Pseudomonas fluorescens BR12-18 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to pseudomonas fluorescens BR12-18 and application thereof.The pseudomonas fluorescens BR12-18 is separated and screened from root endophytes of 12-year-old catalpa bungei and stored in the China General Microbiological Culture Collection Center (CGMCC), the pseudomonas fluorescens is classified and named as pseudomonas fluorescens, and the preservation number is CGMCC No.28475. The pseudomonas fluorescens BR12-18 has the advantages that the pseudomonas fluorescens BR12-18 can be applied to the field of biological engineering, and the application of the pseudomonas fluorescens BR12-18 to the field of biological engineering; the pseudomonas fluorescens BR12-18 and catalpa bungei form a mutualism relationship, and have positive positive effects on stress resistance, growth promotion and the like of catalpa bungei seedlings; when the pseudomonas fluorescens BR12-18 is applied to catalpa bungei seedlings, waterlogging stress of the catalpa bungei seedlings can be effectively relieved, and growth and development of the catalpa bungei seedlings are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Pseudomonas fluorescens BR12-18 and its application. Background Art

[0002] Waterlogging is one of the natural disasters that frequently occur in China, causing serious economic losses to the industries of agriculture, forestry, animal husbandry and fishery. There is a mutual restriction relationship between the water content and oxygen content in the soil, and waterlogging will lead to excessive water and lack of oxygen in the soil, thereby resulting in a decrease in the oxygen concentration in the rhizosphere of plants, forming hypoxic or anoxic stress, affecting the normal physiological metabolism and growth and development of plants, and even causing plant death.

[0003] At present, the prevention and control of waterlogging mainly solves the problem by timely drainage, building water conservancy projects, etc., so as to improve the performance of waterlogged land and wetland. However, the problem of waterlogging caused by waterlogging or other accidental natural conditions and resulting in plant hypoxia has not been fundamentally solved. Catalpa bungei is a deciduous tree belonging to the genus Catalpa of the family Bignoniaceae, and is an excellent wood material, garden ornamental and windbreak and sand fixation tree species, and its cultivation and planting have great economic, social and ecological values. Research shows that Catalpa bungei is relatively sensitive to excessive soil moisture, and the growth of Catalpa bungei in a waterlogged environment is weak, the plants are short, and even die in patches.

[0004] The Chinese invention patent with the publication number of CN118440854B provides a Pseudomonas fluorescens LSOJM27 and its application. The strain provided by it has strong phosphorus-solubilizing ability, can significantly increase the available phosphorus content in cultivated land soil, reduce the application of phosphate fertilizer, and is suitable for the activation of phosphorus in cultivated land soil, but it cannot be used for waterlogging prevention and control. The Chinese invention patent with the publication number of CN118146983B provides an application of a Rhizobium GZHC2-2 strain in alleviating waterlogging stress of sensitive soybeans, but does not mention its application in Catalpa bungei seedlings. The Chinese invention patent application with the publication number of CN116790444A provides a strain of corn rhizobium NF11, which is isolated from the endophytic bacteria of Catalpa bungei tissue culture seedlings and has a positive growth-promoting effect on Catalpa bungei tissue culture seedlings and Catalpa bungei potted seedlings. The corn rhizobium NF11 has nitrogen-fixing ability, phosphorus-solubilizing efficiency and a certain ability to inhibit plant pathogens, but it is still unknown whether the corn rhizobium can enable Catalpa bungei seedlings to grow and develop in a waterlogged environment. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects and deficiencies in the prior art, and provide a Pseudomonas fluorescens BR12-18 and its application.

[0006] The technical solution of the present invention is as follows: A Pseudomonas fluorescens BR12-18, classified and named as Pseudomonas fluorescens, is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is Beijing, China, and the preservation number is CGMCC No. 28475. The preservation date is September 19, 2023. This Pseudomonas fluorescens BR12-18 was isolated and screened from the endophytes in the roots of Catalpa bungei trees with a tree age of 12 years.

[0007] The present invention also provides the application of Pseudomonas fluorescens BR12-18 in alleviating waterlogging stress of Catalpa bungei seedlings, that is, Pseudomonas fluorescens BR12-18 is used for waterlogging resistance and growth promotion of Catalpa bungei seedlings to alleviate waterlogging stress of Catalpa bungei seedlings.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Pseudomonas fluorescens BR12-18 of the present invention is isolated from the endophytes inside the roots of Catalpa bungei trees. The long-term adaptation to the habitat conditions enables it to form a mutually beneficial symbiotic relationship with Catalpa bungei trees, having a positive effect on stress resistance and growth promotion of Catalpa bungei seedlings.

[0009] 2. The Pseudomonas fluorescens BR12-18 of the present invention can effectively alleviate waterlogging stress of Catalpa bungei seedlings and has a broad application prospect.

[0010] 3. Under waterlogging stress conditions, the growth status of Catalpa bungei seedlings in the treatment group applied with Pseudomonas fluorescens BR12-18 is significantly better than that of the blank control group. The plant height, ground diameter, main root, lateral root and fresh weight of Catalpa bungei seedlings applied with Pseudomonas fluorescens BR12-18 increased by 18.79%, 8.65%, 28.37%, 23.77% and 96.93% respectively. At the same time, this Pseudomonas fluorescens BR12-18 reduces the waterlogging stress response of Catalpa bungei seedlings and promotes the growth and development of Catalpa bungei seedlings. Description of the Drawings

[0011] Figure 1 It is the colony morphology diagram of Pseudomonas fluorescens BR12-18 of the present invention; Figure 2 It is the microscopic morphology diagram of Pseudomonas fluorescens BR12-18 of the present invention; Figure 3 It is the metabolic pathway enrichment diagram of Pseudomonas fluorescens BR12-18 of the present invention significantly affecting Catalpa bungei seedlings; Figure 4 It is the metabolic pathway change diagram of Pseudomonas fluorescens BR12-18 of the present invention significantly affecting Catalpa bungei seedlings; Figure 5 It is the potting growth promotion effect diagram of Pseudomonas fluorescens BR12-18 of the present invention alleviating waterlogging stress of Catalpa bungei seedlings (where the left figure is the blank control group and the right figure is the treatment group); Figure 6 This is the root growth promotion effect diagram of Pseudomonas fluorescens BR12-18 of the present invention in alleviating waterlogging stress of Catalpa bungei seedlings (where the left figure is the blank control group and the right figure is the treatment group). Specific implementation manners

[0012] Next, the technical solutions of the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0013] Example 1: Isolation and screening of Catalpa bungei root endophytic bacteria BR12-18 (1) Weigh 2 g of the lateral root tissue of Catalpa bungei with a tree age of 12 years, wash it 3 times with 1×PBS solution, soak it in 75% ethanol for 40 s, and then rinse it 3 times with sterile water.

[0014] (2) Then soak it in 4% (m / v) sodium hypochlorite solution for 10 min, and then rinse it 3 times with sterile water.

[0015] (3) Cut open the epidermis of the Catalpa bungei root tissue, cut the endophytic tissue into pieces and grind it. Take 1 g of the crushed tissue and add it to 100 mL of sterile water. Under the condition of 28 °C, oscillate it at 180 r / min for 30 min, serially dilute it and spread it on the LB solid medium.

[0016] (4) Culture it at 30 °C for 3 days, pick out single colonies and obtain 20 pure strains of Catalpa bungei root endophytic bacteria through multiple streak separations. Among them, the stress resistance and growth promotion effect of strain BR12-18 are relatively prominent.

[0017] The components of the LB solid medium include peptone, yeast extract, sodium chloride and agar. The mass concentrations are as follows: peptone is 10 g / L, yeast extract is 5 g / L, sodium chloride is 10 g / L, and agar is 20 g / L. The pH value of this LB solid medium is 7.0.

[0018] Example 2: Identification of the genus of Catalpa bungei root endophytic bacteria BR12-18 As Figure 1 shown, the growth colonies of Catalpa bungei root endophytic bacteria BR12-18 on the LB solid medium are round, milky white, and the surface is smooth and convex. As Figure 2 shown, the individual morphology of Catalpa bungei root endophytic bacteria BR12-18 under the optical microscope is coccobacillus-shaped, and the Gram stain is red, belonging to Gram-negative bacilli.

[0019] The genomic DNA of the screened strain was extracted using a bacterial genomic DNA extraction kit, and the 16S rRNA universal primers were used for PCR amplification. The amplification procedure was as follows: Pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, for 30 cycles; then extension at 72°C for 10 min. The nucleotide sequence length of the PCR product after sequencing and splicing was 1426 bp. Through Blast sequence alignment analysis, the 16S rRNA sequence similarity between the endophytic bacterium BR12-18 in the root of Catalpa bungei and Pseudomonas fluorescens XT604 (PQ782783.1) was the highest, reaching 99.93%. Therefore, based on the morphological characteristics, the endophytic bacterium BR12-18 in the root of Catalpa bungei was determined to be Pseudomonas fluorescens.

[0020] The obtained Pseudomonas fluorescens BR12-18 was deposited on September 19, 2023, at the China General Microbiological Culture Collection Center, with the deposit address being Beijing, China, and the deposit number being CGMCC No. 28475, and the taxonomic name being Pseudomonas fluorescens.

[0021] The 16S rRNA sequence of the endophytic bacterium BR12-18 in the root of Catalpa bungei measured was:

[0022] Example 3: Stress resistance and growth promotion characteristics of Pseudomonas fluorescens BR12-18 (1) Inoculate Pseudomonas fluorescens BR12-18 into LB liquid medium and culture it at 30 °C at 180 r / min for 24 h. Take 2 mL of the fermentation broth and place it in a centrifuge tube, centrifuge at 10000 rpm for 10 min, collect the bacterial cells, then add 1.5 mL of sterile water to make a bacterial suspension. Pipette 1 mL of this bacterial suspension and place it in a phosphorus-solubilizing liquid medium, and culture it on a shaker at 180 r / min at 28 °C for 7 d, and then measure the available phosphorus content in the culture broth. At the same time, use the addition of an equal volume of sterile water as a control.

[0023] Using the molybdenum antimony anti-colorimetric method, the available phosphorus content in the culture broth of Pseudomonas fluorescens BR12-18 was measured to be 715.89 mg / L, indicating that Pseudomonas fluorescens BR12-18 has strong phosphorus-solubilizing ability.

[0024] (2) Inoculate Pseudomonas fluorescens BR12-18 into LB liquid medium supplemented with L-tryptophan at an inoculation amount of 2% (v / v), and the final concentration of this L-tryptophan is 200 mg / L. Culture it at 28 °C and 180 r / min for 24 h, and then centrifuge at 10000 rpm for 5 min; take the supernatant and mix it with Salkowski colorimetric reagent at a ratio of 1:2, and then dark-treat it at 25 °C for 30 min. Determine the absorbance value at 530 nm by spectrophotometry, and determine that the concentration of biotin indole acetic acid (IAA) in the supernatant is 2.76 mg / L, indicating that Pseudomonas fluorescens BR12-18 has the effects of regulating plant stem growth and promoting root growth, etc.

[0025] (3) Inoculate Pseudomonas fluorescens BR12-18 into TSB liquid medium at an inoculation amount of 2% (v / v), and culture it at 28 °C and 180 r / min for 12 h, and then centrifuge at 10000 rpm for 5 min, collect the bacterial cells and resuspend them in 7.5 mL of DF liquid medium, and continue to culture for 24 h; among them, 45 μL of 1-aminocyclopropane-1-carboxylic acid (ACC) is added to this 7.5 mL of DF liquid medium.

[0026] After the culture, the fermentation broth is centrifuged and resuspended and washed 3 times, then the bacterial cell precipitate is resuspended in 600 μL of Tris-HCl buffer (pH 8.5), add 30 μL of toluene, vortex and break the bacterial cells to obtain a cell lysate. Measure the content of α-ketobutyric acid and bacterial protein in the cell lysate, and determine the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity of Pseudomonas fluorescens BR12-18 to be 8.76 U / mg by referring to the standard curve.

[0027] The method for determining the content of α-ketobutyric acid in the above cell lysate is as follows: Take 200 μL of the cell lysate and 20 μL of 0.5 mol / L ACC solution and mix them, and incubate in a water bath at 30 °C for 15 min. Then add 1 mL of 0.56 mol / L hydrochloric acid solution, mix well and centrifuge. Take 500 μL of the supernatant, 400 μL of 0.56 mol / L hydrochloric acid solution and 150 μL of 2 g / L 2,4-dinitrophenylhydrazine solution and mix well, and incubate in a water bath at 30 °C for 30 min. Then add 2 mL of sodium hydroxide solution for color development, and measure the absorbance at 540 nm to determine the content of α-ketobutyric acid in the cell lysate.

[0028] Stress conditions such as waterlogging can induce the production of excessive ethylene in plants, which can hinder the growth and development of plants and even cause death. The above ACC deaminase can convert the ethylene precursor ACC into α-ketobutyric acid and ammonia, thereby improving the adaptability of plants under stress. Pseudomonas fluorescens BR12-18 has ACC deaminase activity and has a positive effect on alleviating waterlogging stress.

[0029] (4) Inoculate Pseudomonas fluorescens BR12-18 into LB liquid medium, culture it at 30 °C and 180 r / min for 24 h, aspirate the bacterial suspension and apply it to the tissue culture seedlings of Catalpa bungei growing in nutrient agar, 100 μL per plant. At the same time, use sterile water instead of the bacterial suspension to set up a control group.

[0030] Under the conditions of 25 °C light for 15 d, non-targeted metabolomics was used to analyze the metabolic response of Catalpa bungei tissue culture seedlings to Pseudomonas fluorescens BR12-18. Figure 3 It can be seen that compared with the control group, Pseudomonas fluorescens BR12-18 can significantly up-regulate the metabolic pathways such as phenylpropanoid biosynthesis, flavonoid and flavonol biosynthesis, and tyrosine metabolism in the tissue culture seedlings of Catalpa bungei. At the same time, Figure 4 It can be seen that Pseudomonas fluorescens BR12-18 can significantly down-regulate the metabolic pathways such as alanine, aspartate and glutamate metabolism, arginine biosynthesis, aminoacyl biosynthesis, D-amino acid metabolism, protein digestion and absorption, and β-alanine metabolism, indicating that Pseudomonas fluorescens BR12-18 can significantly enhance the antioxidant and stress resistance of Catalpa bungei tissue culture seedlings, can also store amino groups and participate in metabolism, etc., and promote the growth and development of Catalpa bungei tissue culture seedlings.

[0031] The components of the LB liquid medium used in the present invention include peptone, yeast powder and sodium chloride, and the pH value is 7.0; among them, the mass concentrations of each component are as follows: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L.

[0032] The components of the phosphorus-solubilizing culture medium used in the present invention include glucose, tricalcium phosphate, magnesium chloride hexahydrate, magnesium sulfate heptahydrate, potassium chloride, and ammonium sulfate, and the pH value is 7.5. Among them, the mass concentrations of each component are as follows: glucose 10 g / L, tricalcium phosphate 5 g / L, magnesium chloride hexahydrate 5 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium chloride 0.2 g / L, ammonium sulfate 0.1 g / L.

[0033] The components of the TSB liquid culture medium used in the present invention include soy peptone, tryptone, sodium chloride, glucose, and dipotassium hydrogen phosphate, and the pH value is 7.1 - 7.5; among them, the mass concentrations of each component are as follows: soy peptone 3 g / L, tryptone 17 g / L, sodium chloride 5 g / L, glucose 2.5 g / L, dipotassium hydrogen phosphate 2.5 g / L.

[0034] The components of the DF liquid culture medium used in the present invention include disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, gluconic acid, citric acid, ammonium sulfate, magnesium sulfate heptahydrate, and ferrous sulfate heptahydrate, and the pH value is 7.2; among them, the mass concentrations of each component are as follows: disodium hydrogen phosphate 4 g / L, glucose 2 g / L, gluconic acid 2 g / L, citric acid 2 g / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.01 g / L.

[0035] Example 4: Anti-waterlogging and growth-promoting effects of Pseudomonas fluorescens BR12-18 on Catalpa bungei seedlings Select potted Catalpa bungei seedlings with an average plant height of 5.5 cm, and water them twice a day to keep the potted soil in a thoroughly watered and moist and clumped state. Pick the Pseudomonas fluorescens BR12-18 strain preserved on the test tube slant and transfer it to the LB liquid culture medium, culture it at 30 °C and 180 r / min for 24 h, centrifuge at 10000 rpm for 5 min and separate and take out the supernatant, wash the bacterial cell precipitate with sterile water 3 times, and adjust the OD 600 value of the bacterial cell concentration to 0.8 with sterile water. Take 10 mL of the bacterial liquid with an OD 600 value of 0.8 of the bacterial cell concentration, and inoculate it around the rhizosphere of the potted Catalpa bungei seedlings as the treatment group; at the same time, set a blank control group. Apply 10 mL of sterile water to the potted Catalpa bungei seedlings in the blank control group. Apply the bacterial liquid once every 5 days, set 6 replicates for each group, place them in a 25 °C culture greenhouse and continuously treat for 30 days. As Figure 5 and Figure 6 shown, measure the apparent indexes such as the plant height, ground diameter, main root, lateral root length, and fresh weight of the Catalpa bungei seedlings to determine the anti-waterlogging and growth-promoting effects of Pseudomonas fluorescens BR12-18 on the potted Catalpa bungei seedlings, and the measurement results are shown in Table 1.

[0036] As can be seen from Table 1, the plant height, ground diameter, main root, lateral root and fresh weight of the Catalpa bungei seedlings in the treatment group with Pseudomonas fluorescens BR12-18 applied were significantly higher than those in the blank control group, increasing by 18.79%, 8.65%, 28.37%, 23.77% and 96.93% respectively. Pseudomonas fluorescens BR12-18 has a significant effect on waterlogging resistance and growth promotion of Catalpa bungei seedlings.

[0037] Table 1 Determination results of apparent indexes

[0038] Example 5: Waterlogging resistance and growth promotion response of Pseudomonas fluorescens BR12-18 to Catalpa bungei seedlings Collect the leaves of Catalpa bungei seedlings in the blank control group and the treatment group in Example 4 and measure the physiological indexes. Specifically, the spectrophotometer method was used to measure the soluble sugar content, soluble protein content and proline content, as well as the enzyme activities of peroxidase and superoxide dismutase respectively. The measurement results are shown in Table 2.

[0039] As can be seen from Table 2, the soluble sugar and soluble protein contents in the leaves of Catalpa bungei seedlings in the treatment group with Pseudomonas fluorescens BR12-18 applied were significantly higher than those in the blank control group, increasing by 74.66% and 34.74% respectively, indicating that the applied Pseudomonas fluorescens BR12-18 can improve the accumulation of soluble sugar and soluble protein in the leaves of Catalpa bungei seedlings and promote the growth of Catalpa bungei seedlings.

[0040] In addition, the proline content, peroxidase activity and superoxide dismutase activity in the leaves of Catalpa bungei seedlings in the treatment group with Pseudomonas fluorescens BR12-18 applied were significantly lower than those in the blank control group, decreasing by 83.11%, 92.22% and 27.31% respectively, indicating that the applied Pseudomonas fluorescens BR12-18 can reduce the waterlogging stress response of Catalpa bungei seedlings and effectively relieve the waterlogging stress pressure, thus promoting the growth of Catalpa bungei seedlings.

[0041] Table 2 Determination results of physiological indexes

[0042] The method for measuring the soluble sugar content in the present invention is as follows: Take 0.2 g of Catalpa bungei leaves, cut and mix them evenly, add 5 mL of distilled water and grind them in an ice bath, boil them in a water bath for 20 min, take them out and cool them naturally, then make up the volume to 40 mL in a colorimetric tube. Mix the supernatant with anthrone reagent at a ratio of 1:5, boil it in a water bath for 10 min and cool it, measure the absorbance value at 620 nm, and obtain the soluble sugar content by referring to the standard curve.

[0043] The method for measuring the soluble protein content in the present invention is as follows: Take 0.5 g of Catalpa bungei leaves, cut and mix them evenly, add 25 mL of PBS phosphate buffer solution, grind in an ice bath, then centrifuge and take the supernatant. Mix the supernatant with Coomassie Brilliant Blue dye solution at a ratio of 1:5, react for 2 - 5 min, measure the absorbance at 595 nm, and obtain the soluble protein content by referring to the standard curve.

[0044] The method for measuring the proline content in the present invention is as follows: Take 0.2 g of Catalpa bungei leaves, cut and mix them evenly, add 5 mL of 3% sulfosalicylic acid solution, boil in a water bath for 30 min and then cool. Take 2 mL of the supernatant, 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin solution and mix them. After boiling in a water bath for 30 min, add 4 mL of toluene, vortex for 1 min, let it stand and take the upper layer solution, measure the absorbance at 520 nm, and obtain the proline content by referring to the standard curve.

[0045] The method for measuring the peroxidase activity in the present invention is as follows: Take 0.5 g of Catalpa bungei leaves, cut and mix them evenly, add 25 mL of PBS phosphate buffer solution, grind in an ice bath, centrifuge and take the supernatant as the crude enzyme solution. Add 1.5 mL of peroxidase premix (guaiacol and hydrogen peroxide) and 0.5 mL of the crude enzyme solution into the test tube in sequence. After mixing, immediately pour it into the cuvette, and measure the absorbance values A at 1 min and 2 min of the reaction respectively. 470 The enzyme amount that reduces the absorbance by 0.01 within 1 min is defined as 1 enzyme activity unit.

[0046] The method for measuring the superoxide dismutase activity in the present invention is as follows: Take 0.5 g of Catalpa bungei leaves, cut and mix them evenly, add 25 mL of PBS phosphate buffer solution, grind in an ice bath, centrifuge and take the supernatant as the crude enzyme solution. Take 0.05 mL of the crude enzyme solution, 0.25 mL of distilled water, 1.5 mL of PBS buffer solution, 0.3 mL of 130 mmol / L methionine solution, 0.3 mL of 750 μmol / L nitroblue tetrazolium solution, 0.3 mL of 100 μmol / L EDTA - Na 2 solution 0.3 mL and 0.3 mL of 20 μmol / L riboflavin solution and mix them (in the blank control group, 0.05 mL of buffer solution is used instead of 0.05 mL of the crude enzyme solution). After irradiating and reacting for 20 min, measure the absorbance at 560 nm. Taking the inhibition of 50% of the photoreduction of nitroblue tetrazolium as 1 enzyme activity unit.

Claims

1. A Pseudomonas fluorescens BR12-18, characterized in that The Pseudomonas fluorescens BR12-18 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address in Beijing, China. It is classified and named as Pseudomonas fluorescens, and the deposit number is CGMCC No.28475.

2. Use of the fluorescent Pseudomonas BR12-18 described in claim 1 in alleviating waterlogging stress on seedlings of Catalpa ovata.

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