A strain of Pseudomonas kluyveri and its application

Abstract

The present invention belongs to the field of microorganisms, and specifically discloses a strain of Pseudomonas kluyveri and its application. Pseudomonas knackmussii ) strain TYQ5, deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 27214; the applicant found that the above-mentioned Pseudomonas kluyveri can secrete auxin, which can promote the growth of cucumber seedlings, and also has the ability to interfere with the directional migration of nematodes and kill nematodes, with the effect of significantly alleviating the stress of root-knot nematodes. It was also found that it has the ability to inhibit the normal growth of Fusarium oxysporum. Furthermore, it has the ability to degrade vegetable straw and solubilize phosphate. Therefore, the applicant believes that the above-mentioned Pseudomonas kluyveri has significant application prospects in the fields of agriculture and environmental protection.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and specifically discloses a strain of Pseudomonas kluyveri and applications thereof. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) play a key role in the root microecosystem, establishing a mutually beneficial symbiotic relationship with plants and significantly promoting plant growth and soil health. PGPR directly promotes plant growth and development by secreting plant hormones such as indoleacetic acid. They also improve soil nutrient availability and optimize plant nutrient supply by decomposing organic matter and dissolving minerals. Furthermore, PGPR inhibit the growth of pathogens and mitigate disease damage by producing antibiotics and competing for resources. They also induce systemic resistance in plants and enhance immunity. These properties make PGPR crucial for reducing pesticide use, improving crop resistance to pests and diseases, and contributing to sustainable agricultural development. By screening superior PGPR strains and conducting in-depth research on their biological characteristics and functional mechanisms, we can develop highly effective and environmentally friendly microbial agents to promote the green transformation of agriculture.

[0003] Meloidogyne, particularly the southern root-knot nematode, is a very common soil-borne disease in greenhouse cucumber production. Its hidden nature, rapid spread, and severe damage often stunt cucumber growth, resulting in an average annual yield reduction of 10% to 50%, and in severe cases, even total crop failure. It is considered a "terminal disease" in cucumber production. Traditional control methods, such as physical, chemical, and agricultural methods, while somewhat effective, are difficult to eradicate and often lead to increased costs and soil degradation. Fusarium oxysporum is also a highly destructive pathogen to horticultural crops. By infecting roots and vascular systems, it causes plant wilt and death, severely impacting crop yields and increasing control costs, posing a dual threat to the economy and the environment. Given the limited research on the use of beneficial microorganisms in the cucumber rhizosphere to control these two diseases, identifying effective strains for controlling root-knot nematodes and Fusarium oxysporum has become an urgent need and a hot topic of research both domestically and internationally.

[0004] The scale of vegetable cultivation is expanding, and the large amount of vegetable straw produced has become a major problem in the treatment of agricultural waste. If vegetable straw is discarded or burned at will, it will not only cause a waste of resources, but also lead to problems such as environmental pollution. Existing treatment methods, such as composting, are slow to degrade naturally due to the high content of lignin, cellulose and other components in vegetable straw, and are prone to secondary pollution. Microorganisms can decompose the complex organic components in vegetable straw into simple small molecules through the various enzymes they secrete, thereby achieving rapid degradation and conversion of straw. Screening for microorganisms that can degrade vegetable straw has become an effective way to utilize vegetable straw resources.

[0005] In today's complex and ever-changing agricultural production environment, single-function microbial agents struggle to adapt to complex soil conditions and climatic factors, significantly reducing their effectiveness and failing to fully meet agricultural production needs. Developing multifunctional microbial strains is key to improving agricultural production efficiency and quality. These multifunctional strains can take root in different soil types, adapting to diverse growth environments. They can also secrete auxins to stimulate crop root growth, enhance nutrient absorption, efficiently degrade straw, reduce environmental pollution, and increase soil fertility. The use of multifunctional microbial strains can efficiently utilize soil nutrients, reduce the use of chemical fertilizers, lower agricultural production costs, achieve sustainable agricultural development, increase crop yields and quality, and promote the development of high-quality and efficient agriculture. Summary of the Invention

[0006] In order to solve the above problems, the present invention discloses a strain of Pseudomonas kluyveri and applications thereof.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] A strain of Pseudomonas kluyveri, wherein the Pseudomonas kluyveri ( Pseudomonas knackmussii ) strain TYQ5 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 27214.

[0009] The present invention also discloses a microbial agent containing the Pseudomonas kluyveri cells and / or its metabolites and / or its fermentation liquid.

[0010] The invention also discloses use of the bacteria or bacterial agent in preparing a preparation for enhancing the growth promotion of cucumber seedlings.

[0011] The present invention also discloses the use of the bacteria or bacterial agent in preparing a preparation for interfering with the migration of southern root-knot nematodes.

[0012] The present invention also discloses the use of the bacteria or bacterial agent in preparing a preparation for preventing and treating southern root-knot nematodes.

[0013] The invention also discloses the use of the bacteria or bacterial agent in preparing a preparation for inhibiting the growth of Fusarium oxysporum.

[0014] The invention also discloses the use of the bacteria or bacterial agent in preparing a phosphate-dissolving preparation.

[0015] The invention also discloses the use of the bacteria or bacterial agent in preparing a straw degradation preparation.

[0016] The present invention has the following beneficial effects:

[0017] The present invention discloses a strain of Pseudomonas kluyveri and applications thereof. In terms of growth promotion, the Pseudomonas kluyveri TYQ5 disclosed in the present invention has the growth-promoting properties of dissolving inorganic phosphorus and secreting auxin. TYQ5 can utilize tryptophan to produce IAA, with a measured content of 16.75 μg / mL, indicating that it has potential plant growth regulating ability. In addition, TYQ5 exhibits significant effects on the migration and mortality rate of nematodes, especially in terms of lethality to second-instar larvae of root-knot nematodes, and its effect is significantly better than that of the control strain. In an experiment to inhibit the growth of Fusarium oxysporum, TYQ5 demonstrated a strong antagonistic effect. The number of root nodes per unit in cucumber seedlings inoculated with TYQ5 decreased by 31.77%, while total root length, root surface area, and root volume increased by 62.48%, 80.42%, and 96.77%, respectively, compared to the control. The underground dry weight and chlorophyll content increased by 113.33% and 211.44%, respectively. Within two weeks, TYQ5 degraded tomato, cucumber, pepper, and eggplant straw by 68.93%, 58.63%, 31.37%, and 39.24%, respectively. TYQ5's phosphate solubility decreased with increasing salt concentration, reaching its highest phosphate solubility at 75.01 mg / L at 0 g / L NaCl and 74.20 mg / L at pH 6. Biolog analysis of TYQ5 demonstrated significant insensitivity to 70 tested carbon sources and eight chemical reagents, demonstrating strong environmental adaptability and metabolic diversity. In summary, the TYQ5 strain shows good application potential in promoting plant growth, enhancing plant stress resistance and environmental remediation.

[0018] The strain deposit information is as follows:

[0019] Name of depository institution: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0020] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0021] Deposit date: April 27, 2023

[0022] Deposit number CGMCC No. 27214,

[0023] Taxonomic name: Pseudomonas kluyveri Pseudomonas knackmussii . BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony morphology of TYQ5;

[0025] Figure 2 This is the phylogenetic tree diagram of TYQ5;

[0026] Figure 3 A diagram showing the effects of TYQ5 producing auxin;

[0027] Figure 4 Schematic diagram of the tropism migration experiment of southern root-knot nematodes after TYQ5 inoculation;

[0028] Figure 5 This is a diagram showing the effect of TYQ5 inoculation on the tropism and migration of southern root-knot nematodes;

[0029] Figure 6 This is a diagram showing the effect of TYQ5 inoculation on the tropism and migration of southern root-knot nematodes;

[0030] Figure 7 This is a graph showing the effect of TYQ5 fermentation supernatant on the in vitro contact killing of southern root-knot nematodes;

[0031] Figure 8 This is a diagram showing the in vitro contact killing effect of TYQ5 fermentation supernatant on southern root-knot nematodes;

[0032] Figure 9 This is a diagram showing the effect of TYQ5 inoculation on cucumber plants infected with southern root-knot nematodes;

[0033] Figure 10 The effect of TYQ5 inoculation on cucumber roots infected by southern root-knot nematodes

[0034] Figure 11 This is a diagram showing the antagonistic effect of TYQ5 and Fusarium oxysporum on a plate;

[0035] Figure 12 This is the degradation effect diagram of TYQ5 on four types of vegetable straw;

[0036] Figure 13 This is the phosphorus solubility characteristic diagram of TYQ5;

[0037] Figure 14 This is the phosphorus solubilization capacity diagram of TYQ5 under gradient salt concentration and gradient pH;

[0038] Figure 15 This is the fingerprint of TYQ5's ability to utilize different carbon sources. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. The following embodiments and features of the embodiments may be combined with each other unless there is a conflict.

[0040] The detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0041] Example 1

[0042] 1.1 Pseudomonas kluyveri ( Pseudomonas knackmussii )Isolation and purification of TYQ5:

[0043] The present invention Pseudomonas kluyveri ( Pseudomonas knackmussii The TYQ5 strain was isolated from the rhizosphere soil of cucumbers infected with root-knot nematodes in the laboratory of China Agricultural University. The isolation method was soil dilution. The specific steps are as follows:

[0044] Four portions (1 g each) of rhizosphere soil from cucumbers infected with root-knot nematodes in the vegetable laboratory of China Agricultural University were collected and mixed evenly. 1 g of the mixed soil sample was weighed and added to a 50 ml centrifuge tube containing 9 ml of sterile water, and the mixture was shaken at 180 rpm for 20 min. 1 ml of the soil suspension was transferred to a new centrifuge tube, and 9 ml of sterile water was added to make a 10-fold gradient dilution. The operation was repeated until the desired concentration was reached. 100 μL of the appropriately diluted suspension was spread on LB solid culture medium and cultured at 28°C under light for 48 h. A single colony was picked and streaked three times to obtain a pure culture.

[0045] 1.2 Pseudomonas kluyveri ( Pseudomonas knackmussii )Identification of TYQ5 strain

[0046] (1) Microbiological characteristics

[0047] The obtained strain was inoculated on LB medium plates and cultured at 28°C for 2 days. Figure 1 As shown, the single colony of this strain is round, yellow and opaque, with a smooth and moist surface, irregular edges, and a slightly convex center. Gram staining is negative, and the optimal growth temperature is 28-37°C.

[0048] (2) Molecular biological characteristics

[0049] A single colony was picked and placed in a 1.5 mL centrifuge tube containing 1 mL of LB medium. The culture was shaken at 28°C and 180 rpm / min for 24 h. The 16S rRNA sequence was amplified using the bacterial solution as a template using primers 27F and 1492R.

[0050] The PCR amplification reaction system (50 µL) consisted of 25 µL 2xTaq enzyme, 1 µL 27F primer, 1 µL 1492R primer, 1 µL bacterial suspension, and 22 µL ddH₂O. Amplification conditions were as follows: 32 cycles of pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55°C for 25 s, and extension at 72°C for 1 min, followed by 5 min extension at 72°C. The amplified product was stored at 4°C. The amplified product was separated and identified by 1% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The 16S rDNA of strain TYQ5 is shown in SEQ ID NO:1 below:

[0051]

[0052] The results were compared by BLAST homology comparison, and the results showed that the strain was similar to Pseudomonas kluyveri ( Pseudomonas knackmussii ) has a similarity of 99.79%. Figure 2 The strain was identified as Pseudomonas A strain of the genus, named Pseudomonas kluyveri ( Pseudomonas knackmussii )TYQ5, which was deposited in the General Microbiology Center of China Culture Collection Administration with the deposit number CGMCC.27214.

[0053] Example 2

[0054] Qualitative and quantitative analysis of the IAA secretion ability of Pseudomonas kluyveri TYQ5

[0055] The IAA secretion capacity of Pseudomonas kluyveri TYQ5 was determined by the Salkowski colorimetric method.

[0056] 2.1 Qualitative analysis of the IAA secretion capacity of Pseudomonas kluyveri TYQ5

[0057] Preparation of Salkowaki color developer

[0058] Dissolve 0.811 g of FeCl₃ in 10 mL of sterile distilled water to prepare a 0.5 M FeCl₃ solution. Add 1 mL of the prepared 0.5 M FeCl₃ solution to 49 mL of 35% HClO₄ solution to prepare the Salkowaki color developer. Mix thoroughly before use.

[0059] Bacterial culture and fermentation

[0060] Pick a single colony and inoculate it into 30 mL LB liquid medium, shake and culture it overnight at 28°C and 180 rpm / min, and use it as the bacterial solution (OD 600 =0.8). Take 1 mL of TYQ5 bacterial culture and inoculate it at a 1% (v / v) inoculum into LB liquid medium containing 500 µg / mL of 0.22 µm filter-sterilized tryptophan. Set up uninoculated LB liquid medium as a negative control. Cultivate with shaking at 28°C and 180 rpm / min for 3 days.

[0061] IAA qualitative test

[0062] Then centrifuge the fermentation broth at 10,000 rpm / min for 10 minutes and remove the supernatant for later use. Take 500 µL of the fermentation broth supernatant and place it in a 10 mL centrifuge tube. Add 1 mL of Salkowaki colorimetric reagent, mix well, and let it stand at room temperature for 30 minutes to observe the color change. If the solution turns pink, it means that IAA is produced in the fermentation broth. Figure 3 shown.

[0063] 2.2 Quantitative analysis of the IAA secretion capacity of Pseudomonas kluyveri TYQ5

[0064] Preparation of IAA standard curve

[0065] First, prepare 100 mg·L -1 The IAA stock solution was diluted to 0, 10, 20, 30, 40, 50, 60, 80, and 100 mg·L -1 The concentration gradient was measured and the absorbance at 530 nm (OD 530 ), with IAA concentration as the horizontal axis and OD 530 Draw a standard curve for the ordinate.

[0066] IAA content determination

[0067] Take 500 μL of TYQ5 strain fermentation supernatant, add 4 mL of colorimetric reagent, and measure OD after 30 minutes in the dark. 530 The uninoculated culture medium was used as the blank control, and the IAA content was calculated using the standard curve.

[0068] result

[0069] Pseudomonas kluyveri TYQ5 can synthesize IAA from tryptophan with a yield of 16.75 μg / mL.

[0070] Example 3

[0071] Effect of the fermentation supernatant of Pseudomonas kluyveri TYQ5 on the tropism and migration of root-knot nematodes.

[0072] 3.1 Preparation of strain suspension

[0073] Pick a single colony of Pseudomonas kluyveri TYQ5 and inoculate it into 30 mL LB liquid medium. Incubate the culture overnight at 28°C and 180 rpm. Dilute the culture solution to OD 600 = 0.8, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant and retain the cells. Resuspend the cells in 10 mM / LMgSO4 to prepare the bacterial solution for use.

[0074] 3.2 Cucumber root preparation

[0075] Cucumber is a vegetable crop that is sensitive to southern root-knot nematodes. In this example, cucumber was used as a model crop for research.

[0076] Seed disinfection and germination: Select plump, intact cucumber seeds and soak them in 75% ethanol for 30 seconds, then 3% NaClO for 10 minutes. Rinse with sterile water and soak for 6 hours. Place the seeds on moistened sterile filter paper and incubate at 28°C until the cotyledons expand (5-7 days).

[0077] Inoculation and sampling: Inoculate 10 mL of the prepared bacterial solution into the seedlings. When the seedlings have two leaves and one heart, take the roots for subsequent testing.

[0078] 3.3 Preparation of second-instar larvae suspension of root-knot nematodes

[0079] Egg masses were collected from infected water spinach roots, washed with 0.6% sodium hypochlorite by shaking, filtered through a 600-mesh sieve, and rinsed with sterile water. The egg masses were placed in sterile water containing 0.004 mg / mL gentamicin and incubated at room temperature for 2-3 days. Hatched second-instar larvae were collected, counted microscopically, and adjusted to 100 per 100 µL.

[0080] 3.4 23% Pluronic F-127 Gel Preparation

[0081] Dissolve 23 g of F-127 powder in 80 mL of pre-cooled sterile water at 4°C, stir magnetically at low speed until dissolved, and store at 4°C until use.

[0082] 3.5 Migration assay

[0083] Schematic diagram of the tropism migration experiment of southern root-knot nematodes after TYQ5 inoculation is shown in Figure 4 The experiment involved two groups, one without bacterial suspension and one with bacterial suspension, with 20 replicates each. A 0.5 cm diameter Oxford cup was placed in the nematode inoculation area of ​​the migration plate, leaving a gap. Roots from the control and experimental groups were placed on either side, covered with agar gel. After the gel solidified, the Oxford cup was removed, and 100 μL of nematode suspension was added to the center. The plates were then incubated in the dark for 12 hours. The number of nematodes on both sides was counted under a microscope, and the total number was used to calculate migration trends.

[0084] 3.6 Results Analysis

[0085] The effect of TYQ5 inoculation on the tropism and migration of southern root-knot nematodes is shown in Figure 5 and Figure 6 The tactic migration test showed that the application of TYQ5 alone had a significant effect on nematode tactic migration. The percentage of nematodes migrating to the root tips of cucumbers in the CK group reached 54.84%, significantly higher than the side of the cucumber root system with TYQ5. This had a significant impact on nematode tactic migration.

[0086] Example 4

[0087] In vitro contact toxicity of Pseudomonas kluyveri TYQ5 fermentation supernatant against root-knot nematodes.

[0088] 4.1 Preparation of supernatant from fermentation broth

[0089] The test strain was Pseudomonas kluyveri TYQ5, and the bacterial suspension was the same as that used in Example 3. After the bacterial suspension was prepared, it was centrifuged at 10,000 rpm / min for 5 minutes. The supernatant in the centrifuge tube was then aspirated with a syringe and filtered through a 0.22 µm filter membrane. The resulting solution was the supernatant of the fermentation broth of Pseudomonas kluyveri TYQ5.

[0090] 4.2 In vitro contact test

[0091] 400 µL of TYQ5 fermentation supernatant and a control sterile culture medium were transferred to a 48-well plate. Then, 100 µL of a suspension containing 100 second-instar larvae was added (400 µL of sterile water was added to the control). The plates were wrapped in tinfoil and incubated at 28°C. After 12 and 24 hours, the cells were examined microscopically and survival was assessed by adding a drop of 1 mol / L NaOH solution (rigor mortis indicated dead larvae, while wriggling larvae indicated live larvae). The number of dead larvae was counted and the adjusted mortality rate was calculated.

[0092]

[0093] 4.3 Results Analysis

[0094] The effect of TYQ5 fermentation supernatant on the in vitro contact killing of southern root-knot nematodes is shown in Figure 7-8 Treatment with the supernatant of TYQ5 fermentation broth significantly increased the corrected mortality of second-instar larvae of the southern root-knot nematode, with the effect consistently superior to that of the blank control. Time-lapse observation showed that the lethality rate increased continuously from 80.11% at 12 hours to 82.55% at 24 hours, indicating that the metabolites in the TYQ5 fermentation broth have a strong lethal effect on the nematodes.

[0095] Example 5

[0096] Pot culture experiment of Pseudomonas kluyveri TYQ5 against incognita root-knot nematode.

[0097] Cucumber is the most sensitive vegetable crop to southern root-knot nematodes. This example uses cucumber as a model crop for research.

[0098] The treatments in this example are: ① Inoculation with root-knot nematodes (CK+N); ② Inoculation with both TYQ5 and root-knot nematodes (TYQ5+N). The specific implementation steps are as follows:

[0099] 5.1 Seed disinfection

[0100] The specific operation of seed disinfection is the same as that in Example 3.

[0101] 5.2 Strain inoculation

[0102] Sterilized soil preparation: Forest soil was collected and sieved, then sterilized at 120°C for 1 hour, and then sterilized again after 24 hours.

[0103] Preparation of TYQ5 inoculum: inoculate TYQ5 into LB liquid medium and culture at 180 rpm / 28°C until OD 600 = 0.8, centrifuge at 8000 rpm for 5 minutes to collect the cells, discard the supernatant, and resuspend in an equal volume of 10 mM MgSO4 buffer;

[0104] Seedling inoculation treatment: After the radicle of the seeds grows out, sow them in a 50-hole tray with sterilized soil. When the first true leaf appears, start root irrigation: irrigate each plant with 10mL TYQ5 fungicide, once a week for 3 consecutive times.

[0105] 5.3 Inoculation of second-instar larvae of southern root-knot nematode

[0106] When cucumber seedlings have one leaf and one heart, begin inoculating with second-instar larvae of the southern root-knot nematode. Use a pipette to poke two small holes about 1 cm deep around the root circumference of the cucumber seedlings. Add the root-knot nematode suspension into the holes, inoculating 250 root-knot nematodes per seedling.

[0107] 5.4 Sampling and measurement

[0108] On the 21st day after nematode inoculation, cucumber plant growth indicators (plant height, stem diameter, aboveground fresh weight, belowground fresh weight, aboveground dry weight, belowground dry weight, and chlorophyll content) were measured and analyzed. The roots of the cucumber seedlings were scanned with a root scanner and analyzed using WinRHIZO analysis system software.

[0109] 5.4 Results Analysis

[0110] After 21 days of treatment with Pseudomonas kluyveri TYQ5 and infection with root-knot nematodes, the cucumber seedlings were tested. Table 1 shows the effects of Pseudomonas kluyveri TYQ5 of the present invention on the physiological indicators of cucumbers infected with root-knot nematodes. Table 2 shows the effects of Pseudomonas kluyveri TYQ5 of the present invention on the root system of cucumbers infected with root-knot nematodes. Figure 9-10 This is a diagram showing the effects of TYQ5 on the aboveground and underground parts of cucumber infected by root-knot nematodes.

[0111] Analysis showed that in the presence of root-knot nematode infection, the number of root knots per unit in cucumber seedlings inoculated with the TYQ5 strain decreased by 31.77%, while the total root length, root surface area, average root diameter and root volume increased by 62.48%, 80.42%, 9.76% and 96.77% respectively compared with the control. The plant height, underground dry weight and chlorophyll content increased by 20.21%, 113.33% and 211.44% respectively. This indicates that the Pseudomonas kluyveri TYQ5 strain not only slowed down the development process of root-knot nematodes in the cucumber root system and significantly reduced the number of root knots per unit in the cucumber root system, but also promoted root development and growth, which is beneficial to the early prevention and control of root-knot nematodes and helps to achieve safe and pollution-free production of vegetables.

[0112] Table 1 Effects of TYQ5 on physiological parameters of cucumber infected by root-knot nematodes

[0113]

[0114] Table 2 Effects of TYQ5 on cucumber roots infected by root-knot nematodes

[0115]

[0116] Example 6

[0117] Antagonism of TYQ5 against Fusarium oxysporum on plates

[0118] 6.1 Preparation of strain suspension

[0119] The specific operation of preparing the strain suspension is the same as that in Example 3.

[0120] 6.2 Preparation of Fusarium oxysporum plates

[0121] The Fusarium oxysporum used in the present invention is a strain stored in the laboratory's bacterial library. A bacterial mass was picked and inoculated on a solid PDA culture medium plate and cultured at 28° C. for 3 days.

[0122] 6.3 Antagonistic Effect of TYQ5 on Fusarium oxysporum

[0123] Fusarium inoculation: Cut the cultured Fusarium oxysporum plate into 3 × 3 mm pieces and inoculate them on a new PDA plate at 1 / 4 of its diameter.

[0124] TYQ5 streaking: Dip a sterilized toothpick into the TYQ5 bacterial solution with the adjusted OD value and streak perpendicular to the diameter of the Fusarium block 2-3 times;

[0125] Co-culture observation: After culturing at 28°C for 2-3 days, the lengths of the major and minor axes of the Fusarium circle (elliptical morphology) were measured, and the eccentricity was calculated to evaluate the growth inhibition effect.

[0126]

[0127] 6.4 Results Analysis

[0128] The antagonistic effect of TYQ5 and Fusarium oxysporum on plate is shown in Figure 11 The effect of TYQ5 on the growth of Fusarium oxysporum is shown in Table 3. Under the action of TYQ5, the growth of Fusarium oxysporum changed from its original circular shape to an ellipse with an eccentricity greater than 0.5, indicating that TYQ5 significantly inhibited the normal growth of Fusarium oxysporum and blocked its growth direction. In terms of the growth area of ​​Fusarium oxysporum, the area of ​​the colony was reduced by 48.58% compared to the control, which further demonstrates that TYQ5 has an antagonistic effect on Fusarium oxysporum.

[0129] Table 3 Effects of TYQ5 on the growth of Fusarium oxysporum

[0130]

[0131] Note: The eccentricity in the table indicates the growth of Fusarium oxysporum. The higher the eccentricity value, the flatter the ellipse, and the lower the eccentricity value, the closer it is to a circle.

[0132] Example 7

[0133] Degradation characteristics of TYQ5 on vegetable straw

[0134] 7.1 Vegetable straw pretreatment

[0135] This example uses four common fruit and vegetable straws, including cucumber, tomato, pepper, and eggplant straws, as materials. They are cut into 3-4 cm small segments, then placed in an oven for drying, put into a 250 ml triangular flask, sealed with sealing film, and placed in a high pressure sterilizer for sterilization at 121 ° C for 15 minutes.

[0136] 7.2 Determination of vegetable straw degradation rate

[0137] 5 g of straw was added to each of the 125 ml carbon-deficient liquid culture medium in a triangular flask. The formula of the carbon-deficient liquid culture medium was the same as that in Example 3. Then, 5 ml of overnight activated TYQ5 bacterial solution was inoculated into the culture medium. The triangular flask was sealed with a sealing film and placed on a shaker at a temperature of 28°C and a speed of 180 rpm / min for 15 minutes. The straw was then collected, washed, dried, and weighed. The degradation rates of the four types of straw were calculated using the following formula:

[0138]

[0139] 7.3 Degradation effect of TYQ5 on four types of vegetable straw Figure 12After analysis, TYQ5 can degrade four types of vegetable straw to varying degrees, among which the degradation rate of tomato straw is the highest at 68.93%, while the degradation rates of pepper and eggplant straw are lower. This may be because these two types of straw have a higher degree of lignification and contain more lignin, so they are not easy to degrade.

[0140] Table 4 Degradation rate of TYQ5 on four vegetable straws

[0141]

[0142] Example 8

[0143] Phosphate solubilization characteristics of TYQ5

[0144] 8.1 Preliminary evaluation of TYQ5's ability to dissolve poorly soluble phosphate using inorganic phosphate solid medium

[0145] Pipette 10 μL of overnight cultured TYQ1 bacterial solution onto the surface of inorganic phosphorus solid culture medium, seal it with sealing film, and then culture it in a 28°C incubator for 5 days to observe whether there is a phosphate-dissolving zone formed around the colony.

[0146] 8.2 Results

[0147] After 5 days of culture on inorganic phosphorus solid medium, TYQ5 can form a clear transparent phosphate-dissolving circle around the colony ( Figure 13 ).

[0148] Example 9

[0149] Phosphate solubilization ability of TYQ5 under gradient salt concentration and pH

[0150] 9.1 Preparation of Gradient Salt Concentration and pH Inorganic Phosphorus Liquid Culture Medium

[0151] Prepare inorganic phosphorus liquid culture medium, add NaCl to set the gradient salt concentration of 0, 2, 4, 6, and 8 g / L, and use a pH meter to accurately add NaOH and HCl to set the pH gradient of 5, 6, 7, 8, and 9.

[0152] 9.2 Determination of solubility

[0153] TYQ5's ability to solubilize poorly soluble phosphate was evaluated using a gradient salt concentration and inorganic phosphate liquid medium. 50 µL of overnight TYQ5 culture was added to 5 mL of inorganic phosphate liquid medium, sealed with parafilm, and incubated at 28°C for 5 days. The phosphate solubilization capacity of TYQ5 was then quantitatively analyzed using a molybdenum antimony colorimetric assay. The supernatant was collected as the test solution. 5 mL of the supernatant was added to 2.5 mL of molybdenum antimony colorimetric reagent. After incubation at room temperature for 30 minutes, the absorbance was measured at 700 nm.

[0154] 9.3 Results

[0155] like Figure 14 The phosphate solubility of TYQ5 under gradient salinity and gradient pH values ​​showed a decreasing trend with increasing salt concentration. The phosphate solubility was strongest at 75.01 mg / L when the NaCl concentration was 0 g / L, and at 74.20 mg / L when the pH was 6.

[0156] Example 10

[0157] Carbon source utilization ability of TYQ5

[0158] 10.1 Biolog GenIII Microplate Assay

[0159] The ability of TYQ5 strain to utilize or oxidize various pre-selected carbon sources was tested using Biolog GenIII microplates. 590 The absorbance values ​​at nm were used to generate the metabolic fingerprints of TYQ5 to different carbon sources.

[0160] 10.2 Results

[0161] The fingerprint of TYQ5's ability to utilize different carbon sources is shown in Figure 15 Carbon source utilization experiments revealed that TYQ5 exhibited varying degrees of utilization across all 70 tested carbon sources, with OD values ​​directly reflecting its metabolic activity. In chemical resistance testing, the strain demonstrated significant insensitivity to eight of the 21 chemical agents, demonstrating strong environmental adaptability and metabolic diversity.

[0162] Summary: From the above examples, it can be seen that:

[0163] (1) This strain was isolated from cucumber rhizosphere soil in the laboratory of China Agricultural University and its classification name is Pseudomonas kluyveri ( Pseudomonas knackmussii )TYQ5, deposited in the General Microbiology Center of China Culture Collection Administration on April 27, 2023, with the deposit number CGMCC.27214.

[0164] (2) The microbiological characteristics of Pseudomonas kluyveri TYQ5 are as follows: the single colony of this strain is round, opaque yellow, with a smooth and moist surface, irregular edges, a slightly raised center, Gram staining is negative, and the optimal growth temperature is 28-37°C.

[0165] (3) By amplifying the 16S rDNA fragment of the strain and performing BLAST homology comparison, the similarity of the strain to Pseudomonas knackmussii reached 99.79%. Therefore, the strain was identified as a strain of the genus Pseudomonas and named Pseudomonas knackmussii TYQ5.

[0166] (4) Through qualitative and quantitative analysis, it was determined that TYQ5 can secrete auxin. In the presence of tryptophan, the concentration of IAA secreted was 16.75 μg / mL.

[0167] (5) TYQ5 interferes with nematode tropism in plants: When the cotyledons of the seedlings were flattened, TYQ5 fermentation liquid was poured into the soil around the roots, with 10 ml poured per seedling. This application increased the migration of southern root-knot nematodes to the control roots by 54.84%.

[0168] (6) The supernatant of the fermentation liquid of the TYQ5 strain was used to treat isolated root-knot nematodes for 12 hours and 24 hours, and the mortality rate of the southern root-knot nematodes reached 80.11% and 82.55%, respectively.

[0169] (7) The experiment of cultivating cucumbers inoculated with southern root-knot nematodes proved that TYQ5 could significantly alleviate the stress of root-knot nematodes: the number of knots per unit root was reduced by 31.77%, the total root length, root surface area, average root diameter and root volume increased by 62.48%, 80.42%, 9.76% and 96.77% respectively compared with the control, and the plant height, underground dry weight and chlorophyll content increased by 20.21%, 113.33% and 211.44% respectively.

[0170] (8) TYQ5 can effectively inhibit the normal growth of Fusarium oxysporum when confronted with the Fusarium oxysporum plate, and the area of ​​the fungus ring is reduced by 29.02%, which is beneficial for preventing soil-borne diseases caused by Fusarium oxysporum when used.

[0171] (9) Within two weeks, the degradation rates of TYQ5 on tomato, cucumber, pepper and eggplant straw reached 68.93%, 58.63%, 31.37% and 39.24%, respectively.

[0172] (10) The phosphorus solubility of TYQ5 decreases with increasing salt concentration. The phosphorus solubility is strongest at 75.01 mg / L when the NaCl concentration is 0 g / L, and at 74.20 mg / L when the pH is 6.

[0173] (11) Biolog analysis of TYQ5 showed different degrees of utilization ability of 70 tested carbon sources, and 8 of the 21 chemical reagents showed significant insensitivity, indicating strong environmental adaptability and metabolic diversity.

[0174] The description of the limited preferred embodiments of the invention is relatively specific and detailed, but this should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the invention, and such modifications and improvements are within the scope of protection of the invention.

Claims

1. Use of a strain of Pseudomonas kluyveri in preparing a preparation for promoting the growth of cucumber seedlings, characterized in that: The Pseudomonas knackmussii strain TYQ5 is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 27214. The use comprises the following steps: Preparation of TYQ5 inoculum: Inoculate TYQ5 into LB liquid medium and grow at 180 rpm / 28°C until OD600 = 0.

8. Collect the cells by centrifugation at 8000 rpm for 5 minutes, discard the supernatant, and resuspend in an equal volume of 10 mM MgSO4 buffer. After the cucumber seeds have grown radicles, they are sown in 50-hole trays with sterilized soil. When the first true leaf appears, root irrigation begins: 10 mL of TYQ5 inoculant is applied to each plant once a week for three consecutive times.

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