Pseudomonas kirschner and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly discloses a Pseudomonas klackmussii strain TYQ5 and application thereof, the Pseudomonas klackmussii strain TYQ5 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.27214, and the Pseudomonas klackmussii strain TYQ5 is preserved in the China General Microbiological Culture Collection Center (CGMCC). The applicant finds that the Pseudomonas kirschner can secrete auxin, can promote the growth of cucumber seedlings, also has the capabilities of interfering tropism migration of nematodes and killing nematodes, has the effect of remarkably relieving stress of root-knot nematodes, also finds that the Pseudomonas kirschner has the capability of inhibiting normal growth of fusarium oxysporum, and also finds that the Pseudomonas kirschner has the capability of inhibiting normal growth of fusarium oxysporum. Furthermore, the pseudomonas kirschner has the capability of degrading vegetable straws and the capability of solubilizing phosphorus, so that the applicant considers that the pseudomonas kirschner has a remarkable application prospect 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 application thereof. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) play a key role in the root micro-ecosystem, and significantly promote plant growth and soil health by establishing a mutually beneficial symbiotic relationship with plants. PGPR directly promotes plant growth and development by secreting plant hormones such as indoleacetic acid, and improves soil nutrient availability and optimizes plant nutrient supply by decomposing organic matter and dissolving minerals. In addition, PGPR inhibits the growth of pathogens and reduces diseases by producing antibiotics and competing for resources, and induces plants to produce systemic resistance and enhance immunity. These characteristics enable PGPR to play an important role in reducing the use of pesticides and improving the ability of crops to resist diseases and pests, and promote the sustainable development of agriculture. By screening excellent PGPR strains and conducting in-depth research on their biological characteristics and functional mechanisms, efficient and environmentally friendly microbial agents can be developed to promote the green transformation of agriculture.

[0003] Meloidogyne, especially southern root-knot nematode, is a very common soil-borne disease in greenhouse cucumber production. It is highly concealed, spreads rapidly and causes serious damage, often resulting in stunted growth of cucumbers, with an average annual yield reduction of 10% to 50%, and even total crop failure in severe cases. It is considered a "terminal disease" for cucumber production. Although traditional control methods, such as physical, chemical and agricultural methods, have certain effects, they are difficult to cure and are prone to cost increases and soil environmental deterioration. Fusarium oxysporum is also a pathogen that is highly destructive to horticultural crops. By infecting the roots and vascular system, it causes plant wilt and death, seriously affecting crop yields, increasing control costs, and posing a dual threat to the economy and the environment. Given that current research on the use of beneficial microorganisms in the cucumber rhizosphere to control these two diseases is still insufficient, screening for beneficial strains that are highly effective in controlling root-knot nematodes and Fusarium oxysporum has become an urgent need and hot topic for research at home and abroad.

[0004] The scale of vegetable cultivation is expanding day by day, 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 environmental pollution and other problems. 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 various enzymes secreted by themselves, thereby achieving rapid degradation and transformation of straw. Screening for degradable vegetable straw microorganisms 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 are difficult to adapt to complex soil conditions and climatic factors, and their effectiveness is greatly reduced, and they cannot fully meet the needs of agricultural production. The development of microbial strains with diverse functions has become the key to improving agricultural production efficiency and quality. These multifunctional strains can take root in different types of soils, adapt to diverse growth environments, and secrete auxins to stimulate crop root growth, enhance nutrient absorption capacity, 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, reduce 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 view of the above problems, the present invention discloses a strain of Pseudomonas kluyveri and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: A strain of Pseudomonas kluyveri, wherein the Pseudomonas kluyveri ( Pseudomonas knackmussii ) strain TYQ5, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 27214.

[0008] The invention also discloses a microbial agent, which contains the Pseudomonas kluyveri cells and / or its metabolites and / or its fermentation liquid.

[0009] The invention also discloses the use of the bacteria or bacterial agent in preparing a preparation for enhancing the growth promotion of cucumber seedlings.

[0010] The 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.

[0011] The invention also discloses the use of the bacteria or bacterial agent in preparing a preparation for preventing and controlling southern root-knot nematodes.

[0012] The invention also discloses the use of the bacteria or bacterial agent in preparing a preparation for inhibiting the growth of Fusarium oxysporum.

[0013] The invention also discloses the use of the bacteria or bacterial agent in preparing a phosphate-dissolving preparation.

[0014] The invention also discloses the use of the bacteria or bacterial agent in preparing a straw degradation preparation.

[0015] The present invention has the following beneficial effects: The invention discloses a strain of Pseudomonas kluyveri and applications thereof. In terms of growth promotion, the Pseudomonas kluyveri TYQ5 disclosed by the invention has the growth-promoting properties of dissolving inorganic phosphorus and secreting auxin. TYQ5 can utilize tryptophan to produce IAA, and the measured content is 16.75 μg / mL, which indicates 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 the mortality to second-instar larvae of root-knot nematodes, and its effect is significantly better than that of a control strain. It showed a strong antagonistic effect in the growth inhibition experiment of Fusarium oxysporum. After inoculation with TYQ5 strain, the number of root knots per unit of cucumber seedlings decreased by 31.77%, the total root length, root surface area and root volume increased by 62.48%, 80.42% and 96.77% respectively compared with the control, and the underground dry weight and chlorophyll content increased by 113.33% and 211.44% respectively. Within 2 weeks, the degradation rates of tomato, cucumber, pepper and eggplant straw by TYQ5 reached 68.93%, 58.63%, 31.37% and 39.24% respectively. The phosphorus solubility of TYQ5 showed a decreasing trend with the increase of salt concentration. The phosphorus solubility was the strongest at 0 g / L NaCl concentration (75.01 mg / L), and the strongest at pH 6 (74.20 mg / L). The Biolog analysis of TYQ5 can use 70 test carbon sources and showed significant insensitivity to 8 chemical reagents, showing 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 restoration.

[0016] The strain deposit information is as follows: Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration; Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: April 27, 2023 Deposit number: CGMCC No. 27214, Taxonomic name: Pseudomonas kluyveri Pseudomonas knackmussii . BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the colony morphology of TYQ5; Figure 2 This is the phylogenetic tree diagram of TYQ5; Figure 3 This is a diagram showing the effect of TYQ5 producing auxin; Figure 4 This is a schematic diagram of the tropism migration experiment of southern root-knot nematodes after TYQ5 inoculation; Figure 5This is a diagram showing the effect of TYQ5 inoculation on the tropism and migration of southern root-knot nematodes; Figure 6 This is a diagram showing the effect of TYQ5 inoculation on the tropism and migration of southern root-knot nematodes; Figure 7 This is a graph showing the effect of TYQ5 fermentation supernatant on the in vitro contact killing of southern root-knot nematodes; Figure 8 This is a graph showing the in vitro contact killing effect of TYQ5 fermentation supernatant on southern root-knot nematodes; Fig. 9 This is a diagram showing the effect of TYQ5 inoculation on cucumber plants infected by southern root-knot nematodes; Fig.10 The effect of TYQ5 inoculation on cucumber roots infected by southern root-knot nematodes Fig.11 This is a diagram showing the antagonistic effect of TYQ5 and Fusarium oxysporum on a plate; Fig.12 This is the degradation effect of TYQ5 on four types of vegetable straw; Fig.13 This is the phosphorus dissolution characteristic diagram of TYQ5; Fig.14 This is the phosphorus solubilization capacity diagram of TYQ5 under gradient salt concentration and gradient pH; Fig.15 This is the fingerprint of TYQ5's ability to utilize different carbon sources. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0019] The detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1 1.1 Pseudomonas kluyveri ( Pseudomonas knackmussii )Isolation and purification of TYQ5: The Pseudomonas kluyveri of the present invention ( 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 the soil dilution method. The specific steps are as follows: Take 4 portions (1 g each) of rhizosphere soil from cucumbers infected with root-knot nematodes in the Facility Vegetable Laboratory of China Agricultural University and mix them evenly; weigh 1 g of the mixed soil sample, add it to a 50 ml centrifuge tube containing 9 ml of sterile water, and shake it at 180 rpm for 20 minutes; take 1 ml of the soil suspension to a new centrifuge tube, add 9 ml of sterile water to make a 10-fold gradient dilution, and repeat the operation to the required concentration; take 100 μL of the appropriate dilution suspension and spread it on LB solid culture medium, and culture it at 28 °C for 48 hours; pick a single colony and perform three consecutive streaking isolations to obtain a pure culture.

[0021] 1.2 Pseudomonas kluyveri ( Pseudomonas knackmussii )Identification of strain TYQ5 (1) Microbiological characteristics 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, 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.

[0022] (2) Molecular biological characteristics 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 primers 27F and 1492R using the bacterial solution as a template.

[0023] The PCR amplification reaction system was 50 µL, including 25 µL 2xTaq enzyme, 1 µL 27F primer, 1 µL 1492R primer, 1 µL bacterial solution and 22 µL ddH2O. The amplification conditions were: 95°C pre-denaturation for 3 min, 94°C denaturation for 25 s, 55°C annealing for 25 s, 72°C extension for 1 min, 32 cycles, 72°C extension for 5 min, and the amplified product was stored at 4°C. The amplified product was separated and identified by 1% agarose gel electrophoresis, and the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The 16S rDNA of strain TYQ5 is shown in the following SEQ ID NO:1: The results of the assay 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 Microbiological Culture Collection Administration with the deposit number CGMCC.27214.

[0024] Example 2 Qualitative and quantitative analysis of IAA secretion ability of Pseudomonas kluyveri TYQ5 The IAA secretion capacity of Pseudomonas kluyveri TYQ5 was determined by the Salkowski colorimetric method.

[0025] 2.1 Qualitative analysis of the ability of Pseudomonas kluyveri TYQ5 to secrete IAA Preparation of Salkowaki color developer Dissolve 0.811 g FeCl3 in 10 mL sterile distilled water to prepare a 0.5 M FeCl3 solution. Take 1 mL of the prepared 0.5 M FeCl3 solution and add it to 49 mL of 35% HClO4 solution to prepare the Salkowaki colorimetric reagent. Mix thoroughly before use.

[0026] Bacterial culture and fermentation 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 suspension and inoculate it into LB liquid medium containing 500 µg / mL tryptophan that has been sterilized by 0.22 µm filtration at a 1% (v / v) inoculation volume. Set up an LB liquid medium without inoculation as a negative control. Cultivate at 28°C and 180 rpm / min for 3 days.

[0027] IAA qualitative test 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 supernatant of the fermentation broth and place it in a 10 mL centrifuge tube. Add 1 mL of Salkowaki colorimetric reagent, mix well and let 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.

[0028] 2.2 Quantitative analysis of the IAA secretion capacity of Pseudomonas kluyveri TYQ5 Preparation of IAA standard curve First, prepare 100 mg·L -1 The IAA stock solution was diluted to 0, 10, 20, 30, 40, 50, 60, 80, 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 Plot a standard curve for the ordinate.

[0029] IAA content determination Take 500 µL of TYQ5 fermentation supernatant, add 4 mL of colorimetric reagent, and measure OD after 30 minutes of reaction in the dark. 530 The uninoculated culture medium was used as the blank control, and the IAA content was calculated using the standard curve.

[0030] result Pseudomonas kluyveri TYQ5 can synthesize IAA from tryptophan with a yield of 16.75 µg / mL.

[0031] Example 3 Effect of fermentation supernatant of Pseudomonas kluyveri TYQ5 on the tropism and migration of root-knot nematodes.

[0032] 3.1 Preparation of strain suspension Pick a single colony of Pseudomonas kluyveri TYQ5 and inoculate it into 30 mL LB liquid medium. Cultivate overnight at 28°C and 180 rpm. Dilute the bacterial solution to OD 600 =0.8, centrifuge at 10000 rpm for 10 minutes, discard the supernatant and retain the bacteria. 4 Resuspend the bacteria to prepare the bacterial solution for use.

[0033] 3.2 Cucumber root preparation Cucumber is a vegetable crop that is sensitive to southern root-knot nematodes. In this example, cucumber is used as a model crop for research.

[0034] Seed disinfection and germination: Select plump and intact cucumber seeds, soak them in 75% ethanol for 30 seconds, 3% NaClO for 10 minutes, rinse with sterile water and soak for 6 hours. Place the seeds on moist sterile filter paper and culture at 28℃ until the cotyledon flattening stage (5-7 days).

[0035] Inoculation and sampling of bacterial solution: Inoculate 10 mL of the prepared bacterial solution into the seedlings. When the seedlings grow to two leaves and one heart, take the roots for subsequent tests.

[0036] 3.3 Preparation of second-instar larvae suspension of root-knot nematodes Egg masses were picked from the root system of infected water spinach, washed with 0.6% sodium hypochlorite, 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. The hatched second-instar larvae were collected, counted under a microscope, and adjusted to 100 / 100 µL.

[0037] 3.4 23% Pluronic F-127 gel configuration At 4°C, dissolve 23 g of F-127 powder in 80 mL of precooled sterile water, stir with a low-speed magnetic stirrer until dissolved, and store at 4°C for later use.

[0038] 3.5 Migration assay Schematic diagram of the tropism migration experiment of southern root-knot nematodes after TYQ5 inoculation is shown in Figure 4 The experiment set up two groups, CK (no bacterial solution) and bacterial solution, with 20 replicates each. A 0.5 cm diameter Oxford cup was placed in the nematode inoculation area of ​​the migration plate to leave a gap, and the control and experimental group roots were placed on both sides, respectively, and covered with agar gel. After the gel solidified, the Oxford cup was removed, 100 μL of nematode suspension was added to the center, and the mixture was left to stand for 12 h in the dark. The number of nematodes on both sides was counted by microscopy, and the migration trend was calculated based on the total number.

[0039] 3.6 Results Analysis The effect of TYQ5 inoculation on the tropism and migration of southern root-knot nematodes is shown in Figure 5 and Figure 6 The tropism migration test showed that the application of TYQ5 alone had a significant effect on the tropism of nematodes. The percentage of nematodes migrating to the root tip of cucumbers in the CK group reached 54.84%, which was significantly higher than that on the side of the cucumber root system with TYQ5. It had a significant effect on the tropism of nematodes.

[0040] Example 4 In vitro contact killing effect of fermentation supernatant of Pseudomonas kluyveri TYQ5 on root-knot nematodes.

[0041] 4.1 Preparation of supernatant from fermentation broth 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 10000 rpm / min for 5 min, and then the supernatant in the centrifuge tube was aspirated with a syringe and filtered with a 0.22 μm filter membrane. The solution obtained after filtration was the fermentation supernatant of Pseudomonas kluyveri TYQ5.

[0042] 4.2 In vitro contact test Take 400 µL of TYQ5 fermentation supernatant and control sterile culture medium and add them to a 48-well plate, add 100 µL of suspension containing 100 second-instar larvae (add 400 µL sterile water to the control group). Wrap with tin foil and incubate at 28°C. After 12 h and 24 h, examine under a microscope, add 1 mol / L NaOH solution to determine the survival state (rigor mortis means dead worms, wriggling means live worms), count the number of deaths and calculate the corrected mortality rate.

[0043]

[0044] 4.3 Results Analysis The effect of TYQ5 fermentation supernatant on the in vitro contact killing of southern root-knot nematodes is shown in Figure 7-8 TYQ5 fermentation supernatant treatment significantly increased the corrected mortality of second-instar larvae of southern root-knot nematodes, and the effect was consistently better than the blank control. Time-lapse observation showed that the lethality rate continued to increase from 80.11% at 12 h to 82.55% at 24 h, indicating that the metabolites in TYQ5 fermentation broth have a strong lethal effect on nematodes.

[0045] Example 5 Pot culture experiment of Pseudomonas kluyveri TYQ5 against southern root-knot nematode.

[0046] Cucumber is the most sensitive vegetable crop to southern root-knot nematodes. This example uses cucumber as a model crop for research.

[0047] The treatments in this example are as follows: ① inoculation of root-knot nematodes (CK+N); ② inoculation of both TYQ5 bacterial agent and root-knot nematodes (TYQ5+N). The specific implementation steps are as follows: 5.1 Seed disinfection The specific operation of seed disinfection is the same as that in Example 3.

[0048] 5.2 Strain inoculation Sterilized soil preparation: After collecting and sieving forest soil, sterilize it at 120℃ for 1 hour, and repeat the sterilization once after 24 hours; Preparation of TYQ5 inoculant: Inoculate TYQ5 into LB liquid medium and culture at 180rpm / 28℃ 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; 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.

[0049] 5.3 Inoculation of second-instar larvae of southern root-knot nematode When the cucumber seedlings are in the one-leaf-one-heart stage, start inoculating with the second-instar larvae of southern root-knot nematodes. Use a gun tip to poke two small holes about 1 cm deep around the root of the cucumber seedlings, add the root-knot nematode suspension into them, and inoculate 250 root-knot nematodes per seedling.

[0050] 5.4 Sampling and determination On the 21st day after nematode inoculation, samples were taken to measure and count the growth indicators of cucumber plants (plant height, stem diameter, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, chlorophyll content), and the roots of cucumber seedlings were scanned with a root scanner and analyzed using WinRHIZO analysis system software.

[0051] 5.4 Results Analysis After 21 days of treatment with Pseudomonas kluyveri TYQ5 and infection of cucumber seedlings with root-knot nematodes, 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.

[0052] 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%, and 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 showed that the Pseudomonas kluyveri TYQ5 strain not only retarded the development process of root-knot nematodes in the cucumber root system, significantly reduced the number of root knots per unit in the cucumber root system, but also promoted the development and growth of the root system, which is beneficial to the early prevention and control of root-knot nematodes and helps to achieve safe and pollution-free production of vegetables.

[0053] Table 1 Effects of TYQ5 on physiological parameters of cucumber infected by root-knot nematodes

[0054] Table 2 Effects of TYQ5 on cucumber roots infected by root-knot nematodes

[0055] Example 6 Antagonism of TYQ5 against Fusarium oxysporum on plates 6.1 Preparation of strain suspension The specific operation of preparing the strain suspension is the same as that in Example 3.

[0056] 6.2 Preparation of Fusarium oxysporum plates The Fusarium oxysporum used in the present invention is a strain stored in the bacterial bank of the laboratory. A bacterial mass is picked and inoculated on a solid PDA culture medium plate and cultured at 28° C. for 3 days.

[0057] 6.3 Antagonism of TYQ5 against Fusarium oxysporum Fusarium inoculation: Cut the cultured Fusarium oxysporum plate into 3×3mm pieces and inoculate them on a new PDA plate at 1 / 4 of the diameter; TYQ5 streaking: Dip a sterilized toothpick into the TYQ5 bacterial solution with the adjusted OD value, and repeat streaking 2-3 times perpendicular to the diameter of the Fusarium block; 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.

[0058]

[0059] 6.4 Results Analysis The antagonistic effect of TYQ5 and Fusarium oxysporum on the plate is shown in Fig.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 the original approximately circular shape to an ellipse with an eccentricity of >0.5, which shows that TYQ5 significantly inhibited the normal growth of Fusarium oxysporum and blocked its growth direction. From the perspective of the growth area of ​​Fusarium oxysporum, compared with the control, the area of ​​the bacterial circle was reduced by 48.58%, which more intuitively shows that TYQ5 has an antagonistic effect on Fusarium oxysporum.

[0060] Table 3 Effect of TYQ5 on the growth of Fusarium oxysporum

[0061] Note: The eccentricity in the table indicates the growth of Fusarium oxysporum. The higher the eccentricity value, the flatter the ellipse is, and the lower the eccentricity value, the closer it is to a circle.

[0062] Example 7 Degradation characteristics of TYQ5 on vegetable straw 7.1 Vegetable straw pretreatment This embodiment uses four common fruit and vegetable straws including cucumber, tomato, pepper and eggplant straws as materials, which are cut into 3-4 cm small segments respectively, and then placed in an oven for drying, and then put into a 250 ml triangular bottle, which is sealed with a sealing film and placed in a high pressure sterilizer, and sterilized at 121°C for 15 minutes.

[0063] 7.2 Degradation rate of vegetable straw 5 g of straw was added to each of the triangular flasks containing 125 ml of carbon-deficient liquid culture medium, wherein the formula of the carbon-deficient liquid culture medium was the same as that in Example 3, and then 5 ml of TYQ5 bacterial solution activated overnight 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 seconds. The straw was recovered, washed, dried and weighed, and the degradation rates of the four types of straw were calculated respectively. The calculation formula was:

[0064] 7.3 The degradation effect of TYQ5 on four types of vegetable straw is shown in Fig.12 After analysis, TYQ5 can degrade four types of vegetable straws 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 straws have a higher degree of lignification and contain more lignin, so they are not easy to degrade.

[0065] Table 4 Degradation rate of TYQ5 on four vegetable straws

[0066] Example 8 Phosphate dissolving characteristics of TYQ5 8.1 Preliminary evaluation of TYQ5’s ability to dissolve poorly soluble phosphorus using inorganic phosphorus solid medium Pipette 10 µL of overnight cultured TYQ1 bacterial solution onto the surface of inorganic phosphorus solid culture medium, seal it with sealing film, and place it in a 28°C incubator for 5 days to observe whether there is a phosphate-dissolving zone formed around the colony.

[0067] 8.2 Results After 5 days of culture on inorganic phosphorus solid medium, TYQ5 can form an obvious transparent phosphate-dissolving circle around the colony ( Fig.13 ).

[0068] Example 9

[0069] Phosphorus dissolving ability of TYQ5 in gradient salt concentration and pH 9.1 Preparation of Gradient Salt Concentration and pH Inorganic Phosphorus Liquid Culture Medium Prepare inorganic phosphorus liquid culture medium, add NaCl to set the gradient salt concentration of 0, 2, 4, 6, 8 g / L for treatment, and use a pH meter to accurately add NaOH and HCl to set the pH gradient of 5, 6, 7, 8, 9 for treatment.

[0070] 9.2 Determination of solubility The ability of TYQ5 to dissolve poorly soluble phosphorus was evaluated using gradient salt concentration and inorganic phosphorus liquid culture medium: 50 µL of overnight cultured TYQ5 bacterial solution was added to 5 ml of inorganic phosphorus liquid culture medium, sealed with sealing film and placed in a 28°C incubator for 5 days, and then the phosphorus dissolving ability of TYQ5 was further quantitatively analyzed using the molybdenum antimony colorimetric method. The supernatant was taken as the test solution. 5 ml of supernatant was added to 2.5 ml of molybdenum antimony colorimetric agent, and after being placed at room temperature for 30 minutes, its absorbance was measured at a wavelength of 700 nm.

[0071] 9.3 Results like Fig.14 The phosphorus solubilizing ability of TYQ5 under gradient salinity and gradient pH value showed a decreasing trend with the increase of salt concentration. The strongest phosphorus solubilizing ability was 75.01 mg / L when the NaCl concentration was 0 g / L, and the strongest phosphorus solubilizing ability was 74.20 mg / L when the pH was 6.

[0072] Example 10

[0073] Carbon source utilization ability of TYQ5 10.1 Biolog GenIII microplate detection The TYQ5 strain was tested for its ability to utilize or oxidize various pre-selected carbon sources using a Biolog GenIII microplate. 590 The absorbance value at nm generated the metabolic fingerprint of TYQ5 to different carbon sources.

[0074] 10.2 Results The fingerprint of TYQ5's ability to utilize different carbon sources is shown in Fig.15 The carbon source utilization experiment showed that TYQ5 showed different degrees of utilization ability for 70 tested carbon sources, and the OD value directly reflected its metabolic activity. In the chemical resistance test, the strain showed significant insensitivity to 8 of the 21 chemical reagents, showing strong environmental adaptability and metabolic diversity.

[0075] Summary: From the above examples, it can be seen that: (1) This strain was isolated from cucumber rhizosphere soil in the laboratory of China Agricultural University and its taxonomic name is Pseudomonas kluyveri ( Pseudomonas knackmussii )TYQ5, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 27, 2023, with the deposit number CGMCC.27214.

[0076] (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.

[0077] (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.

[0078] (4) Through qualitative and quantitative analysis, it was determined that TYQ5 can secrete auxin, and the concentration of IAA secreted in the presence of tryptophan was 16.75 μg / mL; (5) TYQ5 interferes with nematode tropism in plants: When the cotyledons of the seedlings were flattened, the 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 root system by 54.84%.

[0079] (6) The supernatant of the fermentation liquid of the TYQ5 strain was used to treat isolated root-knot nematodes for 12 h and 24 h, and the mortality rates against southern root-knot nematodes reached 80.11% and 82.55%, respectively.

[0080] (7) The experiment of cultivating cucumbers inoculated with southern root-knot nematodes proved that TYQ5 had the effect of significantly alleviating 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.

[0081] (8) TYQ5 can effectively inhibit the normal growth of Fusarium oxysporum when confronted with Fusarium oxysporum on a plate, and the area of ​​the bacterial circle is reduced by 29.02%, which is beneficial for preventing soil-borne diseases caused by Fusarium oxysporum when used.

[0082] (9) Within two weeks, the degradation rates of tomato, cucumber, pepper and eggplant straw by TYQ5 were 68.93%, 58.63%, 31.37% and 39.24%, respectively.

[0083] (10) The phosphorus solubility of TYQ5 decreased with the increase of salt concentration. The phosphorus 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.

[0084] (11) Biolog analysis of TYQ5 showed that 70 tested carbon sources showed different degrees of utilization ability, and 8 out of 21 chemical reagents showed significant insensitivity, indicating strong environmental adaptability and metabolic diversity.

[0085] The description of the limited preferred embodiments of the invention is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the invention, which all belong to the protection scope of the invention.

Claims

1. A strain of Pseudomonas kluyveri, characterized in that The Pseudomonas kluyveri ( Pseudomonas knackmussii ) strain TYQ5, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.27214.

2. A microbial agent, characterized in that: It contains the Pseudomonas kluyveri cells and / or its metabolites and / or its fermentation liquid as claimed in claim 1.

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

4. Use of the bacterium according to claim 1 or the bacterial agent according to claim 2 in preparing a preparation for interfering with the migration of southern root-knot nematodes.

5. Use of the bacterium according to claim 1 or the bacterial agent according to claim 2 in preparing a preparation for controlling southern root-knot nematodes.

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

7. Use of the bacteria according to claim 1 or the bacterial agent according to claim 2 in preparing a straw degradation preparation.

8. Use of the bacterium according to claim 1 or the bacterial agent according to claim 2 in the preparation of a phosphate-dissolving preparation.

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