Pseudomonas sp. and its application in tomato bacterial wilt and soil health

Abstract

The application discloses a Pseudomonas and application of the Pseudomonas in tomato bacterial wilt and soil health. Pseudomonas moraviensis ) T19G, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 35227. The strain has strong tomato rhizosphere colonization ability, can maintain a high number of bacterial populations for a long time after inoculation, can produce metabolic products with antibacterial activity, effectively inhibits the growth of bacterial wilt, and reduces the accumulation of pathogenic bacteria in tomato tissues. In addition, T19G can improve the tomato rhizosphere soil environment under bacterial wilt stress, and improve the content of part of soil nutrients and the activity of key enzymes. Therefore, the application provides a safe and efficient microbial technical scheme for green prevention and control of tomato bacterial wilt and soil microecological regulation.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, and in particular relates to a strain of Pseudomonas and its application in bacterial wilt of tomatoes and soil health. Background Technology

[0002] tomato( Solanum lycopersicum As a major vegetable crop with the second largest planting area and yield globally after potatoes, tomato production security is directly related to agricultural economy and food supply. However, affected by global climate change, extreme weather events are becoming more frequent, and crop growing environments are becoming increasingly complex. Tomatoes are facing increasingly severe disease pressures, leading to stunted plant development, reduced fruit quality, and sharp yield reductions, resulting in serious economic losses. Among the many diseases, those caused by Ralstonia solanacearum (Ralstonia solanacearum) are particularly prevalent. Ralstonia solanacearum Bacterial wilt caused by bacteria is widely recognized as one of the most destructive soil-borne diseases, severely hindering the sustainable development of the tomato industry. This pathogen has a strong ability to adapt to its environment, primarily spreading through contaminated soil. It can survive for extended periods in saprophytic form within diseased plant debris, with an overwintering period exceeding 14 months. Its infection pathways are diverse, including rain splash, irrigation water, agricultural tools, and mechanical transport by underground pests, entering the host plant through natural wounds or injuries at the root system or stem base.

[0003] Currently, the control strategy for this disease emphasizes integrated management, encompassing planting disease-resistant varieties, implementing crop rotation with non-host crops, adjusting soil physicochemical properties (such as improving structure and pH), and introducing biological control methods such as antagonistic microorganisms (such as specific bacteria and fungi) or predatory organisms (such as parasitic nematodes) to inhibit the proliferation and spread of the pathogen. However, due to R. solanacearu Bacterial wilt possesses the ability to persistently colonize in soil and spreads through a wide range of pathways. Traditional control methods relying on chemical fungicides not only fail to eradicate it but also easily induce drug resistance in the pathogen, while simultaneously causing environmental pollution and ecological risks. Therefore, its application is subject to numerous limitations, necessitating the development of safer, more efficient, and sustainable control technologies. In recent years, biological control has gradually become a hot research area for bacterial wilt. In particular, the application of plant-derived microorganisms and probiotics in control has received widespread attention, and biological control holds promise as an effective alternative to chemical pesticides. Among the many rhizosphere bacteria, Pseudomonas ( Pseudomonas *Pseudomonas molluscum* is one of the dominant groups in soil ecosystems and plant rhizosphere microbiomes. Microorganisms in this genus exert plant growth-promoting and disease-inhibiting effects through multiple mechanisms. Firstly, they limit the colonization space of pathogens by occupying ecological sites and competing for resources such as iron ions; secondly, they synthesize secondary metabolites with antibacterial activity, achieving direct inhibition of pathogens. *Pseudomonas molluscum* (…) Pseudomonas moraviensisOriginally isolated from soil samples along highways in the Czech Republic, this species belongs to the genus *Pseudomonas* in the family Pseudomonasceae, with typical type strains including DSM 16007. This species exhibits strong environmental adaptability and is widely found in various habitats, such as farmland soils and plant endophytic tissues. Previous studies have shown that some strains of this species possess dual functions of promoting plant growth and inhibiting disease.

[0004] Nevertheless, existing P. moraviensis There are still significant shortcomings in related research: (1) There are few verifications of the direct biocontrol effect against bacterial wilt of tomato, and most of them are limited to plate antagonism or short-term pot cultivation; (2) There is a lack of systematic evaluation of the ability of the strain to achieve long-term stable colonization (≥45 days) in the rhizosphere of tomato; (3) There are few reports on the synergistic effect of multiple mechanisms, including "direct antibacterial action + induction of plant defense enzyme systems + improvement of soil physicochemical properties and enzyme activity". The unstable colonization, single function, and short duration of action of biocontrol bacteria are still key bottlenecks restricting their large-scale application. Therefore, it is necessary to screen and identify a strain that has strong rhizosphere colonization ability, synergistic antibacterial action through multiple mechanisms, and significant improvement of soil health (increasing the content of total nitrogen, total phosphorus, total potassium, available potassium, and the activity of key enzymes such as phosphatase, urease, and sucrase). P. moraviensis Superior strains are of great theoretical significance and practical value for constructing an efficient and sustainable biological control and soil health regulation technology system for tomato bacterial wilt. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Pseudomonas and its application in bacterial wilt of tomatoes and soil health.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a strain of *Pseudomonas moravii* (… Pseudomonas moraviensis The Moravian Pseudomonas T19G is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35227 and deposit date of July 14, 2025.

[0007] Preferably, when the Moravian Pseudomonas T19G is inoculated into the rhizosphere of tomatoes, it maintains a viable count of not less than 1×10^7 CFU / g for 60 days after inoculation.

[0008] Secondly, the present invention provides a microbial agent for preventing and controlling bacterial wilt of tomatoes and / or improving the health of tomato rhizosphere soil, the microbial agent comprising Pseudomonas moravinatum T19G as described in claim 1 and agriculturally acceptable adjuvants.

[0009] Preferably, the viable concentration of *Pseudomonas molluscum* T19G in the microbial agent is 1×10^6 to 1×10^10 CFU / mL or CFU / g; The agriculturally acceptable adjuvants are selected from one or more of the following: carriers, protectants, stabilizers, fillers, or auxiliaries. The improvement of tomato rhizosphere soil health includes increasing or restoring at least one of soil organic matter, total nitrogen, and total potassium, and / or increasing the activity of at least one of soil phosphatase, catalase, and succinate dehydrogenase.

[0010] Thirdly, the present invention provides the application of the above-mentioned Pseudomonas Moravianus T19G in the preparation of antibacterial agents for the prevention and control of bacterial wilt of tomatoes.

[0011] Preferably, the tomato bacterial wilt is caused by Ralstonia solanacearum infection; the Moravian Pseudomonas T19G achieves disease control by inhibiting the growth of Ralstonia solanacearum, reducing the colonization of Ralstonia solanacearum in tomato tissues, or limiting the spread of Ralstonia solanacearum. In the antibacterial preparation, the viable bacterial concentration of Pseudomonas Moravia T19G is 1×10^6~1×10^10 CFU / mL or CFU / g.

[0012] Fourthly, the present invention provides the application of the above-mentioned Pseudomonas Moravia T19G in the preparation of regulators that improve rhizosphere soil health.

[0013] Preferably, the improvement of rhizosphere soil health includes increasing the content of rhizosphere soil organic matter, total nitrogen, and total potassium, and / or increasing the activity of rhizosphere soil phosphatase, catalase, succinate dehydrogenase A, urease, sucrase, or protease.

[0014] Fifthly, the present invention provides a method for preventing and controlling bacterial wilt of tomatoes, comprising the following steps: The bacteria containing Pseudomonas moravii as described in claim 1 ( Pseudomonas moraviensis The bacterial suspension of T19G was applied to the rhizosphere soil of tomato seedlings at the 2-3 leaf stage. The concentration of viable bacteria of Pseudomonas moravii T19G in the bacterial suspension was not less than 1×10^7 CFU / mL. The amount of bacterial suspension applied was 10 mL per tomato seedling. The bacterial suspension prevents bacterial wilt of tomatoes caused by Ralstonia solanacearum by inhibiting its growth and reproduction and reducing its accumulation in the rhizosphere and plant tissues.

[0015] Sixthly, the present invention provides a method for regulating the rhizosphere soil environment of tomato under Ralstonia solanacearum stress, comprising the following steps: After tomato seedlings are infected with Ralstonia solanacearum, the rhizosphere soil of the tomato seedlings is irrigated with a fungicide containing Pseudomonas moraceae as described in claim 1. Pseudomonas moraviensisThe bacterial suspension of Pseudomonas morawusta T19G, wherein the viable bacterial concentration of Pseudomonas morawusta T19G in the bacterial suspension is not less than 1×10^7 CFU / mL, and the irrigation amount is 10 mL per tomato seedling; The regulation of the rhizosphere soil environment of tomatoes under Ralstonia solanacearum stress includes increasing at least one of the organic matter, total nitrogen and total potassium in the rhizosphere soil, and / or increasing the activity of at least one of the phosphatase, catalase, succinate dehydrogenase A, urease, sucrase and protease in the rhizosphere soil.

[0016] The beneficial effects of this invention are as follows: This invention screened and obtained a strain of *Pseudomonas morawica* with excellent biocontrol properties. Pseudomonas moraviensis The invention describes T19G and its application in controlling bacterial wilt of tomato and improving the rhizosphere soil environment. Compared with existing biocontrol strains that have problems such as insufficient rhizosphere colonization ability, short duration of action, and single function, T19G of this invention has significant rhizosphere adaptability and long-term colonization advantages. After inoculation into the tomato rhizosphere, it can maintain a high level of bacterial population, providing a stable basis for its sustained biocontrol effect.

[0017] The T19G strain described in this invention exhibits a significant inhibitory effect on *Ralstonia solanacearum* (bacterial wilt fungus) of tomato. It inhibits *Ralstonia solanacearum* growth by producing extracellular metabolites with antibacterial activity, bacteriocin-related active substances, and various secondary metabolites. Furthermore, treatment with live T19G bacteria demonstrates a synergistic antibacterial effect exceeding the theoretical additive effect of single bacterial components and extracellular products, indicating a multi-component synergistic mechanism that effectively reduces the accumulation of *Ralstonia solanacearum* in the rhizosphere and plant tissues of tomato.

[0018] Furthermore, the T19G microbial inoculant provided by this invention can not only be used for the control of bacterial wilt in tomatoes, but also improve the rhizosphere microecological environment under the stress of Ralstonia solanacearum. After applying T19G, it can promote the recovery of nutrient indicators such as organic matter, total nitrogen, and total potassium in the rhizosphere soil, and increase the activity of key enzymes such as phosphatase, catalase, succinate dehydrogenase A, urease, sucrase, and protease, thereby enhancing the ecological function of the rhizosphere soil.

[0019] In summary, the *Pseudomonas molluscum* T19G described in this invention possesses advantages such as stable rhizosphere colonization, direct antibacterial activity, multi-mechanism synergistic control, and rhizosphere environment regulation. It can effectively overcome the problems of resistance and ecological risks associated with traditional chemical control, providing a safe, efficient, and sustainable microbial technology solution for green control of tomato bacterial wilt and soil health regulation. Attached Figure Description

[0020] Figure 1This study observes the morphology, tissue localization, and antibacterial effect of T19G on Ralstonia solanacearum. A shows a scanning electron microscope (SEM) image of T19G, B shows a SEM image of Ralstonia solanacearum (scale bar 5 μm), C shows a fluorescence in situ hybridization (FISH) image of tomato stem tissue after inoculation with Ralstonia solanacearum, D shows a FISH image of tomato stem tissue after inoculation with Ralstonia solanacearum and application of T19G (arrow "RS" indicates Ralstonia solanacearum signal, scale bar 100 μm), E shows the agar perforation antibacterial experiment (1 is sterile water control, 2 is LB medium control, 3 is T19G cell-free fermentation supernatant), and F shows the change in the number of Ralstonia solanacearum after adding different volumes of T19G treatment solution. Figure 2 A phylogenetic tree based on 16S rRNA gene sequences; Figure 3 A genomic circle diagram of strain T19G is provided, showing genomic characteristics such as chromosome coding sequences, functional classification, GC content, and GC shift. Figure 4 The antibacterial results of the T19G bacteriocin-related gene and its expression product; Wherein, A represents the gene location, length, and annotation information of gene1779 and gene5326; B represents the SDS-PAGE results of gene1779 expression product; C represents the SDS-PAGE results of gene5326 expression product; D represents the antibacterial rate of gene1779 expression product at doses of 5–80 μg; and E represents the antibacterial rate of gene5326 expression product at doses of 5–80 μg. Figure 5 The effects of T19G on the physicochemical properties of tomato rhizosphere soil were investigated. In this study, A represents pH, B represents organic matter (OM), C represents total nitrogen (TN), D represents total phosphorus (TP), E represents total potassium (TK), F represents available nitrogen (AN), G represents available phosphorus (AP), and H represents available potassium (AK). Figure 6 The effects of T19G on enzyme activities in tomato rhizosphere soil were investigated. A represents urease activity, B represents phosphatase activity, C represents sucrase activity, D represents catalase (CAT) activity, E represents protease activity, F represents succinate dehydrogenase A (SDHA) activity, G represents nitrate reductase (NR) activity, H represents alanine aminotransferase (ALT) activity, and I represents aspartate decarboxylase (ASPD) activity. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various modifications, substitutions, or variations to the technical solutions of this invention without departing from the spirit and substance of this invention, and all such modifications should fall within the scope of protection of this invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0022] In the following experiments, CK represents the healthy control group that was not inoculated with Ralstonia solanacearum and was irrigated with sterile water; RS represents the pathogen treatment group that was inoculated with Ralstonia solanacearum and irrigated with sterile water; and RS+T19G represents the treatment group that was inoculated with Ralstonia solanacearum and then irrigated with T19G bacterial suspension. Different lowercase letters on the bar chart of the same indicator indicate significant differences between groups (P < 0.05), and ns indicates no significant difference; "—" in the table indicates below the detection limit or not detected.

[0023] Example 1: Isolation and Identification of Strains (1) Fifteen sampling points were randomly selected from multiple tomato cultivation fields in Zhejiang Province, China. Approximately 500 g of fresh tomato rhizosphere soil was collected from each sampling point. The rhizosphere soil was a soil layer of about 1–2 mm close to the root surface. The samples were placed in sterile bags, transported at 4 ℃, and processed within 24 hours.

[0024] (2) Take 10 g of rhizosphere soil and add 90 mL of sterile phosphate buffer (PBS, pH 7.4). Shake for 30 min and then perform a tenfold serial dilution. Spread 100 μL of the diluted solution onto LB agar plates and incubate at 30 ℃ for 48–72 hours. Select colonies with different morphologies and streak them continuously for purification 3–5 times to obtain multiple candidate isolates of Pseudomonas, one of which was named T19G.

[0025] (3) The morphology of T19G and Ralstonia solanacearum was observed using scanning electron microscopy. T19G cells were rod-shaped, often distributed singly or in clusters, with a relatively rough cell surface; Ralstonia solanacearum cells were also rod-shaped, but with a smoother surface. The results are shown in the figures below. Figure 1 A and Figure 1 B.

[0026] (4) Extract T19G genomic DNA using universal primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) The 16S rRNA gene was amplified using 1492R (5′-GGTTACCTTGTTACGACTT-3′).

[0027] (5) The amplified sequences were compared with sequences in publicly available databases, and phylogenetic analysis was performed using MEGA 11 software. The results are as follows: Figure 2 As shown.

[0028] The results showed that the 16S rRNA gene sequence of T19G was similar to... Pseudomonas moraviensis The similarity to WFdJ (HG000013) was 100%. Based on morphological characteristics and subsequent whole-genome analysis, this strain was identified as *Pseudomonas moravii* (…). Pseudomonas moraviensis )T19G.

[0029] Example 2: Detection of the colonization ability of T19G in the rhizosphere of tomatoes For evaluation Pseudomonas moraviensis To investigate the colonization ability and duration of T19G in the tomato rhizosphere, other representative Pseudomonas isolates obtained from the same batch were used as controls in a tomato rhizosphere inoculation experiment under the same conditions.

[0030] (1) Preparation of test bacterial solution Will P. moraviensis T19G and P. moraviensis T2P, Pseudomonas sp. T3D, P. moraviensis T4G, P. moraviensis T4H, P. moraviensis T6R, P. moraviensis T14T, P. moraviensis T1K, P. moraviensis Z10L and Pseudomonas sp. T3A was inoculated into LB liquid medium and cultured with shaking at 30°C and 180 rpm until the logarithmic growth phase.

[0031] The obtained culture medium was centrifuged at 4 ℃ and 6000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile water and resuspended. The viable cell concentration was determined by plate count method, and all bacterial suspensions were adjusted to approximately 1×10^7 CFU / mL with sterile water for later use.

[0032] (2) Tomato rhizosphere inoculation Tomato seedlings with uniform growth, free from pests and diseases, and at the 2-3 leaf stage were selected for the experiment. Each strain was set up as an independent treatment group, with 3 biological replicates for each treatment, and each replicate including 5 tomato seedlings.

[0033] Before inoculation, tomato seedlings were irrigated normally to ensure a consistent moisture content in the rhizosphere. During inoculation, 10 mL of the appropriate bacterial suspension was slowly applied around the rootstock of each seedling, ensuring the suspension fully penetrated the rhizosphere. The inoculation amount per seedling was approximately 1 × 10⁸ CFU. After inoculation, the seedlings were cultured under the same temperature, light, irrigation, and fertilization conditions. No further inoculation with the test strain was performed during the experiment.

[0034] (3) Rhizosphere soil sampling Tomato rhizosphere soil was collected on days 10, 20, 30, 40, 50, and 60 after inoculation. During sampling, the tomato plants were removed from the substrate, and the soil that was not tightly bound to the roots was gently shaken off. The soil still attached to the root surface and around the roots was collected as rhizosphere soil.

[0035] Tomato plants were randomly selected for each biological replicate, and the collected rhizosphere soil was thoroughly mixed. 10.0 g of rhizosphere soil was accurately weighed and added to an Erlenmeyer flask containing 90 mL of sterile physiological saline. The flask was shaken at 180 rpm for 30 min to fully release the bacteria, yielding a 10^-1 soil suspension. This suspension was then serially diluted 10-fold using sterile physiological saline.

[0036] (4) Determination of colony size and verification of strains Take 100 μL of soil suspension at appropriate dilutions and spread it evenly on a selective medium suitable for Pseudomonas growth. Prepare three parallel plates for each dilution and incubate at 30 °C for 24–48 h. Select plates with colony counts of 30–300 CFU for counting, and calculate the colony density of the tested strain in the tomato rhizosphere soil based on the dilution factor. The calculation formula is as follows: Colony size (CFU / g) = number of colonies on a plate × dilution factor × 10 ÷ rhizosphere soil mass.

[0037] Based on the colony morphology, color, edge characteristics, and growth characteristics of the tested strains, suspected target colonies were initially screened. At least five representative colonies were randomly selected from each sample and verified by strain-specific PCR or 16S rRNA gene sequence comparison to eliminate interference from other Pseudomonas species in the rhizosphere soil. The detection limit of this method was 1.0 × 10^1 CFU / g rhizosphere soil; samples below the detection limit or not detected were indicated by "—". The colonization results for each treatment are shown in Table 1.

[0038] Table 1. Results of constant values ​​for each treatment

[0039] Table 1 shows that the colonization capacity of different *Pseudomonas* isolates in the tomato rhizosphere varied significantly. The T19G strain achieved a rhizosphere colonization of 1.37 × 10⁷ CFU / g on day 10 post-inoculation, indicating that this strain could adapt to the tomato rhizosphere environment and establish a high initial colonization population within a short period after inoculation. By day 20, the T19G colonization increased to 7.75 × 10⁷ CFU / g, approximately 5.66 times that of day 10, demonstrating that T19G not only survived in the rhizosphere but also proliferated using root exudates and rhizosphere nutrients.

[0040] From day 20 to 60 post-inoculation, the colonization rate of T19G remained consistently between 6.94 × 10^7 and 7.99 × 10^7 CFU / g, without showing a continuous decline with prolonged culture time. Specifically, the colonization rate was 7.99 × 10^7 CFU / g on days 30 and 40, and still reached 7.75 × 10^7 CFU / g on day 60, indicating that after establishing a stable rhizosphere population, T19G could maintain colonization in the tomato rhizosphere for at least 60 days.

[0041] In contrast, the colonization levels of the other tested isolates all showed varying degrees of rapid decline. On day 10 post-inoculation, the strain with the highest colonization level besides T19G was T6R, at 7.23 × 10⁵ CFU / g, only about 1 / 19th that of T19G; by day 20, the colonization level of T19G was approximately 1126 times that of T6R. On day 30, only T19G, T2P, and T6R remained detectable, with T19G's colonization level approximately 1.16 × 10⁶ times that of T2P and 5.15 × 10⁵ times that of T6R, respectively. By day 40, only T19G and T6R remained detectable, but T19G's colonization level was approximately 1.13 × 10⁶ times that of T6R; on days 50 and 60, only T19G maintained a detectable high level of colonization, while the other strains were below the detection limit.

[0042] Most strains, including T2P, T3D, T4G, T4H, T14T, T1K, Z10L, and T3A, decreased to below the detection limit within 20–30 days after inoculation. Although T6R had relatively good early survival ability, its colonization rate still decreased from 7.23 × 10^5 CFU / g on day 10 to 7.10 × 10^1 CFU / g on day 40, a decrease of more than four orders of magnitude, indicating that this strain could not form a stable and continuous rhizosphere dominant population.

[0043] The results above indicate that, compared with other Pseudomonas isolates obtained in the same batch, T19G not only has stronger early rhizosphere adaptation and proliferation capabilities, but also outstanding long-term survival and population maintenance capabilities. T19G maintained a colonization level of 10^7 CFU / g throughout the 60-day experimental period, demonstrating a significant strain-specific rhizosphere colonization advantage, which can provide a stable inoculum basis for its sustained role in tomato growth promotion, disease control, or rhizosphere microecological regulation.

[0044] Example 3: Inhibitory effect of T19G on Ralstonia solanacearum and evaluation using the Bliss independent model. (1) The antibacterial activity of T19G was preliminarily evaluated using the agar diffusion method. *Ralstonia solanacearum* (Tomato Ralstonia solanacearum) was used as a preliminary method. R. solanacearum Add 1% inoculum to LB solid medium, mix well, pour into a plate, and after the medium solidifies, punch wells and add 20 μL of the sample to be tested to each well. Figure 1 In E, 1 represents the sterile water control, 2 represents the LB medium control, and 3 represents the T19G cell-free fermentation supernatant. Observations after culture revealed that a clear, transparent inhibition zone formed around the wells of the T19G cell-free fermentation supernatant, while no obvious inhibition zone was formed in the sterile water and LB medium controls. This indicates that T19G produced extracellular metabolites with antibacterial activity during fermentation.

[0045] (2) Further, the inhibitory ability of different Pseudomonas isolates on Ralstonia solanacearum was compared using a liquid co-culture system. The fermentation broth of each strain was added to the culture system containing Ralstonia solanacearum. After the culture was completed, the number of Ralstonia solanacearum was detected, and the inhibition rate was calculated according to the formula: inhibition rate = (number of Ralstonia solanacearum in the control group - number of Ralstonia solanacearum in the treatment group) / number of Ralstonia solanacearum in the control group × 100%. The results are shown in Table 2.

[0046] Table 2. Inhibitory effect of *Pseudomonas rhizosphereus* isolates on *Ralstonia solanacearum*.

[0047] As shown in Table 2, among the 10 tested Pseudomonas isolates, only P. moraviensis T19G exhibited significant inhibitory activity against Ralstonia solanacearum, with an inhibition rate of 90.61±4.57%, while the inhibition rates of other isolates were 0%. Further increasing the amount of cell-free fermentation supernatant added to T19G (100–500 μL) revealed that the number of Ralstonia solanacearum bacteria gradually decreased with increasing supernatant addition, showing a clear dose-dependent inhibitory trend. Figure 1 F).

[0048] (3) To analyze the contribution relationship among different components of T19G, the T19G treatment system was divided into three categories: live bacterial culture, cell-free fermentation supernatant, and inactivated bacterial cells. The Bliss independent model was used to evaluate the synergistic effect. Among them, EA is the cell-free supernatant effect, EB is the inactivated bacterial cell effect, EBliss = EA + EB - EA × EB / 100, ΔE = Eobs - EBliss. The results are shown in Table 3.

[0049] Table 3. Evaluation of the effects of live T19G bacteria using the Bliss independent model.

[0050] Note: Except for ΔE, all other effect values ​​are in percentage. The 95% confidence interval for ΔE is calculated using a t-distribution, and the statistical test is a two-tailed one-sample t-test comparing ΔE with 0.

[0051] Table 3 shows that the cell-free supernatant effect (EA) of T19G was 34.83±2.93%, the inactivated bacterial cell effect (EB) was 22.91±2.83%, and the calculated Bliss theoretical expected effect (EBliss) was 49.77±2.75%. However, the actual inhibitory effect (Eobs) of the live T19G culture reached 90.61±4.57%, significantly higher than the theoretical value. The Bliss bias ΔE was 40.84±2.83, with a 95% confidence interval of 37.32–44.35, and the difference was statistically significant (P<0.001). The results indicate that there is a significant synergistic effect among the continuous metabolism, extracellular antibacterial substance production, and bacterial cell component effects of live T19G, collectively enhancing its inhibitory ability against *Ralstonia solanacearum*.

[0052] Example 4: Localization observation of T19G's inhibition of Ralstonia solanacearum in tomato tissue To further verify the antibacterial effect of T19G in the infected environment of tomato plants, fluorescence in situ hybridization (FISH) was used to observe the distribution of Ralstonia solanacearum in tomato tissues after T19G treatment.

[0053] (1) Select tomato seedlings with uniform growth and at the 2-3 leaf stage for the experiment, and set up a Ralstonia solanacearum treatment group (RS group) and a Ralstonia solanacearum + T19G treatment group (RS+T19G group): The RS group was inoculated into the rhizosphere with Ralstonia solanacearum suspension, followed by irrigation with 10 mL of sterile water; After inoculating the RS+T19G group with Ralstonia solanacearum under the same conditions, 10 mL of T19G bacterial suspension was applied to the rhizosphere, with the T19G live bacteria concentration adjusted to 1×10^7 CFU / mL. Samples were taken from each treatment after culturing under the same environmental conditions for 3 days.

[0054] (2) Tomato stem tissue was taken, fixed, dehydrated, and sectioned. In situ hybridization was performed using oligonucleotide probes designed with fluorescent groups specifically targeting the Ralstonia solanacearum sequence. After hybridization, non-specific binding signals were removed by washing. The distribution of Ralstonia solanacearum in the tomato tissue was observed and recorded using a fluorescence microscope. The results are as follows: Figure 1 C and Figure 1 As shown in D.

[0055] The results showed that a large number of Ralstonia solanacearum-specific fluorescent signals were observed in the vascular tissue region of tomato stems in the RS group, and the signals were continuously distributed, indicating that Ralstonia solanacearum had formed a significant infection and accumulation in the tomato tissue. Figure 1 C). In contrast, the Ralstonia solanacearum-specific fluorescence signal was significantly reduced in the RS+T19G group, with only a few scattered fluorescent areas observed. Figure 1 (D) This indicates that T19G treatment can effectively reduce the colonization level of Ralstonia solanacearum in tomato tissues. These results demonstrate that T19G not only exhibits significant antagonistic ability against Ralstonia solanacearum in in vitro culture systems, but also limits the spread and accumulation of Ralstonia solanacearum in tissues in actual plant infection environments, thus playing a potential role in biocontrol.

[0056] Example 5: Whole-genome characterization of strain T19G To elucidate the genetic basis and potential functional genome composition of strain T19G, whole-genome sequencing and bioinformatics analysis were performed on T19G.

[0057] (1) T19G was inoculated into LB liquid medium and cultured at 30 ℃ and 180 rpm for 16–20 h. After the culture was completed, the cells were collected by centrifugation at 6000×g for 3 min, and high-quality genomic DNA was extracted after cell lysis.

[0058] (2) DNA integrity was detected by agarose gel electrophoresis, and DNA purity was detected by micro-spectrophotometer. Genomic DNA samples that met the sequencing requirements were sequenced in real time using the PacBio RS II platform.

[0059] (3) After quality filtering, the raw sequencing data were assembled to obtain the complete T19G genome sequence. Glimmer software was used to predict protein-coding sequences (CDS), and Tandem Repeats Finder was used to identify tandem repeat sequences. Barrnap and tRNAscan-SE were used to predict rRNA and tRNA genes, respectively. BLAST and HMMER were used to perform functional annotation of the predicted genes, and classification analysis was performed using NR, Pfam, COG, GO, and KEGG databases. At the same time, antiSMASH software was used to predict the gene clusters for the biosynthesis of secondary metabolites.

[0060] See the complete genome map of T19G. Figure 3 The genome annotation results are shown in Table 4, and the analysis results of secondary metabolite synthesis gene clusters are shown in Table 5.

[0061] Table 4. Genome annotation features of strain T19G

[0062] Table 5. Secondary metabolite synthesis gene clusters of strain T19G

[0063] The results showed that the T19G genome is a complete circular chromosome with a size of 6,434,684 bp and a G+C content of 60.52%. A total of 5,642 protein-coding genes, 73 tRNA genes, 19 rRNA genes, and 90 sRNA genes were predicted. Functional annotation results indicate that T19G possesses abundant gene resources related to metabolic regulation and environmental adaptation.

[0064] Further antiSMASH analysis revealed 14 potential secondary metabolite synthesis gene clusters in the T19G genome, including NRPS, RiPP-like, arylpolyene, β-lactone, siderophore, and redox cofactor-related gene clusters. Cluster 6 showed 100% similarity to the anikasin-related gene cluster, while clusters 4 and 12 showed high homology to the pyoverdin-related gene cluster, suggesting that T19G may have the ability to synthesize antimicrobial metabolites and iron-competitive molecules.

[0065] Combined with the significant inhibitory effect of T19G on Ralstonia solanacearum shown in Example 3, the above genomic analysis results indicate that T19G has the potential to synthesize multiple types of secondary metabolites, and its rich functional gene composition may provide a genetic basis for its rhizosphere competition, biocontrol effects and sustained antagonistic ability.

[0066] Example 6: Validation of the antibacterial activity of the T19G bacteriocin-related gene expression product (1) To further elucidate the molecular basis of T19G's inhibition of Ralstonia solanacearum, potential antibacterial functional genes were screened based on the genome-wide annotation results obtained in Example 5. The results showed that two bacteriocin-related genes exist in the T19G genome, named gene1779 and gene5326, respectively. Gene1779 is located in the region of chromosome 1967157–1967525 bp, with a coding region length of 369 bp, and is predicted to encode a bacteriocin-related protein; gene5326 is located in the region of chromosome 5997714–5998700 bp, with a coding region length of 987 bp, and is predicted to encode a lipid II-degrading bacteriocin-related protein. The location and structural information of the relevant genes are shown in [reference needed]. Figure 4 A.

[0067] (2) Using T19G genomic DNA as a template, the complete coding sequences of gene1779 and gene5326 were amplified and ligated into prokaryotic expression vectors, which were then transformed into expression host bacteria for induced expression. The expression cells were collected, and the corresponding recombinant expression products were obtained through disruption, centrifugation, and protein purification. SDS-PAGE was used to detect the purified products. The results showed that specific protein bands corresponding to the theoretical molecular weight appeared in both the gene1779 and gene5326 expression systems, and the purified bands were clear, indicating that the target expression proteins (…) were obtained. Figure 4 B and Figure 4 C).

[0068] (3) Further evaluate the direct inhibitory effect of the two bacteriocin-associated proteins on Ralstonia solanacearum. The purified expression products of gene1779 and gene5326 were added to the Ralstonia solanacearum culture system to make the final concentrations 5, 10, 20, 40 and 80 μg, respectively. After the culture was completed, the number of Ralstonia solanacearum was detected and the inhibition rate was calculated.

[0069] The results are as follows Figure 4 D and Figure 4 As shown in Figure E, with increasing amounts of the expression product, the inhibition rates of both gene1779 and gene5326 protein treatment groups against *Ralstonia solanacearum* gradually increased, exhibiting a clear dose-dependent effect. Among them, the gene5326 expression product showed a higher inhibitory effect at the same concentration, suggesting that lipid II-degradable bacteriocin-associated proteins may be an important active factor in the anti-*Ralstonia solanacearum* activity of T19G.

[0070] The above results indicate that the bacteriocin-associated gene encoded in the T19G genome can produce a protein product with direct antibacterial activity. Its expression product participates in the antagonistic process of T19G against Ralstonia solanacearum, providing a theoretical basis for elucidating the molecular antibacterial mechanism of T19G and developing novel microbial control agents.

[0071] Example 7: Effects of T19G on the physicochemical properties and enzyme activity of tomato rhizosphere soil (1) To evaluate the ecological regulation effect of T19G in the rhizosphere environment of tomatoes, tomato seedlings with the same growth status and in the 2-3 leaf stage were selected for pot experiment. Normal control group (CK), Ralstonia solanacearum treatment group (RS) and Ralstonia solanacearum + T19G treatment group (RS+T19G) were set up.

[0072] In the CK group, no bacterial wilt was inoculated, and each plant was irrigated with 10 mL of sterile water. The RS group was inoculated into the rhizosphere with Ralstonia solanacearum suspension, followed by irrigation of each plant with 10 mL of sterile water; After inoculation with Ralstonia solanacearum, each plant in the RS+T19G group was irrigated with 10 mL of T19G bacterial suspension, and the concentration of live bacteria in the suspension was adjusted to 1×10^7 CFU / mL.

[0073] After each treatment was cultured for another 10 days under the same environmental conditions, tomato rhizosphere soil was collected for testing.

[0074] (2) The pH, organic matter (OM), total nitrogen (TN), total phosphorus (TP), total potassium (TK), available nitrogen (AN), available phosphorus (AP), and available potassium (AK) contents of the rhizosphere soil in each treatment were determined using soil agricultural chemical analysis methods. The results are shown in the table below. Figure 5 .

[0075] The results showed that after Ralstonia solanacearum infection, some nutrient indicators in the rhizosphere soil of the RS group changed, with organic matter, total nitrogen, and total potassium levels decreasing compared to the CK group. After T19G application, the soil organic matter content in the RS+T19G group showed an increasing trend compared to the RS group, the total nitrogen content increased significantly, and the total potassium content recovered to near the CK group level; the total phosphorus content was between that of the CK and RS groups. In contrast, pH, available nitrogen, available phosphorus, and available potassium did not show significant differences among the treatment groups, indicating that T19G mainly affected rhizosphere soil nutrient transformation processes without causing significant changes in the basic soil pH environment.

[0076] (3) The activities of key enzymes in rhizosphere soil were further determined using commercially available detection kits, including urease, phosphatase, sucrase, catalase (CAT), protease, succinate dehydrogenase A (SDHA), nitrate reductase (NR), alanine aminotransferase (ALT), and aspartate decarboxylase (ASPD). The results are shown in […]. Figure 6 .

[0077] The results showed that Ralstonia solanacearum stress led to a decrease in the activity of various soil enzymes in the RS group. Compared with the RS group, after T19G application, the activities of rhizosphere soil phosphatase, catalase, and succinate dehydrogenase A were significantly increased; the activities of urease, sucrase, and protease showed a clear recovery trend; while the activities of nitrate reductase, alanine aminotransferase, and aspartate decarboxylase did not differ significantly among the three groups.

[0078] comprehensive Figure 5 and Figure 6 The results showed that T19G could alleviate the decline in rhizosphere ecological function caused by Ralstonia solanacearum infection, improve the rhizosphere microenvironment of tomatoes by regulating soil nutrient status and promoting the recovery of key metabolic enzyme activity, and provide a favorable ecological foundation for its continuous colonization and biological control.

Claims

1. A strain of Pseudomonas moravii ( Pseudomonas moraviensis T19G, characterized in that, The *Pseudomonas molluscum* T19G strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35227 and deposit date of July 14, 2025.

2. The *Pseudomonas molluscum* T19G according to claim 1, characterized in that, When the Moravian Pseudomonas T19G was inoculated into the rhizosphere of tomatoes, it maintained a viable count of not less than 1×10^7 CFU / g for 60 days after inoculation.

3. A microbial inoculant for controlling bacterial wilt of tomatoes and / or improving the health of tomato rhizosphere soil, characterized in that, The microbial agent includes Pseudomonas Moravia T19G as described in claim 1 and agriculturally acceptable adjuvants.

4. The microbial agent according to claim 3, characterized in that, The viable concentration of *Pseudomonas molluscum* T19G in the microbial agent is 1×10^6 to 1×10^10 CFU / mL or CFU / g. The agriculturally acceptable adjuvant is selected from one or more of the following: carrier, protectant, stabilizer, filler, or auxiliary agent; The improvement of tomato rhizosphere soil health includes increasing or restoring at least one of soil organic matter, total nitrogen, and total potassium, and / or increasing the activity of at least one of soil phosphatase, catalase, and succinate dehydrogenase.

5. The application of Pseudomonas Moravia T19G as described in claim 1 in the preparation of an antibacterial agent for controlling bacterial wilt of tomato.

6. The application according to claim 5, characterized in that, The bacterial wilt of tomato is caused by infection with Ralstonia solanacearum; the Pseudomonas moravii T19G strain controls the disease by inhibiting the growth of Ralstonia solanacearum, reducing the colonization of Ralstonia solanacearum in tomato tissues, or limiting the spread of Ralstonia solanacearum. In the antibacterial preparation, the viable bacterial concentration of *Pseudomonas molluscum* T19G is 1×10^6 to 1×10^10 CFU / mL or CFU / g.

7. The use of Pseudomonas Moravia T19G as described in claim 1 in the preparation of a regulator to improve rhizosphere soil health.

8. The application according to claim 7, characterized in that, The improvement of rhizosphere soil health includes increasing the content of rhizosphere soil organic matter, total nitrogen, and total potassium, and / or increasing the activity of rhizosphere soil phosphatase, catalase, succinate dehydrogenase A, urease, sucrase, or protease.

9. A method for preventing and controlling bacterial wilt in tomatoes, characterized in that, Includes the following steps: The bacteria containing the Pseudomonas moravii strain described in claim 1 ( Pseudomonas moraviensis The bacterial suspension of T19G was applied to the rhizosphere soil of tomato seedlings at the 2-3 leaf stage. The concentration of viable bacteria of Pseudomonas moravii T19G in the bacterial suspension was not less than 1×10^7 CFU / mL. The amount of bacterial suspension applied was 10 mL per tomato seedling. The bacterial suspension prevents bacterial wilt of tomatoes caused by Ralstonia solanacearum by inhibiting its growth and reproduction and reducing its accumulation in the rhizosphere and plant tissues.

10. A method for regulating the rhizosphere soil environment of tomato under Ralstonia solanacearum stress, characterized in that, Includes the following steps: After tomato seedlings are infected with Ralstonia solanacearum, the rhizosphere soil of the tomato seedlings is irrigated with a fungicide containing Pseudomonas moravae as described in claim 1. Pseudomonas moraviensis The bacterial suspension of Pseudomonas molluscum T19G, wherein the viable bacterial concentration of Pseudomonas molluscum T19G in the bacterial suspension is not less than 1×10^7 CFU / mL, and the irrigation amount is 10 mL per tomato seedling; The regulation of the rhizosphere soil environment of tomatoes under Ralstonia solanacearum stress includes increasing at least one of the organic matter, total nitrogen and total potassium in the rhizosphere soil, and / or increasing the activity of at least one of the phosphatase, catalase, succinate dehydrogenase A, urease, sucrase and protease in the rhizosphere soil.

Images