Defense pseudomonas and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly discloses a strain of defensive pseudomonas and application thereof, the strain of defensive pseudomonas TYQ2 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.27211, and the strain of defensive pseudomonas TYQ2 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.27211. The inventor finds that the pseudomonas defensinoides has a growth promoting function of secreting siderophore and solubilizing phosphorus, has the capabilities of interfering tropism migration of nematodes and killing nematodes, and further finds that the pseudomonas defensinoides has the capability of inhibiting normal growth of fusarium oxysporum and the capability of degrading straws of different crops, so that the inventor considers that the pseudomonas defensinoides has an important application prospect in the fields of agriculture and environmental protection. The anti-pseudomonas has a remarkable application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and specifically discloses a strain of Pseudomonas protegens and its applications. Background Art

[0002] Modern agriculture is facing the superposition effect of multiple challenges: the rampant soil-borne diseases, the continuous decline of soil fertility, and the inefficient treatment of agricultural waste have become the core bottlenecks restricting sustainable development. Soil-borne pathogens represented by root-knot nematodes (Meloidogynespp.) and Fusarium oxysporum seriously affect the growth and yield of crops. Although traditional chemical and physical control methods can temporarily relieve diseases, their residual toxicity and destructive impact on the soil microbial community cannot be underestimated, and environmentally friendly alternative solutions are urgently needed. At the same time, a large amount of agricultural waste (such as tomato and cucumber straws) generated during vegetable production is usually treated by in-situ returning to the field, but this practice has significant drawbacks: the straw that has not been fully degraded not only becomes the overwintering place for pathogens and pests, but also the allelochemicals released during its slow decomposition process will inhibit crop growth, forming a vicious cycle of "waste - pathogen accumulation - crop yield reduction".

[0003] Currently, there are still significant limitations in the practical application of biological control technologies. Commercial microbial agents generally have the problem of single function and are difficult to meet multiple requirements such as disease prevention and control, soil improvement, and waste degradation at the same time. This limitation leads to the need to combine and use multiple special microbial agents in agricultural production, which not only greatly increases the use cost, but also may reduce the overall control effect due to the mutual competition among different microbial populations. More critically, most existing strains have insufficient adaptability to complex field environments. Under conditions such as soil pH fluctuations, salt changes, or organic matter differences, their survival rate and functional activity often decrease significantly, seriously restricting the actual application effect. These dual defects of single function and insufficient environmental adaptability make the existing microbial technologies difficult to meet the needs of modern agriculture for comprehensive solutions.

[0004] Industrial practices have shown that it has become an urgent task to develop microbial strains with broad-spectrum environmental adaptability and multi-functional integration characteristics. An ideal biocontrol strain should have the following characteristics: (1) inhibiting the infection of various pathogens in production by secreting metabolites; (2) activating soil nutrients; (3) accelerating the harmless degradation of agricultural waste; (4) adapting to diverse field environments (such as pH and salinity fluctuations); (5) promoting crop growth. Some studies have shown that Pseudomonas protegens, as a potential plant protection microorganism, has a wide application prospect in agriculture, especially in controlling soil-borne diseases, promoting crop growth, and increasing crop yields. Based on this background, the present invention isolated and purified strain TYQ2, and verified its potential in promoting growth, interfering with nematode migration, inhibiting the growth of Fusarium oxysporum, degrading straw, etc. through a series of experiments, and deeply explored its potential in practical applications. It provides a new idea for modern agricultural biological control technology. Summary of the Invention

[0005] In view of the above problems, the present invention discloses a Pseudomonas protegens and its application.

[0006] In order to achieve the above invention object, the technical solution of the present invention is as follows:

[0007] A Pseudomonas protegens, the Pseudomonas protegens strain TYQ2 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 27211.

[0008] The present invention also discloses a microbial inoculum containing the above-mentioned Pseudomonas protegens cells and / or its metabolites and / or its fermentation broth.

[0009] The present invention also discloses the use of the above-mentioned bacterium or bacterium agent in the preparation of a preparation for interfering with the migration of Meloidogyne incognita.

[0010] The present invention also discloses the use of the above-mentioned bacterium or bacterium agent in the preparation of a preparation for controlling Meloidogyne incognita.

[0011] The present invention also discloses the use of the above-mentioned bacterium or bacterium agent in the preparation of a preparation for inhibiting the growth of Fusarium oxysporum.

[0012] The present invention also discloses the use of the above-mentioned bacterium or bacterium agent in the preparation of a preparation for degrading the straw of different vegetable crops.

[0013] The present invention also discloses the use of the above-mentioned bacterium or bacterium agent in the preparation of a preparation for dissolving insoluble phosphorus.

[0014] The present invention also discloses the use of the above-mentioned bacterium or bacterium agent in the preparation of a preparation for chelating Fe elements in the soil.

[0015] The present invention has the following beneficial effects:

[0016] The present invention discloses a Pseudomonas protegens and its application. The Pseudomonas protegens TYQ2 disclosed by the present invention exhibits the characteristics of significantly promoting the growth of cucumber seedlings and enhancing their resistance to adversity. TYQ2 has the ability to dissolve insoluble phosphorus and chelate Fe ions in the soil. In addition, TYQ2 shows a significant effect on the migration and lethality of nematodes, especially in terms of the lethality to the second-stage larvae of Meloidogyne incognita, and its effect is significantly better than that of the control. In addition, TYQ2 shows a strong antagonistic effect in the growth inhibition test of Fusarium oxysporum. In terms of straw degradation, the degradation rates of tomato, cucumber, pepper, and eggplant straw by this strain reach 38.43%, 38.04%, 29.89%, and 22.98% respectively within two weeks. In summary, the TYQ2 strain shows good application potential in promoting plant growth, enhancing plant stress resistance, and straw degradation and other aspects.

[0017] The information on the preservation of the strain is as follows:

[0018] Name of the preservation unit: China General Microbiological Culture Collection Center;

[0019] Address of the preservation unit: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0020] Date of preservation: April 27, 2023;

[0021] Preservation number: CGMCC No. 27211,

[0022] Taxonomic nomenclature: Pseudomonas protegens. Description of the drawings

[0023] Figure 1 is the colony morphology of TYQ2;

[0024] Figure 2 is the phylogenetic tree diagram of TYQ2;

[0025] Figure 3 is the fingerprint diagram for the determination of the ability of TYQ2 to utilize different carbon sources and its chemical sensitivity

[0026] Figure 4 is the phosphorus-solubilizing characteristic diagram of TYQ2;

[0027] Figure 5 is the phosphorus-solubilizing characteristic diagram of TYQ2 under different NaCl concentrations and pH gradients;

[0028] Figure 6 is the siderophore production characteristic diagram of TYQ2;

[0029] Figure 7 Growth promotion phenotype diagram of TYQ2 on cucumber plant growth and biomass

[0030] Figure 8 Schematic diagram of the tropotactic migration test of Meloidogyne incognita after inoculation with TYQ2;

[0031] Figure 9 Influence diagram of the tropotactic migration of Meloidogyne incognita after inoculation with TYQ2;

[0032] Figure 10 Influence diagram of the in vitro contact-killing effect of the original fermentation supernatant of TYQ2 on Meloidogyne incognita;

[0033] Figure 11 Salvage phenotype diagram of cucumber plant growth under the infection of Meloidogyne incognita by TYQ2;

[0034] Figure 12 Effect diagram of the confrontation antagonism between TYQ2 and Fusarium oxysporum on the plate;

[0035] Figure 13 Effect diagram of TYQ2 degrading straws of different vegetable crops. Detailed implementation manners

[0036] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0037] The following detailed description of the embodiments of the present invention provided is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0038] Example 1

[0039] 1.1 Isolation and purification of Pseudomonas protegens TYQ2:

[0040] The Pseudomonas protegens TYQ2 strain used in the present invention was isolated from a cucumber rhizosphere soil sample infected with Meloidogyne incognita provided by the Laboratory of Regulation of Facility Vegetable Growth and Development, China Agricultural University. The gradient dilution method was used for strain isolation, and the specific steps are as follows:

[0041] After collecting 4 rhizosphere soil samples of diseased cucumbers (1 g each) and thoroughly mixing them, 1 g of the mixed sample was added to 9 mL of sterile phosphate buffer (PBS, 0.1 M, pH 7.4), and the soil suspension was prepared by shaking at 180 rpm and 28 °C for 20 min; subsequently, it was serially diluted 10-fold to 10 -6 , and 100 μL of the 10 -4 -10 -6 gradient dilution was taken and spread on the surface of LB solid medium (1.5% agar). After culturing at a constant temperature of 28 °C for 48 h, single colonies were picked and continuously purified 3 times by the three-zone streaking method. Finally, pure cultures were obtained and stored in LB medium containing 20% glycerol at -80 °C. All operations were completed under sterile conditions in a laminar flow hood.

[0042] 1.2 Identification of Pseudomonas protegens strain TYQ2

[0043] (1) Microbiological characteristics

[0044] The obtained strain was inoculated on an LB medium plate and cultured at 28 °C for 2 days. As Figure 1 shown, the single colonies of this strain were round, milky white and opaque, with a colony morphology size of 2-3 mm, a smooth and moist surface, regular edges, slightly raised in the center, and clear biofilms formed around some colonies. Gram staining was negative, with typical Pseudomonas characteristics.

[0045] (2) Molecular biological characteristics

[0046] Single colonies were picked into 1.5 mL centrifuge tubes containing 1 mL of LB medium and cultured with shaking at 28 °C and 180 rpm / min for 24 h. Using this bacterial solution as a template, 16S rRNA sequence amplification was performed with primers 27F and 1492R.

[0047] The PCR amplification reaction system was 50 μL, including 25 μL of 2x Taq enzyme, 1 μL of 27F primer, 1 μL of 1492R primer, 1 μL of bacterial solution, and 22 μL of ddH2O. Amplification conditions: pre-denaturation at 95 °C for 3 min, denaturation at 94 °C for 25 s, annealing at 55 °C for 25 s, extension at 72 °C for 1 min, 32 cycles, extension at 72 °C for 5 min, and the amplification product was stored at 4 °C. The amplification product was separated and identified by 1% agarose gel electrophoresis, and the PCR product was sent to Beijing Tsingke Biotechnology Co., Ltd. for bidirectional sequencing. The 16S rDNA of strain TYQ2 is shown as SEQ ID NO: 1 below:

[0048]

[0049]

[0050] The determination results were subjected to BLAST homology alignment, and the results showed that the similarity of this strain to Pseudomonas protegens reached 99.93%. Therefore, as Figure 2 shown, this strain was identified as a strain of the genus Pseudomonas, named Pseudomonas protegens TYQ2, and it was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC.27211.

[0051] Example 2

[0052] Determination of the ability of Pseudomonas protegens TYQ2 to utilize different carbon sources and its chemical sensitivity

[0053] 2.1 Preparation of the strain inoculum

[0054] Take a fresh culture (≤24 h) of TYQ2 single colonies, scrape and inoculate them with a sterile cotton swab into the IFA inoculum (Biolog, USA) adapted to the GEN III MicroPlate TM identification plate, and vortex (1,000 rpm, 30 s) to obtain a uniform bacterial suspension. Subsequently, transfer the bacterial suspension to the MicroPlate TM identification plate at an inoculation volume of 100 μL / well. This identification plate contains 71 carbon source metabolism and 23 chemical sensitivity detection systems, which can comprehensively characterize the carbon source utilization spectrum and environmental adaptability of the strain. The inoculated identification plate was cultured in the dark at 33 °C for 24 h, and the absorbance values of each well at a wavelength of 590 nm were measured using a fully automatic microplate reader, and then the carbon source metabolism fingerprint map of TYQ2 was constructed.

[0055] 2.2 Result analysis

[0056] The results of metabolic phenotype analysis ( Figure 3 ) showed that TYQ2 could effectively utilize 62 carbon sources (utilization rate > 80%), and at the same time showed tolerance to 12 chemical reagents (relative growth inhibition rate < 20%). The sensitivity test further revealed that TYQ2 showed significantly enhanced growth adaptability in a slightly acidic environment (pH 5.0 - 6.5) and was resistant to antibiotics such as vancomycin and lincomycin. These results suggest that TYQ2 has broad metabolic diversity and strong environmental stress resistance, providing potential advantages for its colonization and function in complex ecological niches.

[0057] Example 3

[0058] Phosphate-solubilizing characteristics of Pseudomonas protegens TYQ2

[0059] 3.1 Qualitative analysis of phosphorus solubilization by Pseudomonas protegens TYQ2

[0060] The phosphorus solubilization ability of TYQ2 on insoluble phosphorus was initially evaluated using an inorganic phosphorus solid medium: 10 μL of the TYQ2 bacterial solution cultured overnight was pipetted onto the surface of the inorganic phosphorus solid medium, sealed with a sealing film, and cultured in an incubator at 28 °C for 5 days. Whether a phosphorus solubilization circle was formed around the colonies was observed.

[0061] The results showed that after culturing TYQ2 on the inorganic phosphorus solid medium for 5 days, a distinct transparent phosphorus solubilization circle could be formed around the colonies ( Figure 4 ), indicating that TYQ2 has the ability to solubilize insoluble phosphorus.

[0062] 3.2 Quantitative analysis of phosphorus solubilization by Pseudomonas protegens TYQ2 at different NaCl concentrations and pH values

[0063] In Example 2, based on the results of the Microplate identification plate, the strain TYQ2 showed adaptive growth characteristics in a slightly acidic and low-salt environment. To further explore its phosphorus solubilization ability, a liquid PVK medium system with different NaCl concentration gradients (0%, 2%, 4%, 6%, 8%) and different pH gradients (5, 6, 7, 8, 9) was designed in the present invention. The specific test method was as follows: TYQ2 was inoculated into the above treatment group media at an inoculation amount of 1% (v / v), and at the same time, a non-inoculated liquid PVK medium with the corresponding NaCl concentration and pH conditions was set as a blank control. All cultures were shaken at 28 °C and 180 rpm for 72 h.

[0064] After the culture was completed, the content of soluble phosphorus in the culture solution was determined by the standard molybdenum antimony anti-colorimetric method. The specific operation process was as follows: 10 mL of the centrifuged supernatant was taken as the test sample, 5 mL of the molybdenum antimony anti-color reagent was added, and after color development in the dark at room temperature for 30 min, the absorbance value was measured at a wavelength of 700 nm using a spectrophotometer. The absolute content of soluble phosphorus in each treatment group was calculated by establishing a standard curve to quantitatively evaluate the phosphorus solubilization efficiency of TYQ2 under different environmental conditions.

[0065] 3.3 Result analysis

[0066] The change in the soluble phosphorus concentration directly characterized the activation efficiency of the strain TYQ2 on insoluble phosphorus under different environmental conditions. As Figure 5As shown, the results of the pH gradient test indicated that TYQ2 exhibited the best phosphorus solubilization efficiency in a weakly acidic environment (pH = 6), and the concentration of soluble phosphorus released by it was significantly higher than that of other treatment groups (71.42 mg / L). This result was consistent with the acidic environment adaptability characteristics shown by the Biolog metabolic phenotype analysis. The salinity stress test showed that the phosphorus solubilization ability of TYQ2 was significantly negatively correlated with the NaCl concentration, reaching the highest phosphorus solubilization efficiency (76.83 mg / L) under salt-free stress conditions (0% NaCl), while when the NaCl concentration increased to 8%, the soluble phosphorus concentration decreased to 50.99 mg / L. These data indicated that the phosphorus solubilization function of TYQ2 had significant environmental dependence, and it demonstrated the optimal phosphorus activation potential under slightly acidic and low salinity conditions.

[0067] Example 4

[0068] Characteristics of siderophore production by Pseudomonas protegens TYQ2

[0069] 4.1 Qualitative analysis of siderophore production by Pseudomonas protegens TYQ2

[0070] The improved CAS siderophore detection medium was used to preliminarily evaluate whether TYQ2 had the ability to produce siderophore: 10 μL of the overnight cultured TYQ2 bacterial solution was pipetted and inoculated into the CAS detection medium (pH = 6.8), sterilized at 115 °C for 20 min, and each treatment group had 5 replicates. During the cultivation process, the presence of a yellow transparent circle around the colonies was continuously observed. If there was, it proved that the antagonistic bacterium had the ability to produce siderophore.

[0071] 4.2 Result analysis

[0072] The detection results were as Figure 6 shown. TYQ2 formed an orange-yellow transparent circle on the CAS medium. Thus, it could be known that TYQ2 had a strong ability to secrete siderophore, which could integrate iron in the soil by producing siderophore, thereby helping plants absorb Fe elements and indirectly improving the nutritional status, health status of plants, as well as their ability to resist diseases and environmental stresses.

[0073] Example 5

[0074] Growth promotion effect of Pseudomonas protegens TYQ2 on plants

[0075] Using cucumber as the test material, the ability of TYQ2 to promote plant growth was measured through a pot experiment. The specific implementation steps were as follows:

[0076] 5.1 Seed disinfection

[0077] Select seeds with full grains and intact seed coats, disinfect them with 75% ethanol surface for 30 seconds, rinse them with sterile deionized water 3 times (1 minute each time), then soak them in 3% sodium hypochlorite solution for 10 minutes for thorough sterilization, and rinse them with sterile deionized water again until there is no irritating odor. Soak the sterilized seeds at 25°C for 6 hours. Finally, evenly arrange the seeds in a culture dish covered with sterile moist filter paper and place them in a constant temperature incubator at 28°C for dark culture.

[0078] 5.2 Preparation and inoculation of Pseudomonas TYQ2 suspension

[0079] A single colony of TYQ2 was picked and inoculated into 30 mL of LB liquid medium, and cultured at 28 ° C and 180 rpm for 16 h until the logarithmic growth phase. The OD600 value of the bacterial solution was measured by ultraviolet spectrophotometer, and the bacterial solution was diluted to OD600 = 0.8 using sterile MgSO4 solution (10 mM). Subsequently, the bacteria were collected by centrifugation at 10,000 × g for 10 min at 4 ° C, and the supernatant was discarded and the bacteria were resuspended with MgSO4 solution, vortexed (1,500 rpm, 30 s) to fully disperse them, and finally prepared into a uniform bacterial suspension for use. When the cotyledon flattening period (about 5-7 days) was entered, 10 mL of the prepared bacterial solution was inoculated, and the plants were harvested 21 days after inoculation, and the aboveground growth index, biomass and root morphology index were measured, and the seedling index was calculated. In this experiment, an uninoculated control group (CK) and a TYQ2 treatment group were set up, and 20 biological replicates were set up in each group.

[0080]

[0081] 5.3 Results Analysis

[0082] The experimental results showed that inoculation of Pseudomonas tyq2 had a significant growth-promoting effect on cucumber seedlings ( Figure 7 , Table 1-2). The plant height and stem diameter of the TYQ2-treated group increased significantly by 9.60% and 9.21% respectively compared with the control group; biomass measurement showed that the dry weight of the aboveground and underground parts increased by 35.24% and 32.43% respectively, and the seedling index increased by 33.33%. Through the WinRHIZO root analysis system, TYQ2 treatment significantly improved the root system architecture, and the total root length (63.25%), root surface area (103.47%), average root diameter (26.31%) and root volume (156.41%) were significantly higher than those of the control. These data confirm that TYQ2 achieves an improvement in overall growth performance by synergistically promoting the growth of the aboveground part of the plant and the development of the root system.

[0083] Table 1. Effects of TYQ2 on aboveground growth indexes and biomass of cucumber plants

[0084]

[0085] Table 2. Effects of TYQ2 on cucumber root morphological indexes

[0086]

[0087] Example 6

[0088] Effect of fermentation supernatant of Pseudomonas protegens TYQ2 on the chemotactic migration of Meloidogyne incognita

[0089] 6.1 Preparation and inoculation of Pseudomonas protegens TYQ2 strain suspension

[0090] The specific operation for preparing the strain suspension is the same as that in Example 5

[0091] 6.2 Preparation of second-stage juvenile suspension of Meloidogyne incognita

[0092] The second-stage juveniles (J2) of Meloidogyne incognita used in the present invention were isolated from the roots of water spinach infected with nematodes. First, the roots of water spinach were washed, and the egg masses on the root surface were picked with forceps. Subsequently, the egg masses were washed by shaking with 0.6% sodium hypochlorite solution and filtered through a 600-mesh sieve. The egg masses on the sieve were repeatedly rinsed with sterile water, and finally transferred to a round petri dish filled with sterile water, and 4 mg / L gentamicin solution was added to prevent the growth of miscellaneous bacteria. After hatching at room temperature for 2 to 3 days, the hatched second-stage juveniles were collected, observed and counted under a microscope, and finally the larval density was adjusted to 1×10 3 larvae / mL

[0093] 6.3 Preparation of 23% Pluronic F-127 gel

[0094] Preparation of 23% Pluronic F-127 gel: The solution was prepared in a 4°C environment. 23 g of Pluronic F-127 gel powder was added to 80 mL of sterile water pre-cooled to 4°C, and placed on a magnetic stirrer to stir at a low speed. The dissolved gel was stored at 4°C

[0095] 6.4 Chemotactic migration test

[0096] As Figure 8As shown in the figure, in this experiment, an uninoculated control group (CK) and a TYQ2 treatment group were established, with 20 biological replicates in each group. A modified two-way selection migration test device was used, and the specific operation was as follows: A sterile Oxford cup (Φ = 0.5 cm) was embedded in the center of a square gel migration plate with a diameter of 14 cm. After the gel solidified, the Oxford cup was removed to form an inoculation area. Cucumber seedling roots treated with TYQ2 and untreated (control group) were placed on both sides of the migration plate, with a distance of 4 cm between the two roots and equidistant from the central inoculation area. 100 μL of suspension of Meloidogyne incognita J2 larvae was injected into the central inoculation area, covered with tin foil to avoid light, and left to stand at 25 °C for 12 h. Subsequently, the partition counting method was used to observe the migration of nematodes on both sides under a stereomicroscope, calculate the number of nematodes in different areas of the square culture dish, and calculate the movement trend of nematodes based on the total number of nematodes in the culture dish.

[0097] 6.5 Result analysis

[0098] As Figure 9 , the TYQ2 strain significantly inhibited the tropotactic migration of the second-stage larvae (J2) of Meloidogyne incognita to plant roots. Under the condition of single-strain treatment with TYQ2, the migration percentage of nematodes to the cucumber root tips in the control group (CK) reached 56.94%, which was significantly higher than the migration rate of 43.06% on the side of the TYQ2-treated group roots.

[0099] Example 7

[0100] Contact-killing effect of the fermentation supernatant of Pseudomonas protegens TYQ2 on root-knot nematodes in vitro.

[0101] 7.1 Preparation of the supernatant of the strain fermentation broth

[0102] The tested strain was Pseudomonas protegens TYQ2, and the control strain and the bacterial suspension were the same as those used in Example 5. After the preparation of the bacterial suspension was completed, it was centrifuged at 10,000 rpm for 5 min, and then the supernatant in the centrifuge tube was aspirated with a syringe and filtered through a 0.22 μm filter membrane. The solution obtained after filtration was the supernatant of the fermentation broth of Pseudomonas protegens TYQ2.

[0103] 7.2 Contact-killing test of Meloidogyne incognita in vitro

[0104] Take 400 μL of the supernatant of the fermentation broth of strain TYQ2 and the control strain and place them in a 48-well cell culture plate. Then add 100 μL of the suspension of second-stage root-knot nematode larvae (containing 100 second-stage larvae). Add 400 μL of sterile water to the control group. Wrap the cell culture plate with tin foil and place it in an incubator at 28 °C for static incubation. After 12 h and 24 h of treatment respectively, observe the survival status of the second-stage larvae under a stereomicroscope. The judgment criterion is as follows: after dropping 1 mol / L NaOH solution into each treatment, the larvae that remain rigid and motionless are dead, and the larvae that bend and wriggle are alive. Count the number of dead nematodes and calculate the corrected mortality rate of the nematodes.

[0105] Add 400 μL of the supernatant of the TYQ2 fermentation broth (experimental group) and sterile water (negative control) to the 48-well cell culture plate respectively. Then inoculate 100 μL of the suspension containing 100 J2 larvae (1×10 3 J2 / mL) into each well. After wrapping it with tin foil to avoid light, place it in a constant temperature incubator at 28 °C for static culture; after 12 h and 24 h of treatment respectively, add 10 μL of 1 mol / L NaOH solution to each well, and observe and count the nematode mortality rate under a stereomicroscope. Among them, the larvae with rigid bodies and no response to mechanical stimulation are determined to be dead.

[0106]

[0107] 7.3 Result analysis

[0108] As Figure 10 shown, the supernatant of the TYQ2 fermentation broth showed a significant lethal effect on the second-stage larvae (J2) of Meloidogyne incognita. The timeliness analysis showed that the nematode mortality rate in the TYQ2 treatment group increased in a time-dependent manner. The corrected mortality rate was 79.35% at 12 h and increased significantly to 96.52% at 24 h. This result indicated that the metabolites contained in the TYQ2 fermentation broth had strong nematicidal activity.

[0109] Example 8

[0110] In vivo experiment on the control of Meloidogyne incognita by Pseudomonas protegens TYQ2.

[0111] In this example, cucumber was used as a model crop for research. The treatments set in the example were: inoculating with root-knot nematodes (CK+N); inoculating with TYQ2 and root-knot nematodes simultaneously (TYQ2+N).

[0112] 8.1 Seed disinfection

[0113] The specific operation of seed disinfection was the same as that in Example 5.

[0114] 8.2 Preparation and inoculation of the suspension of Pseudomonas protegens TYQ2 strain

[0115] The preparation and inoculation of the suspension of Pseudomonas protegens strain TYQ2 were the same as in Example 5.

[0116] 8.3 Inoculation of second-stage juveniles of Meloidogyne incognita

[0117] In the nematode inoculation experiment of the present invention, cucumber seedlings grown to the one-leaf and one-heart stage were selected as the test materials. Three wound points with a depth of 1 cm were made in the root zone 1 cm away from the base of the stem using a sterile pipette tip, and 100 μL of the suspension containing 100 J2 larvae was inoculated at each point. Samples were taken 21 days after inoculation for determination, and the above-ground growth indexes, biomass, and root morphology indexes were measured, and the number of root knots per unit root was counted. In this experiment, an uninoculated control group (CK) and a TYQ2 treatment group were set up, and 20 biological replicates were set in each group.

[0118] 8.4 Result analysis

[0119] The experimental results showed ( Figure 11 , Table 3-4) that Pseudomonas protegens strain TYQ2 showed a significant growth rescue effect on cucumber seedlings under the infection of Meloidogyne incognita. Quantitative analysis showed that compared with the control group infected with nematodes alone, the TYQ2 treatment significantly promoted plant morphogenesis, and the plant height and stem diameter increased by 39.84% and 12.69% respectively; the analysis of biomass accumulation showed that the above-ground and underground dry weights increased by 6.06% and 68.88% respectively (P < 0.05), and the SPAD value of leaf chlorophyll increased by 154.24%. The WinRHIZO root architecture analysis revealed that the TYQ2 treatment significantly improved the total root length (71.64%), root surface area (84.46%), average root diameter (26.82%), and root volume (145.16%). Particularly importantly, the TYQ2 treatment reduced the number of root knots per unit root weight by 14.72%, indicating that it has both growth-promoting and disease-resistant dual functions.

[0120] Table 3. Effects of TYQ2 on the above-ground growth indexes and biomass of cucumber plants under the infection of Meloidogyne incognita

[0121]

[0122] Table 4. Effects of TYQ2 on the root morphology parameters and root knot formation of cucumber under the infection of Meloidogyne incognita

[0123]

[0124] Example 9

[0125] Plate antagonism of TYQ2 against Fusarium oxysporum

[0126] 9.1 Preparation of strain suspension

[0127] The specific operation of preparing the strain suspension was the same as in Example 5.

[0128] 9.2 Preparation of Fusarium oxysporum Plates

[0129] The Fusarium oxysporum used in this invention is the strain stored in the laboratory's strain bank. Pick hyphae and inoculate them on a solid PDA medium plate, and culture at 28°C for 3 days.

[0130] 9.3 Test of TYQ2 Antagonizing Fusarium oxysporum

[0131] The inhibition effect of Pseudomonas protegens TYQ2 on Fusarium oxysporum was determined by the plate confrontation method. The specific method is as follows: Use a sterile scalpel to cut the activated Fusarium oxysporum colony into 3×3 mm blocks, and inoculate them at a distance of 1 / 4 radius from the edge of a fresh PDA plate; Take another sterilized toothpick and dip it into the TYQ2 bacterial suspension (OD 600 = 0.8), and make a vertical streak inoculation 2 cm away from the pathogen inoculation point (repeat the streaking 2 - 3 times). After culturing the plate in a 28°C constant temperature incubator for 72 h, measure the major and minor axis diameters of the Fusarium oxysporum colony, and calculate the inhibition rate and the eccentricity of the colony. This experiment set 12 biological replicates.

[0132]

[0133] 9.4 Result Analysis

[0134] As shown in Table 5 and Figure 12 shown, the treatment with TYQ2 significantly changed the colony morphological characteristics and growth pattern of Fusarium oxysporum. The eccentricity of the colony in the TYQ2 treatment group reached 0.27, and the colony morphology changed from nearly circular in the control group to elliptical, indicating that TYQ2 interfered with the radial expansion of the pathogen through a directional inhibition mechanism. The measurement of the colony area showed that the treatment with TYQ2 reduced the growth area of Fusarium oxysporum by 60.45%, and this result confirmed that TYQ2 could effectively inhibit the growth of the pathogen. These data together proved that TYQ2 had a significant antagonistic effect on Fusarium oxysporum.

[0135] Table 5. Effects of TYQ2 on the Growth of Fusarium oxysporum

[0136]

[0137]

[0138] Note: The eccentricity in the table represents the growth situation of Fusarium oxysporum. The higher the eccentricity value, the flatter the ellipse; the lower the eccentricity value, the more circular it is.

[0139] Example 10

[0140] In this example, four common fruit and vegetable straws, including cucumber, tomato, pepper, and eggplant straws, were used as materials. They were shaken and cultured with TYQ2 in a liquid carbon-deficient medium to explore whether TYQ2 has the degradation characteristics of vegetable straws. The specific method is as follows:

[0141] 10.1 Vegetable straw pretreatment

[0142] The tomato, pepper, eggplant, and cucumber straws were respectively cut into small sections of 3 - 4 cm, then dried in an oven, placed in 250 ml Erlenmeyer flasks, sealed with sealing film, and put into an autoclave for sterilization at 121 °C for 15 minutes.

[0143] 10.2 Determination of vegetable straw degradation rate

[0144] In this study, a carbon-deficient liquid culture system was used to evaluate the degradation ability of TYQ2 strain on straws. The specific method is as follows: Add 80 mL of carbon-deficient basal medium (components: MgSO4·7H2O 0.4 g / L, K2HPO4 1.3238 g / L, KH2PO4 0.3266 g / L, CaCl2·2H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·H2O 1 mg / L, ZnSO4·7H2O 0.25 mg / L, pH 7.2 ± 0.1) into a 250 mL conical flask. Add 3.2 g of straw sample to each bottle. After sterilization at 121 °C for 20 min, inoculate 3.2 mL of TYQ2 overnight culture broth (OD600 = 0.8). Seal the bottle mouth with sealing film and place it in a constant temperature shaker at 28 °C and 180 rpm for 15 days. After the culture is completed, collect the residual straw, repeatedly rinse it with deionized water, and dry it at 60 °C to constant weight.

[0145] Set 5 biological replicates for each group.

[0146]

[0147] 10.3 Result analysis

[0148] The test results show that ( Figure 13 and Table 6), Pseudomonas protegens TYQ2 shows different degradation abilities for the four vegetable straws. Among them, TYQ2 has the highest degradation efficiency for tomato straw, reaching 38.43%, which is significantly higher than other treatment groups. In contrast, the degradation rates of this strain for pepper and eggplant straws are relatively low (29.89% and 22.98% respectively). It may be because the lignification degree of these two straws is relatively high and they contain more lignin, so they are not easily degraded.

[0149] Table 6 Degradation rates of TYQ2 for four vegetable straws

[0150]

[0151]

[0152] Summary: As can be seen from the above examples:

[0153] (1) This strain was isolated from the rhizosphere soil of cucumbers in the laboratory of China Agricultural University. Its taxonomic name is Pseudomonas protegens TYQ2, and it was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on April 27, 2023, with the deposit number CGMCC No. 27211.

[0154] (2) The microbiological characteristics of Pseudomonas protegens TYQ2 are as follows: The single colony of this strain is round, milky white and opaque, with a colony morphology size of 2-3 mm, a smooth and moist surface, regular edges, and a slightly raised center. The cells are rod-shaped and Gram-negative.

[0155] (3) By amplifying the 16S rDNA fragment of this strain and performing BLAST homology alignment, the similarity between this strain and Pseudomonas protegens reaches 99.93%. Therefore, this strain is identified as a strain of the genus Pseudomonas, named Pseudomonas protegens TYQ2.

[0156] (4) Pseudomonas protegens TYQ2 was identified by the GENIII MicroPlate TM system to show broad-spectrum carbon source utilization ability (62 carbon sources) and multiple environmental adaptabilities, including a preference for slightly acidic environments (pH 5.0-6.5), tolerance to 12 chemical reagents, and antibiotic resistance. These characteristics lay a metabolic foundation for its colonization and function in complex habitats.

[0157] (5) Pseudomonas protegens TYQ2 has the best phosphorus solubilization effect in a slightly acidic environment with a pH of 6 and under salt-free conditions (reaching 71.42 mg / L and 76.83 mg / L respectively). Its characteristics of forming obvious phosphorus solubilization circles and releasing soluble phosphorus confirm that this strain has high phosphorus activation potential in low-salt and slightly acidic environments.

[0158] (6) Through qualitative analysis, it was identified that TYQ2 has the characteristic of secreting siderophores.

[0159] (7) Pseudomonas protegens TYQ2 significantly promotes the growth of cucumber seedlings, increasing plant height and stem diameter by 9.60% and 9.21% respectively, and increasing the dry weights of the aboveground and underground parts by 35.24% and 32.43%, achieving a 33.33% increase in the strong seedling index. At the same time, it can improve the root architecture, confirming its significant plant growth-promoting potential.

[0160] (8) Pseudomonas protegens TYQ2 significantly changes the chemotactic behavior of second-stage juveniles of Meloidogyne incognita. In the two-way migration experiment, the migration rate of nematodes to the control group (56.94%) is significantly higher than that of the treatment group (43.06%), indicating that its metabolites can effectively interfere with the nematodes' ability to locate plant roots.

[0161] (9) The fermentation supernatant of Pseudomonas protegens TYQ2 has a significant contact-killing effect on second-stage juveniles of Meloidogyne incognita. The corrected mortality rate reaches 96.52% after 24 hours of treatment, and it shows time-dependence (79.35% at 12 hours), confirming that its metabolites have strong nematicidal activity.

[0162] (10) Pseudomonas protegens TYQ2 has a significant rescue effect on nematode-infected cucumber seedlings, increasing plant height and stem diameter by 39.84% and 12.69% respectively, increasing the dry weight of the underground part by 68.88%, improving the root architecture at the same time and reducing the root knot number by 14.72%, confirming its dual effects of promoting growth and resisting nematodes.

[0163] (11) Pseudomonas protegens TYQ2 significantly inhibits the growth of Fusarium oxysporum, increasing the colony eccentricity to 0.27 (the control group is nearly circular), and reducing the colony area by 60.45%, confirming that it interferes with the radial expansion of the pathogen through a directional inhibition mechanism, showing a strong antagonistic effect.

[0164] (12) The strain Pseudomonas protegens TYQ2 has the function of degrading agricultural waste straw. The strain was shaken and cultured with different vegetable straws as the sole carbon source. The degradation rates of the strain on tomato, cucumber, pepper, and eggplant straws reached 38.43%, 38.04%, 29.89%, and 22.98% respectively in 15 days.

[0165] The above embodiments are several limited preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A strain of Pseudomonas protegens, characterized in that, The Pseudomonas protegens strain TYQ2 is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 27211.

2. A microbial inoculant, characterized in that, Containing the Pseudomonas protegens cells and / or its metabolites and / or its fermentation broth as described in claim 1.

3. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for promoting the growth of cucumber seedlings.

4. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for interfering with the migration of Meloidogyne incognita.

5. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for controlling Meloidogyne incognita.

6. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for inhibiting the growth of Fusarium oxysporum.

7. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for degrading the straws of different vegetable crops.

8. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for dissolving insoluble phosphorus.

9. Use of the bacterium as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a preparation for chelating Fe element in soil.

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