The *Pseudomonas aeruginosa* synbiotic for controlling tobacco black shank according to claim 1, characterized in that, In the formulation of *Pseudomonas aeruginosa* synbiotic, the final concentration of glycolic acid was 0.1 mM, and the final concentration of *Pseudomonas aeruginosa* YM3-423 was 1 × 10⁻⁶ mM. 8 CFU / mL.

4. The *Pseudomonas aeruginosa* synbiotic for controlling tobacco black shank disease according to claim 2, characterized in that, The *Pseudomonas aeruginosa* YM3-423 bacterial suspension was prepared by the following method: *Pseudomonas aeruginosa* YM3-423 strain was activated using LB solid medium; single colonies were picked and cultured in LB liquid medium at 28–30°C with shaking for 14–18 h; centrifuged, the supernatant was discarded, the bacterial cells were collected, and sterile water or sterile physiological saline was added to obtain the *Pseudomonas aeruginosa* YM3-423 bacterial suspension.

5. The application of the *Pseudomonas aeruginosa* synbiotic according to claim 1, characterized in that, The *Pseudomonas aeruginosa* synbiotic was used to control tobacco black shank disease.

6. The application according to claim 5, characterized in that, The aforementioned tobacco black shank disease is caused by Phytophthora indica. Phytophthora nicotianae Tobacco black shank caused by [the disease].

7. The application according to claim 5, characterized in that: The application described is to use a root irrigation method to apply glycolic acid in combination with Pseudomonas aeruginosa YM3-423 to control tobacco black shank disease.

8. A method for controlling tobacco black shank disease, characterized in that, The *Pseudomonas donghuensis* synbiotic as described in claim 1 or 2 is applied to the tobacco rhizosphere soil by root irrigation.

9. The method for preventing and controlling tobacco black shank disease according to claim 8, characterized in that, The volume of the *Pseudomonas donghuensis* synbiotic applied to each tobacco seedling is 10-30 mL.

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