Pseudomonas fluorescens BR12-18 and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a fluorescent Pseudomonas BR12-18 and its application. The fluorescent Pseudomonas BR12-18 was isolated and screened from the root endophytes of 12-year-old catalpa trees and preserved in the General Microbiology Center of the China Microorganism Culture Collection Administration. It is classified and named Pseudomonas fluorescens and has a preservation number of CGMCC No. 28475. The fluorescent Pseudomonas BR12-18 forms a mutually beneficial symbiotic relationship with the catalpa trees, and has positive effects on the stress resistance and growth promotion of catalpa seedlings. The application of the fluorescent Pseudomonas BR12-18 in catalpa seedlings can effectively alleviate the waterlogging stress of the catalpa seedlings and promote their growth and development.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Pseudomonas fluorescens BR12-18 and applications thereof. Background Art

[0002] Waterlogging is a frequent natural disaster in my country, causing severe economic losses to agriculture, forestry, animal husbandry, and fishery. Water and oxygen levels in the soil are mutually restrictive. Waterlogging can lead to excess water and a lack of oxygen in the soil, which in turn reduces oxygen concentration in the rhizosphere of plants, causing hypoxic or anaerobic stress, impacting plant metabolism and growth, and even causing plant death.

[0003] Currently, waterlogging is primarily prevented and controlled through timely drainage and water conservancy projects, thereby improving the performance of waterlogged areas and wetlands. However, the problem of waterlogging and plant hypoxia caused by waterlogging or other unexpected natural conditions has not been fundamentally resolved. Catalpa chinensis, a deciduous tree of the Bignoniaceae family, is an excellent timber, ornamental, and windbreak and sand fixation species. Its cultivation has significant economic, social, and ecological value. Studies have shown that catalpa chinensis is sensitive to excessive soil moisture. Trees grown in waterlogged environments experience weak growth, dwarfed plants, and even die en masse.

[0004] The Chinese invention patent with publication number CN118440854B provides a fluorescent Pseudomonas LSOJM27 and its application. The strain provided has a strong ability to solubilize phosphorus, can significantly increase the content of available phosphorus in cultivated soil, reduce the application of phosphorus fertilizer, and is suitable for activating phosphorus in cultivated soil, but cannot be used for waterlogging prevention and control. The Chinese invention patent with publication number CN118146983B provides an application of a rhizobium GZHC2-2 strain in alleviating sensitive soybean waterlogging stress, but does not mention its application in catalpa seedlings. The Chinese invention patent application with publication number CN116790444A provides a strain of corn rhizobium NF11, which is isolated from endophytic bacteria of catalpa tissue culture seedlings and has a positive effect on promoting the growth of catalpa tissue culture seedlings and potted catalpa seedlings. The corn rhizobium NF11 has nitrogen fixation ability, phosphate solubilization efficiency and a certain ability to inhibit plant pathogens, but it is still unknown whether the corn rhizobium can enable catalpa 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 shortcomings in the prior art and provide a Pseudomonas fluorescens BR12-18 and its application.

[0006] The technical solutions of the present invention are as follows:

[0007] A strain of Pseudomonas fluorescens BR12-18, classified as Pseudomonas fluorescens, was deposited at the General Microbiology Center of the China Culture Collection Administration in Beijing, China, with the deposit number CGMCC No. 28475 and the deposit date of September 19, 2023. Pseudomonas fluorescens BR12-18 was isolated and screened from the root endophytes of a 12-year-old catalpa tree.

[0008] The present invention also provides an application of Pseudomonas fluorescens BR12-18 in alleviating waterlogging stress on catalpa chinensis seedlings, that is, Pseudomonas fluorescens BR12-18 is used for waterlogging resistance and growth promotion of catalpa chinensis seedlings, thereby alleviating waterlogging stress on catalpa chinensis seedlings.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] 1. The fluorescing Pseudomonas BR12-18 of the present invention is isolated from an endophyte inside the root of the catalpa tree. Long-term adaptation to the habitat conditions enables it to form a mutually beneficial symbiotic relationship with the catalpa tree, and has a positive effect on the stress resistance and growth promotion of the catalpa tree seedlings.

[0011] 2. The fluorescent Pseudomonas BR12-18 of the present invention can effectively alleviate the waterlogging stress of catalpa ovata seedlings and has broad application prospects.

[0012] 3. Under waterlogging stress, the growth of catalpa seedlings in the group treated with Pseudomonas fluorescens BR12-18 was significantly better than that in the blank control group. The plant height, ground diameter, taproot, lateral root, and fresh weight of catalpa seedlings treated with Pseudomonas fluorescens BR12-18 increased by 18.79%, 8.65%, 28.37%, 23.77%, and 96.93%, respectively. Furthermore, Pseudomonas fluorescens BR12-18 reduced waterlogging stress in catalpa seedlings and promoted their growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a colony morphology diagram of Pseudomonas fluorescens BR12-18 of the present invention;

[0014] Figure 2 This is a microscopic morphology of Pseudomonas fluorescens BR12-18 of the present invention;

[0015] Figure 3 This is an enrichment diagram of the metabolic pathways that the fluorescent Pseudomonas BR12-18 of the present invention significantly affects catalpa ovata seedlings;

[0016] Figure 4 This is a diagram showing changes in metabolic pathways significantly affected by the fluorescent Pseudomonas BR12-18 of the present invention in catalpa ovata seedlings;

[0017] Figure 5This is a diagram showing the potted growth-promoting effect of the present invention's Pseudomonas fluorescens BR12-18 on alleviating waterlogging stress in catalpa oleifera seedlings (wherein the left figure is a blank control group and the right figure is a treatment group);

[0018] Figure 6 This is a diagram showing the root growth-promoting effect of the fluorescent Pseudomonas BR12-18 of the present invention on alleviating waterlogging stress of catalpa oleifera seedlings (wherein the left figure is the blank control group and the right figure is the treatment group). DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] Example 1: Isolation and screening of endophytic bacteria BR12-18 from catalpa roots

[0021] (1) Weigh 2 g of lateral root tissue from a 12-year-old Catalpa elata tree, wash it three times with 1× PBS solution, soak it in 75% ethanol for 40 seconds, and then rinse it three times with sterile water.

[0022] (2) Soak in 4% (m / v) sodium hypochlorite solution for 10 minutes, and then rinse with sterile water three times.

[0023] (3) Cut open the epidermis of the root tissue of the Chinese catalpa tree and cut and grind the endophytic tissue. Take 1 g of the broken tissue and add it to 100 mL of sterile water. Oscillate at 180 r / min for 30 min at 28°C, dilute it step by step, and spread it on LB solid culture medium.

[0024] (4) After culturing at 30°C for 3 days, single colonies were picked and separated by multiple streaking to obtain 20 pure strains of endophytic bacteria from the roots of catalpa trees. Among them, the strain BR12-18 had a more prominent stress-resistant and growth-promoting effect.

[0025] LB solid medium consists of peptone, yeast extract, sodium chloride, and agar. The mass concentrations are as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and agar 20 g / L. The pH of the LB solid medium is 7.0.

[0026] Example 2: Identification of the genus of endophytic bacteria BR12-18 from catalpa roots

[0027] like Figure 1 As shown in Figure 1, the colonies of endophytic bacteria BR12-18 from catalpa trees grown on LB solid medium are round, milky white, and have smooth and raised surfaces. Figure 2As shown in the figure, the individual morphology of the endophytic bacteria BR12-18 in the roots of catalpa trees under an optical microscope is coccus-shaped, Gram-stained red, and belongs to Gram-negative rods.

[0028] The genome of the screening strain was extracted using a bacterial genome extraction kit, and PCR amplification was performed using universal primers for 16S rRNA. The amplification procedure was as follows:

[0029] The PCR product was pre-denatured at 94°C for 4 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes, followed by extension at 72°C for 10 minutes. The PCR product was sequenced and assembled to yield a 1426-bp nucleotide sequence. Blast sequence alignment analysis revealed that the 16S rRNA sequence of BR12-18, an endophytic bacterium of catalpa tree roots, shared the highest similarity with that of Pseudomonas fluorescens XT604 (PQ782783.1), reaching 99.93%. Therefore, based on morphological characteristics, BR12-18 was identified as Pseudomonas fluorescens.

[0030] The obtained Pseudomonas fluorescens BR12-18 was deposited in the General Microbiology Center of China Culture Collection Administration on September 19, 2023, with the deposit address in Beijing, China, the deposit number is CGMCC No.28475, and the classification name is Pseudomonas fluorescens.

[0031] The 16S rRNA sequence of the endophytic bacteria BR12-18 from the roots of catalpa ovata was determined to be:

[0032]

[0033] Example 3: Stress-resistant and growth-promoting properties of Pseudomonas fluorescens BR12-18

[0034] (1) Pseudomonas fluorescens BR12-18 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 24 h. 2 mL of the fermentation broth was collected in a centrifuge tube and centrifuged at 10,000 rpm for 10 min to collect the cells. 1.5 mL of sterile water was then added to prepare a bacterial suspension. 1 mL of this suspension was then placed into phosphate-solubilizing liquid medium and cultured in a shaker at 28°C and 180 rpm for 7 days. The available phosphorus content in the culture medium was determined. An equal volume of sterile water was added as a control.

[0035] The available phosphorus content in the culture medium of Pseudomonas fluorescens BR12-18 was determined by molybdenum antimony colorimetry and was 715.89 mg / L, indicating that Pseudomonas fluorescens BR12-18 has a strong ability to solubilize phosphorus.

[0036] (2) Fluorescent Pseudomonas BR12-18 was inoculated at a 2% (v / v) inoculum into LB liquid medium supplemented with L-tryptophan to a final concentration of 200 mg / L. The culture was incubated at 28°C and 180 rpm for 24 h, followed by centrifugation at 10,000 rpm for 5 min. The supernatant was mixed with a Salkowski colorimetric reagent in a 1:2 ratio and then treated in the dark at 25°C for 30 min. The absorbance at 530 nm was measured by spectrophotometry, and the concentration of biotin indoleacetic acid (IAA) in the supernatant was determined to be 2.76 mg / L, indicating that fluorescent Pseudomonas BR12-18 has the ability to regulate plant stem growth and promote rooting.

[0037] (3) Pseudomonas fluorescens BR12-18 was inoculated into TSB liquid medium at a 2% (v / v) inoculation rate, cultured at 28°C and 180 rpm for 12 h, and then centrifuged at 10,000 rpm for 5 min. The cells were collected and resuspended in 7.5 mL of DF liquid medium and cultured for another 24 h; wherein, 45 μL of 1-aminocyclopropane-1-carboxylic acid (ACC) was added to the 7.5 mL of DF liquid medium.

[0038] After three washes of centrifugation and resuspension, the fermentation broth was pelleted and resuspended in 600 μL of Tris-HCl buffer (pH 8.5). 30 μL of toluene was added, and the cells were vortexed to disrupt the cells to obtain a cell lysate. The α-ketobutyric acid and bacterial protein contents in the cell lysate were determined. The 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity of Pseudomonas fluorescens BR12-18 was determined to be 8.76 U / mg by comparison with a standard curve.

[0039] The method for determining the α-ketobutyric acid content in the cell lysate is as follows: 200 μL of the cell lysate and 20 μL of 0.5 mol / L ACC solution are mixed and incubated at 30°C in a water bath for 15 minutes. Then, 1 mL of 0.56 mol / L hydrogen chloride solution is added, mixed thoroughly, and centrifuged. 500 μL of the supernatant, 400 μL of 0.56 mol / L hydrogen chloride solution, and 150 μL of 2 g / L 2,4-dinitrophenylhydrazine solution are mixed and incubated at 30°C in a water bath for 30 minutes. Then, 2 mL of sodium hydroxide solution is added and color developed. The absorbance at 540 nm is measured to determine the α-ketobutyric acid content in the cell lysate.

[0040] Adverse conditions such as waterlogging can induce excessive ethylene production in plants, hindering their growth and development, or even leading to death. ACC deaminase, as mentioned above, converts ACC, the precursor for ethylene synthesis, into α-ketobutyric acid and ammonia, thereby improving plants' adaptability to adverse stress. Pseudomonas fluorescens BR12-18 exhibits ACC deaminase activity and has a positive effect in alleviating waterlogging stress.

[0041] (4) Pseudomonas fluorescens BR12-18 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 24 h. The bacterial suspension was aspirated and applied to Catalpa sylvatica tissue culture seedlings grown on nutrient agar, with 100 μL per plant. At the same time, sterile water was used instead of the bacterial suspension to establish a control group.

[0042] The metabolic response of Catalpa chinensis seedlings to Pseudomonas fluorescens BR12-18 was analyzed by non-targeted metabolomics under 25℃ light conditions for 15 days. Figure 3 It can be seen that compared with the control group, Pseudomonas fluorescens BR12-18 can significantly upregulate metabolic pathways such as phenylpropanoid biosynthesis, flavonoid and flavonol biosynthesis, and tyrosine metabolism in tissue culture seedlings of Catalpa ovata. Figure 4 It can be seen that Pseudomonas fluorescens BR12-18 can significantly downregulate metabolic pathways such as alanine, aspartic acid 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 tissue culture seedlings, and can also store amino acids and participate in metabolism, and promote the growth and development of catalpa tissue culture seedlings.

[0043] The LB liquid culture medium used in the present invention comprises peptone, yeast powder and sodium chloride, with a pH value of 7.0; wherein the mass concentration of each component is as follows: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L.

[0044] The phosphate-solubilizing medium used in the present invention comprises glucose, tricalcium phosphate, magnesium chloride hexahydrate, magnesium sulfate heptahydrate, potassium chloride, and ammonium sulfate, with a pH value of 7.5. The mass concentrations of the components 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, and ammonium sulfate 0.1 g / L.

[0045] The TSB liquid culture medium used in the present invention comprises soy peptone, tryptone, sodium chloride, glucose and dipotassium hydrogen phosphate, with a pH value of 7.1-7.5; wherein the mass concentrations of the components are as follows: soy peptone 3 g / L, tryptone 17 g / L, sodium chloride 5 g / L, glucose 2.5 g / L, and dipotassium hydrogen phosphate 2.5 g / L.

[0046] The DF liquid culture medium used in the present invention includes disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, gluconic acid, citric acid, ammonium sulfate, magnesium sulfate heptahydrate and ferrous sulfate heptahydrate, with a pH value of 7.2; wherein the mass concentrations of the components 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, and ferrous sulfate heptahydrate 0.01 g / L.

[0047] Example 4: Effect of Pseudomonas fluorescens BR12-18 on waterlogging resistance and growth promotion of Catalpa chinensis seedlings

[0048] Select potted catalpa seedlings with an average plant height of 5.5 cm and water them twice a day to ensure that the pot soil is always thoroughly watered and moist. Pick the fluorescent Pseudomonas BR12-18 strain preserved on the test tube slant and transfer it to LB liquid medium. Incubate at 30°C and 180 rpm for 24 hours, centrifuge at 10,000 rpm for 5 minutes, separate and remove the supernatant, wash the bacterial precipitate three times with sterile water, and adjust the bacterial concentration to OD 600 The value was adjusted to 0.8. Take the bacterial concentration OD 600 10 mL of bacterial solution with a value of 0.8 was inoculated around the rhizosphere of potted catalpa seedlings and used as the treatment group; at the same time, a blank control group was set up. 10 mL of sterile water was applied to the potted catalpa seedlings in the blank control group. The bacterial solution was applied once every 5 days, with 6 replicates set up for each group, placed in a 25°C culture greenhouse and treated continuously for 30 days. Figure 5 and Figure 6 As shown in the figure, the plant height, ground diameter, main root, lateral root length and fresh weight of the seedlings of Catalpa elata were measured to determine the waterlogging resistance and growth-promoting effect of Pseudomonas fluorescens BR12-18 on the potted seedlings of Catalpa elata. The test results are shown in Table 1.

[0049] As shown in Table 1, the plant height, ground diameter, taproot, lateral root, and fresh weight of catalpa chinensis seedlings in the treatment group with Pseudomonas fluorescens BR12-18 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 significantly protected catalpa chinensis seedlings from waterlogging and promoted their growth.

[0050] Table 1 Measurement results of apparent indicators

[0051]

[0052] Example 5: Fluorescent Pseudomonas BR12-18 on waterlogging-resistant and growth-promoting responses of catalpa chinensis seedlings

[0053] Leaves of catalpa ovata seedlings from the blank control group and the treatment group in Example 4 were collected and subjected to physiological index measurements. Specifically, the soluble sugar content, soluble protein content, and proline content, as well as the enzyme activities of peroxidase and superoxide dismutase, were measured using a spectrophotometer. The results are shown in Table 2.

[0054] As can be seen from Table 2, the soluble sugar and soluble protein contents in the leaves of Catalpa elata seedlings in the treatment group with Pseudomonas fluorescens BR12-18 were significantly higher than those in the blank control group, increasing by 74.66% and 34.74%, respectively. This indicates that the application of Pseudomonas fluorescens BR12-18 can increase the accumulation of soluble sugar and soluble protein in the leaves of Catalpa elata seedlings and promote the growth of Catalpa elata seedlings.

[0055] In addition, the proline content, peroxidase activity and superoxide dismutase activity of the leaves of catalpa elata seedlings in the treatment group with fluorescing Pseudomonas BR12-18 were significantly lower than those in the blank control group, decreasing by 83.11%, 92.22% and 27.31% respectively, indicating that the applied fluorescing Pseudomonas BR12-18 can reduce the waterlogging stress of catalpa elata seedlings, effectively alleviate the waterlogging stress, and thus promote the growth of catalpa elata seedlings.

[0056] Table 2 Measurement results of physiological indicators

[0057]

[0058] The method for determining the soluble sugar content in the present invention is as follows: 0.2 g of catalpa leaves are cut into pieces and mixed, 5 mL of distilled water is added, and the mixture is ground in an ice bath, and then boiled in a boiling water bath for 20 minutes. After being taken out and naturally cooled, the volume in a colorimetric tube is adjusted to 40 mL, and the supernatant is mixed with an anthrone reagent in a ratio of 1:5, and the mixture is boiled in a boiling water bath for 10 minutes and cooled. The absorbance at 620 nm is measured, and the soluble sugar content is obtained by comparing with the standard curve.

[0059] The method for determining the soluble protein content in the present invention is as follows: 0.5 g of catalpa leaves are cut into pieces and mixed evenly, 25 mL of PBS phosphate buffer is added and ground in an ice bath, and then centrifuged to obtain the supernatant, the supernatant is mixed with Coomassie brilliant blue dye in a ratio of 1:5, reacted for 2-5 minutes, and the absorbance value is measured at 595 nm. The soluble protein content is obtained by comparing with the standard curve.

[0060] The proline content determination method of the present invention is as follows: 0.2 g of catalpa elata leaves are chopped and mixed, 5 mL of 3% sulfosalicylic acid solution is added, and the mixture is cooled after being boiled in a water bath for 30 minutes. 2 mL of supernatant, 2 mL of glacial acetic acid, and 2 mL of acidic ninhydrin solution are taken and mixed. After being boiled in a water bath for 30 minutes, 4 mL of toluene is added, and the mixture is vortexed for 1 minute. The mixture is allowed to stand and the upper layer solution is collected. The absorbance at 520 nm is measured, and the proline content is obtained by comparing the solution with the standard curve.

[0061] The method for determining peroxidase activity in the present invention is as follows: 0.5 g of catalpa leaves are chopped and mixed, 25 mL of PBS phosphate buffer is added and ground in an ice bath, and the supernatant is centrifuged to obtain a crude enzyme solution. 1.5 mL of a peroxide premix (guaiacol and hydrogen peroxide) and 0.5 mL of the crude enzyme solution are added to a test tube in sequence, mixed, and immediately poured into a cuvette. The absorbance values ​​A after 1 minute and 2 minutes of reaction are measured respectively. 470 The amount of enzyme that decreased the absorbance by 0.01 within 1 min was defined as one unit of enzyme activity.

[0062] The method for determining superoxide dismutase activity in the present invention is as follows: 0.5 g of catalpa leaves are chopped and mixed, then 25 mL of PBS phosphate buffer is added and ground in an ice bath. The mixture is centrifuged and the supernatant is used as a crude enzyme solution. 0.05 mL of the crude enzyme solution, 0.25 mL of distilled water, 1.5 mL of PBS buffer, 0.3 mL of a 130 mmol / L methionine solution, 0.3 mL of a 750 µmol / L nitroblue tetrazolium solution, 0.3 mL of a 100 µmol / L EDTA-Na2 solution, and 0.3 mL of a 20 µmol / L riboflavin solution are mixed (a blank control group is treated with 0.05 mL of buffer instead of 0.05 mL of the crude enzyme solution). After 20 minutes of illumination, the absorbance at 560 nm is measured, with 50% inhibition of nitroblue tetrazolium photoreduction being defined as one unit of enzyme activity.

Claims

1. A Pseudomonas fluorescens BR12-18, characterized in that The Pseudomonas fluorescens BR12-18 is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, 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 Pseudomonas fluorescens BR12-18 according to claim 1 in alleviating waterlogging stress on catalpa chinensis seedlings.

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