Pseudomonas hormaeckii, microbial agent prepared from pseudomonas hormaeckii and application of pseudomonas hormaeckii
By using high nitrogen fixation ability and synthetic iron carriers as microbial agents, the soil and water pollution caused by the use of fertilizers in corn and wheat was solved, and the soil fertility improvement and plant growth promotion effect was achieved.
Patent Information
- Application Number
- CN202510427764.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-06-20
AI Technical Summary
The existing technology relies on chemical fertilizers during the growth of corn and wheat to lead to soil pollution, water pollution, crop quality decline and health risks, and lacks a green and environmentally friendly production method to promote the sustainable development of agriculture.
Pseudomonas rhodesiae (CGMCC No. 33691), which has high nitrogen fixation ability, synthetic iron carriers and secreted auxin, is used as a microbial agent to promote plant growth and improve soil fertility through its fermentation broth.
It significantly improves the root length, plant height and wet and dry root weight of corn and wheat, improves the content of soil organic matter and nitrogen and phosphorus, reduces soil pH, and enhances the nitrogen metabolism level of plants.
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Abstract
Description
Technical Field
[0001] The present invention relates to microbial strains and their applications, particularly Pseudomonas hoi, a microbial inoculant prepared therefrom, and its applications. Background Art
[0002] Corn and wheat are two major important food crops in China. During the growth process of corn and wheat, the demand for nutrient elements is relatively large. Currently, chemical fertilizers are the main way to provide crop nutrient elements. However, the increase in the use of chemical fertilizers leads to the residues of chemical molecules, resulting in a series of problems such as soil pollution, water pollution, decline in crop quality, increased human health risks, increased economic costs, and energy consumption. Therefore, it is extremely important to develop a green and environment-friendly production method to promote the green and sustainable development of agriculture for the production of high-yield and high-quality corn and wheat. Currently, the technical measures mainly for promoting crop growth usually adopt rhizosphere microorganisms. Common rhizosphere microorganisms include Agrobacterium, Bacillus, Klebsiella, Priestia megaterium, Pseudomonas, Sphingomonas, etc.
[0003] Soil nitrogen is an essential key nutrient element for plant growth and development, and has a crucial impact on the yield and quality of crops. Nitrogen metabolism is one of the important physiological metabolic processes in plants. Nitrogen is not only an essential basic element for plant growth and development, but also an important structural substance, playing an irreplaceable role in the synthesis of amino acids, proteins, chlorophyll, and various nitrogen-containing metabolites. The forms of nitrogen that plants can directly absorb and utilize include ammonium nitrogen (NH4 + -N) and nitrate nitrogen (NO3-N), which are mainly obtained from the soil. Nitrogen metabolism key enzymes are closely related to nitrogen absorption. The activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) are positively correlated with the absorption of ammonium nitrogen; the absorption of nitrate nitrogen is completed under the action of nitrate reductase (NR). The nitrogen metabolism status is positively correlated with the formation of carbohydrates and the accumulation of dry matter. Good nitrogen supply promotes the growth and development of crops.
[0004] There are abundant microorganisms in plants, which play an important role in the activation and recycling of nutrient elements. Endophytic bacteria form a symbiotic relationship with host plants during the long-term evolution process. Endophytic diazotrophs are a type of microorganisms that can colonize in plants and carry out nitrogen fixation in association with host plants. They can provide the nitrogen source required for the growth of host plants through nitrogen fixation and help the host adapt to the external environment. In addition to nitrogen fixation, endophytic diazotrophs also have functions such as producing IAA, dissolving phosphorus, producing siderophores, and antagonizing pathogenic bacteria. At the same time, because endophytic diazotrophs can colonize in plants, they have a positional advantage over rhizosphere microorganisms and can play beneficial roles such as promoting plant growth and biological control more directly and efficiently. And they do not need to form nodules with plants, which has greater potential and convenience in agricultural applications. Pseudomonas rhodesiae LM2-47 has been reported to have the effects of dissolving phosphorus and promoting growth, but there is no report on Pseudomonas rhodesiae with nitrogen fixation and growth promotion effects and improving the soil of medium- and low-yield fields. Summary of the Invention
[0005] One of the objectives of the present invention is to provide a Pseudomonas rhodesiae with good nitrogen fixation ability, capable of secreting auxin and synthesizing siderophores. Its Latin name is Pseudomonas rhodesiae, and the deposit number is CGMCC No. 33691.
[0006] The strain in the present invention was isolated from tomato roots. The strain was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 3, 2025. The taxonomic name is Pseudomonas rhodesiae, and the deposit number is CGMCC No. 33691. The address of the deposit institution is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing (abbreviated as CGMCC).
[0007] The colony morphology and physiological and biochemical characteristics of the strain are as follows:
[0008] The colonies of the strain are small, beige, and the surface is moist. It can synthesize siderophores and has a high ability to produce siderophilin. It has the ability to secrete auxin (IAA) and has a high nitrogen fixation ability. The similarity with Pseudomonas rhodesiae RL-M2-1 is 99.93%.
[0009] Another objective of the present invention is to provide a microbial inoculant containing Pseudomonas rhodesiae with the Latin name Pseudomonas rhodesiae and the deposit number CGMCC No. 33691.
[0010] A third object of the present invention is to provide the application of Pseudomonas rhodesiae with the Latin name and deposit number CGMCC No. 33691, its fermentation broth or microbial inoculum in promoting plant growth, wherein the plant is corn or wheat.
[0011] A fourth object of the present invention is to provide the application of Pseudomonas rhodesiae with the Latin name and deposit number CGMCC No. 33691, its fermentation broth or microbial inoculum in the preparation of soil conditioners for medium- and low-yield farmland and / or alkaline soil.
[0012] A fifth object of the present invention is to provide the application of Pseudomonas rhodesiae with the Latin name and deposit number CGMCC No. 33691, its fermentation broth or microbial inoculum in increasing the content of nitrogen-containing organic matter in corn seedlings.
[0013] Specifically, the fermentation broth containing Pseudomonas rhodesiae is obtained by inoculating Pseudomonas rhodesiae with the Latin name and deposit number CGMCC No. 33691 into a sterilized medium containing a carbon source, a nitrogen source and inorganic salts, and then performing shake culture.
[0014] Specifically, sucrose is selected as the carbon source in the medium, soybean powder is selected as the nitrogen source, and calcium nitrate is selected as the inorganic salt.
[0015] The medium consists of the following raw material components:
[0016] Sucrose 7.5 g / L; soybean powder 10.0 g / L; calcium nitrate 12.5 g / L; the balance is water; the initial pH of the medium is 7.0.
[0017] The shake culture conditions are as follows:
[0018] The medium filling amount is 20%, the capacity of the shake flask containing the medium is 50 mL - 250 mL, the inoculation amount is 15.0%, and shake culture is performed at a temperature of 30 °C and a rotation speed of 170 revolutions per minute for 36 hours.
[0019] The substantial features and remarkable technical progress achieved by the present invention are as follows:
[0020] The screening method of the present invention has obtained a strain of Pseudomonas rhodesiae with the preservation number of CGMCC No. 33691. It has been experimentally verified by the applicant that this strain can synthesize siderophores, has a relatively high ability to produce siderophilin, has the ability to secrete auxin (IAA), and has a relatively high nitrogen fixation ability. When applied to the production of corn and wheat, it can promote root length, plant height, and root dry and wet weights. After treating medium- and low-yield farmland soils with scarce soil nitrogen and phosphorus, low organic matter content, and high soil alkalinity, it can significantly increase the content of soil organic matter, nitrogen, and phosphorus elements in the rhizosphere of corn, improve soil fertility, reduce soil pH value, and increase soil conductivity. It can promote the absorption of nitrogen by corn plants, increase the total nitrogen content in the roots, stems, and leaves of corn, significantly enhance the activities of nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT), and enhance the nitrogen metabolism level of corn seedlings.
[0021] The strain in the present invention was deposited on March 3, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the taxonomic name of Pseudomonas rhodesiae and the preservation number of CGMCC No. 33691. The address of the preservation institution is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing (abbreviated as CGMCC). Brief Description of the Drawings
[0022] The drawings of the present invention are as follows:
[0023] Figure 1 It is a schematic diagram of the colony of Pseudomonas rhodesiae CGMCC No. 33691 cultured in a flat culture dish.
[0024] Figure 2 It is the effect diagram of the qualitative determination plate test of siderophilin synthesis of Pseudomonas rhodesiae CGMCC No. 33691.
[0025] Figure 2 After inoculating Pseudomonas rhodesiae CGMCC No. 33691 into the CAS solid medium, the ability to produce siderophores is judged according to whether there is an orange halo. It has been verified that the strain can produce an orange light circle on the CAS medium, indicating that the strain has a relatively high ability to produce siderophores.
[0026] Figure 3 It is the effect diagram of the qualitative determination plate test of the nitrogen fixation ability of Pseudomonas rhodesiae CGMCC No. 33691.
[0027] Figure 3 After inoculating Pseudomonas rhodesiae CGMCC No.33691 on Ashby solid medium, the nitrogen fixation ability of the strain was judged according to the presence and size of the clear zone. After verification, the strain could produce a clear zone on Ashby solid medium and the clear zone was relatively large, indicating that the nitrogen fixation ability of the strain was relatively high.
[0028] Figure 4 It is the effect diagram of the qualitative determination test of the ability of Pseudomonas rhodesiae CGMCC No.33691 to secrete indole-3-acetic acid (IAA).
[0029] Figure 4 Pseudomonas rhodesiae CGMCC No.33691 was inoculated into 1 mL of LB liquid medium containing 200 mg / L of L-tryptophan, and 3 replicates were set. After culturing for 2 days at a temperature of 30 °C and a rotation speed of 180 r / min, 500 μL of the bacterial suspension was taken onto a 48-well plate, and 500 μL of Salkowski color-developing solution was added. Taking 500 μL of uninoculated LB liquid medium and 500 μL of color-developing solution as a control. After standing at room temperature in the dark for 20 minutes, turning pink was positive, indicating the ability to secrete indole-3-acetic acid (IAA), and the darker the color, the stronger the secretion intensity. On the left side of the figure is the control, and on the right side is Pseudomonas rhodesiae CGMCC No.33691. After verification, the strain showed a light pink color, indicating that the strain had the ability to secrete indole-3-acetic acid (IAA).
[0030] Figure 5 It is a comparative bar chart of the effect of carbon sources on the fermentation of Pseudomonas rhodesiae CGMCC No.33691.
[0031] Figure 6 It is a comparative bar chart of the effect of nitrogen sources on the fermentation of Pseudomonas rhodesiae CGMCC No.33691.
[0032] Figure 7 It is a comparative bar chart of the effect of inorganic salts on the fermentation of Pseudomonas rhodesiae CGMCC No.33691.
[0033] From Figure 5 、 Figure 6 、 Figure 7It can be seen that the OD values of the strains in the present invention are different in different combinations of carbon sources, nitrogen sources, and inorganic salt media. 600 The optimal carbon source, nitrogen source, and inorganic salt for the fermentation of the strains in the present invention are sucrose, soybean powder, and calcium nitrate, respectively.
[0034] Figure 8 Figure is a comparative bar chart showing the effect of initial pH on the fermentation of Pseudomonas rhodesiae CGMCC No. 33691.
[0035] Figure 9 Figure is a comparative bar chart showing the effect of temperature on the fermentation of Pseudomonas rhodesiae CGMCC No. 33691.
[0036] Figure 10 Figure is a comparative bar chart showing the effect of inoculum size on the fermentation of Pseudomonas rhodesiae CGMCC No. 33691.
[0037] Figure 11 Figure is a comparative bar chart showing the effect of liquid volume on the fermentation of Pseudomonas rhodesiae CGMCC No. 33691.
[0038] Figure 12 Figure is a comparative bar chart showing the effect of rotation speed on the fermentation of Pseudomonas rhodesiae CGMCC No. 33691.
[0039] Figure 13 Figure is a comparative bar chart showing the effect of culture time on the fermentation of Pseudomonas rhodesiae CGMCC No. 33691.
[0040] From Figures 8 - 13 the optimal parameters for the fermentation of Pseudomonas rhodesiae CGMCC No. 33691 in the culture conditions can be screened out.
[0041] Figure 14 Figure Figures 8 - 13 is a bar chart showing the effect of Pseudomonas rhodesiae CGMCC No. 33691 on the total nitrogen content in the middle roots of maize plants.
[0042] Figure 15 Figure is a bar chart showing the effect of Pseudomonas rhodesiae CGMCC No. 33691 on the total nitrogen content in the leaves of maize plants.
[0043] From Figure 14 and 15 it can be seen that applying Pseudomonas rhodesiae CGMCC No. 33691 can significantly promote the total nitrogen content in the roots and leaves of maize (P < 0.05), with increases of 48.96% and 59.80% respectively. The total nitrogen content range in the leaves of maize treated with Pseudomonas rhodesiae CGMCC No. 33691 is higher than that in the roots, indicating that Pseudomonas rhodesiae CGMCC No. 33691 is beneficial to the absorption and transportation of nitrogen from the roots to the leaves.
[0044] Figure 16 This is the effect of Pseudomonas rhodesiae CGMCC No. 33691 on the activity of nitrate reductase (NR) in the nitrogen metabolism of maize seedlings.
[0045] From Figure 16 it can be seen that Pseudomonas rhodesiae CGMCC No. 33691 significantly increased the activity of nitrate reductase (NR) in maize leaves (P < 0.01), with an increase of 80.82% compared to the control treatment.
[0046] Figure 17 This is the effect of Pseudomonas rhodesiae CGMCC No. 33691 on the activity of glutamate synthase (GOGAT) in the nitrogen metabolism of maize seedlings.
[0047] From Figure 17 it can be seen that the activity of glutamate synthase (GOGAT) in the leaves of the inoculated treatment group is higher than that of the non-inoculated treatment, with an increase of 39.33% compared to the control treatment.
[0048] Figure 18 This is the effect of Pseudomonas rhodesiae CGMCC No. 33691 on the activity of glutamine synthetase (GS) in the nitrogen metabolism of maize seedlings.
[0049] From Figure 18 it can be seen that the activity of glutamine synthetase (GS) in the leaves of the inoculated treatment group is higher than that of the non-inoculated treatment, and there is a significant difference between the two (P < 0.05). Specific implementation method
[0050] The present invention will be further described below in conjunction with embodiments, but it is not intended to limit the present invention. The protection scope of the present invention shall be subject to the content recorded in the claims. Any equivalent means substitution made according to the description of the present invention's specification shall not depart from the protection scope of the present invention.
[0051] Example 1
[0052] Isolation and Identification of Pseudomonas rhodesiae CGMCC No. 33691
[0053] 1. Isolation of Strains
[0054] Rinse the surface of tomato roots thoroughly under running water, dry with sterile filter paper, take 1 g of main roots and lateral roots, transfer them into a laminar flow hood for surface disinfection. First, soak them in 75% alcohol with shaking for 1 minute, then soak them in 1% sodium hypochlorite solution with shaking for 5 - 10 minutes, and then rinse them 3 times with sterile water. Spread the last rinse solution on the rich nutrient medium NR plate respectively. After 72 hours, observe whether colonies grow. If no colonies grow, it means the surface sterilization is complete. Put 1 g of surface-disinfected material into a sterile mortar, add 9 ml of sterile normal saline, and grind it into a homogenate, that is, obtain a dilution solution with a concentration of 10 -1 dilution solution. Pipette 10 ml of the dilution solution with a concentration of 10 -1 and perform gradient dilution to 10 -3 . Take 10 -1 , 10 -2 , 10 -3 concentration dilution solutions and spread them on the NR medium. Set 3 replicates for each gradient of each medium. Culture at 30 °C for 48 hours, and verify by combining colony morphology observation and 16S rDNA gene sequence amplification and sequencing.
[0055] Rich Nutrient Medium NR (g / L): Yeast extract 0.50 g; Tryptone 0.50 g; Glucose 0.50 g; Soluble starch 0.50 g; Dipotassium hydrogen phosphate 0.30 g; Magnesium sulfate 0.02 g; Sodium pyruvate 0.30 g; Agar 15.00 g.
[0056] Media for Strain Culture
[0057] LB Liquid Medium: Add distilled water to 10 g of tryptone, 5 g of yeast extract, and 3 g of sodium chloride, adjust the pH = 7.2, adjust the volume to 1 L, sterilize at 121 °C for 15 minutes, and cool for use.
[0058] LB Solid Medium: Add agar to the LB liquid medium to make its concentration 15 g / L; sterilize at 121 °C for 15 minutes. Pour the LB liquid medium cooled to about 55 °C into petri dishes and let it cool naturally.
[0059] Strain preservation: Inoculate a single colony into LB liquid medium and culture it at 30 °C for 16 hours to obtain a cultured bacterial solution. Mix 1 volume of the cultured bacterial solution with 1 volume of 50% (v / v) glycerol aqueous solution and store it at -80 °C. The obtained strain is named M11.
[0060] 2. Morphological characteristics of the strain
[0061] The colonies of M11 are small, beige, and the surface is moist. The specific results are shown in Figure 1 .
[0062] 3. Physiological and biochemical characteristics of the strain
[0063] (1) Qualitative determination of siderophore synthesis
[0064] The CAS medium consists of the following raw materials: 0.060 g of chrome azurol; 0.073 g of cetyltrimethylammonium bromide; 2.645 mg of ferric chloride hexahydrate; 0.295 g of sodium dihydrogen phosphate dihydrate; 1.213 g of disodium hydrogen phosphate dodecahydrate; 0.125 g of ammonium chloride; 0.0375 g of potassium dihydrogen phosphate; 0.0625 g of sodium chloride; 0.1 g of magnesium sulfate heptahydrate; 10 g of glucose; 10 g of peptone; 10 g of acid-hydrolyzed casein; 15 - 20 g of agar.
[0065] Spot inoculate the strain M11 that has been activated for 24 hours on the CAS medium, repeat each treatment 3 times, and incubate it in an inverted position at 30 °C for 3 days. Observe the color change of each plate, whether a transparent halo is formed, and record the results. If the strain can grow on the CAS medium and form an orange-yellow halo, it indicates that the strain has the function of synthesizing siderophores. The specific results are shown in Figure 2 and its description.
[0066] (2) Analysis of the nitrogen fixation ability of the strain
[0067] Each liter of liquid Ashby medium consists of the following raw materials: 0.2 g of potassium dihydrogen phosphate; 0.2 g of sodium chloride; 0.2 g of magnesium sulfate heptahydrate; 0.2 g of potassium sulfate dihydrate; 5 g of calcium carbonate; 5 g of glucose; 5 g of mannitol; the balance is water, pH = 7.0.
[0068] Spot inoculate the selected strain M11 on the Ashby solid medium (10% agar is added to the above liquid Ashby medium), set up 3 replicates, place it in an incubator and incubate it in an inverted position at 28 °C, and observe whether the strain grows and produces a transparent halo. The specific results are shown in Figure 3 and the description.
[0069] (3) Analysis of the ability of the strain to secrete indole-3-acetic acid (IAA)
[0070] The 250 - milliliter Salkowski color - developing solution contains the following raw materials: 150 milliliters of concentrated sulfuric acid and 37.5 milliliters of a ferric chloride solution with a concentration of 0.5 M.
[0071] Inoculate the isolated and purified strain into 1 milliliter of LB liquid medium containing 200 milligrams per liter of L - tryptophan, and set up 3 replicates. After culturing for 2 days at a temperature of 30 °C and a rotation speed of 180 revolutions per minute, take 500 microliters of the bacterial suspension onto a 48 - well plate, add 500 microliters of the Salkowski color - developing solution, and use the addition of 500 microliters of uninoculated LB liquid medium and 500 microliters of the color - developing solution as a control. After standing at room temperature in the dark for 20 minutes, if the color turns pink, it is positive, indicating the ability to secrete indole - 3 - acetic acid (IAA). The darker the color, the greater the secretion intensity. The specific results are shown in Figure 4 and the description.
[0072] 4. Identification and preservation of the strain
[0073] The similarity between the isolated strain and Pseudomonas rhodesiae RL - M2 - 1 is 99.93%. The specific results are shown in Table 1. By comprehensively analyzing the cell morphology, physiological and biochemical characteristics, and 16S rDNA gene sequence, it can be known that the strain in this application is Pseudomonas rhodesiae. This strain was preserved on March 3, 2025, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (the abbreviation of this preservation institution is CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101), and the preservation number is CGMCC No. 33691, and the taxonomic name is Pseudomonas rhodesiae.
[0074] Table 1 Comparison results of the isolated strain and the typical strains in the database
[0075] Strain number Closest strain with 16S rDNA similarity Similarity (%) M11 Pseudomonas rhodesiae RL - M2 - 1 99.93％
[0076] Example 2
[0077] Optimization of the culture medium components
[0078] First, prepare the seed solution, draw the growth curves of each dominant strain, and then screen the carbon source, nitrogen source, and inorganic salts in the fermentation medium. The components of the fermentation medium are as follows: soluble starch, malt extract powder, glucose, sucrose, and corn starch are carbon sources; urea, soybean powder, ammonium sulfate, and beef extract are nitrogen sources; zinc sulfate, dipotassium hydrogen phosphate, magnesium chloride, calcium nitrate, and ferrous sulfate heptahydrate are inorganic salts.
[0079] 1. Preparation of the seed solution
[0080] After activating each test strain, inoculate it into a 250 - milliliter Erlenmeyer flask containing 100 milliliters of LB medium, and shake - culture it under dark conditions at a temperature of 28°C and a rotation speed of 180 revolutions per minute to prepare a seed solution. Measure the OD of the fermentation broth with a spectrophotometer every 6 hours. 600 value. Using the culture time as the abscissa and the OD 600 value as the ordinate, plot the growth curves of each strain.
[0081] 2. Screening and optimization of the components of carbon source, nitrogen source, and inorganic salts in the liquid fermentation medium
[0082] Using the standard - formula LB liquid medium as a control, replace the yeast extract (carbon source) in the LB liquid medium with equal amounts (5.0 g / L) of soluble starch, malt extract, glucose, sucrose, and corn flour respectively to prepare fermentation culture broths with different carbon sources. The volume of the fermentation culture broth in each 250 - milliliter Erlenmeyer flask is 100 milliliters. Inoculate the seed solutions of each strain into the fermentation culture broths with different carbon sources according to an inoculation amount of 5%, and shake - culture them under dark conditions at a temperature of 28°C and a rotation speed of 180 revolutions per minute for 24 hours. Measure the OD 600 value, repeat 3 times, and screen out the best carbon source. Respectively use equal amounts (10 g / L) of urea, soybean powder, ammonium sulfate, and beef extract to replace the tryptone (nitrogen source) in the LB medium. The fermentation reaction conditions are the same as those for carbon - source screening to screen out the best nitrogen source. Respectively use equal amounts (10.0 g / L) of zinc sulfate, dipotassium hydrogen phosphate, magnesium chloride, calcium nitrate, and ferrous sulfate heptahydrate to replace the sodium chloride (inorganic salt) in the LB medium. The fermentation reaction conditions are the same as those for carbon - source screening to screen out the best inorganic salt.
[0083] 3. Optimization of the ratio of carbon source, nitrogen source, and inorganic salts in the liquid fermentation medium
[0084] Based on the screening data of the best carbon source, nitrogen source, and inorganic salts, conduct a multi - factor orthogonal analysis to study the best ratio of the three components of carbon source, nitrogen source, and inorganic salts.
[0085] As Figures 5 - 7 can be seen, the OD 600 values of the strains in different combinations of carbon - source, nitrogen - source, and inorganic - salt media are different. The best carbon source, nitrogen source, and inorganic salt suitable for the fermentation of Pseudomonas rhodesiae CGMCC No.33691 are sucrose, soybean powder, and calcium nitrate respectively.
[0086] On the basis of screening out the best carbon source, nitrogen source, and inorganic salt suitable for the fermentation of each strain, use L9(3 3)Orthogonal design analysis was performed on 3 factors and 3 levels (3 different weights, see Table 2) to determine the optimal ratio of each substance. L9(3 3 )The results of the orthogonal design analysis (see Table 3) show that the larger the R, the greater the influence on the test results. From the corresponding OD 600 values of each column, the optimal combination of Pseudomonas rhodesiae CGMCC No. 33691 was determined to be A2B1C2, that is, 7.5 g / L of sucrose, 10.0 g / L of soybean powder, and 12.5 g / L of calcium nitrate.
[0087] Table 2 Orthogonal test factors and levels of the culture medium components of Pseudomonas rhodesiae CGMCC No. 33691
[0088]
[0089] Table 3 Orthogonal test for optimizing the ratio of each component of the culture medium of Pseudomonas rhodesiae CGMCC No. 33691
[0090]
[0091]
[0092] According to the nine treatments in the orthogonal design table 3, the treatment with the largest OD 600 value in the nine treatments is the optimal ratio. The optimal combination was determined to be A2B1C2, that is, 7.5 g / L of sucrose, 10.0 g / L of soybean powder, and 12.5 g / L of calcium nitrate.
[0093] Example 3
[0094] Results of fermentation condition optimization
[0095] Orthogonal tests were carried out with factors such as culture temperature, initial pH value, rotation speed, inoculation amount, and liquid loading amount to determine the optimal fermentation conditions for each dominant strain.
[0096] (1) Screening of initial pH
[0097] Using the optimized medium of each component as the liquid fermentation medium, the initial pH was set to 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The fermentation medium loading in a 250 - milliliter Erlenmeyer flask was 100 milliliters. The seed liquid was inoculated at 5%, the rotation speed was 180 revolutions per minute, the temperature was 28 °C, and it was shake - fermented for 24 hours. Each treatment had 3 replicates. According to the OD 600 value of the fermentation reaction solution, the optimal initial pH was determined. The specific results are shown in Figure 8 .
[0098] (2) Fermentation temperature screening
[0099] After determining the optimal initial pH of the fermentation reaction, the fermentation reaction temperatures were set at 28.0 °C, 30.0 °C, 32.0 °C, and 34.0 °C. The specific fermentation method was the same as that for the initial pH screening to screen the optimal fermentation reaction temperature. The specific results are shown in Figure 9 .
[0100] (3) Inoculum size of the seed culture
[0101] After determining the optimal initial pH and fermentation temperature of the fermentation reaction, the inoculum sizes of the seed culture were set at 5.0%, 10.0%, 15%, and 20.0% respectively. The specific cultivation and fermentation method was the same as that for the initial pH screening to screen the optimal inoculum size. The specific results are shown in Figure 10 .
[0102] (4) Screening of liquid loading volume
[0103] After determining the optimal initial pH, reaction temperature, and inoculum size of the seed culture, the liquid loading volumes in 250 - milliliter Erlenmeyer flasks were set as: 50 milliliters (20%), 100 milliliters (40%), 150 milliliters (60%), and 200 milliliters (80%). The specific cultivation and fermentation method was the same as that for the initial pH screening to screen the optimal liquid loading volume. The specific results are shown in Figure 11 .
[0104] (5) Screening of shaker speed
[0105] After determining the optimal initial pH, reaction temperature, inoculum size, and liquid loading volume, the shaker speeds were set at 140 revolutions per minute, 170 revolutions per minute, 200 revolutions per minute, and 220 revolutions per minute. The specific cultivation and fermentation method was the same as that for the initial pH screening to screen the optimal shaker speed. The specific results are shown in Figure 12 .
[0106] (6) Optimization of fermentation time
[0107] After determining the optimal initial pH, reaction temperature, inoculum size, liquid loading volume, and shaker speed, the fermentation culture times were set at 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, and 56 hours. Each treatment had 3 replicates. According to the OD 600 values, the optimal fermentation culture time was screened. The specific results are shown in Figure 13 .
[0108] On the basis of screening and obtaining the optimal fermentation medium, single factors such as the initial pH of the medium, the loading volume of the liquid, the rotation speed, and the temperature were screened and optimized. The optimal fermentation reaction conditions for Pseudomonas rhodesiae CGMCC No. 33691 were an initial pH = 7.0, a fermentation temperature of 30 °C, a loading volume of 20% for the fermentation medium filled in a shake flask with a capacity of 50 mL to 250 mL, an inoculum size of 15.0%, a rotation speed of 170 rpm, and a cultivation time of 36 hours.
[0109] Example 4
[0110] Effect of the dominant strain on the growth of potted wheat and corn
[0111] Select corn and wheat seeds of uniform size, disinfect them with 75% ethanol solution for 10 minutes, rinse them with water and place them in a beaker. Soak the corn and wheat seeds in the fermentation broth of Pseudomonas rhodesiae CGMCC No. 33691 for 12 hours respectively, with sterile water as the control. Then cultivate the corn and wheat seedlings by the potted method. The soil used is medium and low-yield farmland soil. After 30 days, measure the plant height, root length, stem dry weight, and root dry weight of the wheat and corn seedlings. The specific results are shown in Table 4 and Table 5, and each treatment is as follows:
[0112] Treatment No. -0: Water with tap water; Treatment No. -1: Water with bacterial liquid M11; Treatment 1: Field soil; Treatment 2: Field soil + biochar; Treatment 3: Field soil + diatomite; Treatment 4: Field soil + biochar + diatomite.
[0113] Table 4 Effects of different treatments on the root length, plant height, root dry weight, and root wet weight of corn
[0114]
[0115] Table 5 Effects of different treatments on the root length, plant height, root dry weight, and root wet weight of wheat
[0116]
[0117] Experimental results: Pseudomonas rhodesiae CGMCC No. 33691 has a significant promoting effect on the growth of corn and wheat, specifically manifested in the increase of root length, plant height, and dry and wet weights. Among different treatment combinations, the application of bacterial agent M11 alone or in combination with biochar and diatomite can effectively promote the root length, plant height, dry and wet weights of the roots and stems of corn and wheat. Among them, the combination of Pseudomonas rhodesiae CGMCC No. 33691 and biochar has the most significant growth-promoting effect on corn and wheat.
[0118] Example 5
[0119] Effect of Pseudomonas rhodesiae CGMCC No. 33691 on Physicochemical Properties of Maize Rhizosphere Soil
[0120] 1. Collection and Treatment of Soil Samples
[0121] Collect the rhizosphere soil of maize in Example 4, pass it through a 2-mm sieve, spread the fresh soil sample on a newspaper and air-dry it indoors. The air-dried soil sample is used for the determination of soil physicochemical properties.
[0122] 2. Determination of Soil Physicochemical Properties
[0123] Potassium dichromate oxidation method (external heating method) is used for soil organic matter; Kjeldahl method is used for total nitrogen in soil; acid digestion-inductively coupled plasma emission spectrometry is used for total phosphorus in soil; sodium bicarbonate extraction-molybdenum antimony anti-colorimetry is used for available phosphorus in soil; alkali-hydrolyzable diffusion method is used for alkali-hydrolyzable nitrogen in soil; the determinations of soil electrical conductivity (EC) and pH are carried out according to the water-soil ratios of 5:1 and 2.5:1 respectively, and measured with a conductivity meter and a pH meter. The specific results are shown in Table 6.
[0124] Table 6 Effect of Pseudomonas rhodesiae CGMCC No. 33691 on Physicochemical Properties of Maize Rhizosphere Soil
[0125] Physical and chemical parameters M11 treatment group Control group Total nitrogen (g / kg) 0.387±0.01 0.309±0.01 Total phosphorus (g / kg) 0.681±0.02 0.541±0.01 Organic matter (g / kg) 8.34±0.13 7.817±0.01 Alkaline hydrolyzable nitrogen (mg / kg) 33.31±0.81 22.568±0.01 Available phosphorus (mg / kg) 10.74±0.09 9.267±0.04 pH 7.86±0.07 8.067±0.02 EC (us / cm) 263.33±3.51 187.667±2.08
[0126] Experimental results: The analysis of the control group soil shows that the original soil belongs to medium and low-yield farmland soil, with problems of lack of nitrogen and phosphorus elements, low organic matter content and relatively high soil alkalinity. After treatment with Pseudomonas rhodesiae CGMCC No. 33691, the contents of organic matter, nitrogen and phosphorus elements in the maize rhizosphere soil are significantly increased. The organic matter content is 0.52 g / kg higher than that of the control, and the total phosphorus and available phosphorus are increased by 25.88% and 15.90% respectively compared with the control. Among them, the contents of total nitrogen and alkali-hydrolyzable nitrogen are increased most significantly, by 25.24% and 47.60% respectively compared with the control, and the soil fertility is significantly improved. At the same time, the treatment with Pseudomonas rhodesiae CGMCC No. 33691 also reduces the soil pH value, effectively alleviates the degree of soil alkali stress, and at the same time, increases the soil electrical conductivity. To sum up, Pseudomonas rhodesiae CGMCC No. 33691 can effectively improve the physicochemical properties of soil, has a good soil conditioning effect, and can be popularized and applied as a conditioner for medium and low-yield farmland and alkalized soil.
[0127] Example 6
[0128] Effect of Pseudomonas rhodesiae CGMCC No. 33691 on the total nitrogen content of maize plants
[0129] Take the roots and leaves of the maize plants in Example 4, rinse them clean under running water. For the roots, take the main roots and lateral roots, and for the leaves, take the second fully expanded leaf. Crush the samples, dry them to a constant weight at a temperature of 70 °C, pass through a 40-mesh sieve, accurately weigh 0.3 g of each part, use the H2SO4-H2O2 nitrification method, and then use the Kjeldahl method to detect the total nitrogen content of the roots and leaves of the maize plants. Each treatment is set with 3 replicates. The specific results are shown in Figure 14 、 15 。
[0130] Experimental results: It can be seen from Figure 14 、 15 that applying Pseudomonas rhodesiae CGMCC No. 33691 can significantly promote the increase in the total nitrogen content of maize roots and leaves (P < 0.05), with increases of 48.96% and 59.80% respectively. The range of the total nitrogen content of maize leaves treated with Pseudomonas rhodesiae CGMCC No. 33691 is higher than that of maize roots, indicating that Pseudomonas rhodesiae CGMCC No. 33691 is beneficial to the absorption and transportation of nitrogen from roots to leaves.
[0131] Example 7
[0132] Effect of Pseudomonas rhodesiae CGMCC No. 33691 on the activities of key enzymes in nitrogen metabolism of maize seedlings
[0133] Take the second fully expanded leaf of the maize plants in Example 4 and weigh about 0.5 g of the fresh sample. The activities of glutamine synthetase (GS) (product number: GS-1-Y), glutamate synthase (GOGAT) (product number: GOGAT-1-W), and nitrate reductase (NR) (product number: NR-1-W) are detected using a kit from Suzhou Keming Biotechnology Co., Ltd. Each treatment is set with 3 replicates. The specific results are shown in Figures 16 - 18 。
[0134] Experimental results: Nitrate reductase (NR) is a key enzyme for the conversion of plant nitrate nitrogen to ammonium nitrogen, and the activity of NR is closely related to the form of nitrogen supplied. Under normal temperature conditions, Pseudomonas rhodesiae CGMCC No. 33691 significantly increased the activity of NR (P < 0.01), with an 80.82% increase compared to the control treatment, promoting the conversion of nitrogen to ammonium nitrogen. Glutamine synthetase (GS) is a key enzyme for ammonia assimilation in plants and can catalyze the synthesis of glutamine from ammonium ions and glutamate. The GS activity in the leaves of the treatment group with Pseudomonas rhodesiae CGMCC No. 33691 was higher than that of the control treatment, and there was a significant difference between the two (P < 0.05). Glutamate synthase (GOGAT) and GS together constitute the GS / GOGAT cycle, which is involved in the regulation of ammonia assimilation in plants. The GOGAT activity in the leaves of the treatment group with Pseudomonas rhodesiae CGMCC No. 33691 was significantly higher than that of the control treatment, with a 39.33% increase compared to the control treatment. Applying Pseudomonas rhodesiae CGMCC No. 33691 can increase the activities of NR, GS, and GOGAT, induce the conversion of inorganic nitrogen to organic nitrogen, and thus increase the content of nitrogen-containing organic matter in maize seedlings.
Claims
1. Pseudomonas rhodesiae, whose Latin name is Pseudomonas rhodesiae, and whose deposit number is CGMCC No.33691.
2. A microbial agent containing the Pseudomonas rhodesiae described in claim 1, whose Latin name is Pseudomonas rhodesiae and whose deposit number is CGMCC No. 33691.
3. Use of the Pseudomonas rhodesiae with a Latin name of Pseudomonas rhodesiae and a preservation number of CGMCC No. 33691 as claimed in claim 1 or a fermentation liquid containing the same or the microbial agent as claimed in claim 2 in promoting plant growth, wherein the plant is corn or wheat.
4. Use of the Pseudomonas rhodesiae with Latin name Pseudomonas rhodesiae and deposit number CGMCC No. 33691 as claimed in claim 1 or a fermentation liquid containing the same or the microbial agent as claimed in claim 2 as a soil conditioner for medium- and low-yield fields and / or alkaline soils.
5. Use of the Pseudomonas rhodesiae with Latin name Pseudomonas rhodesiae and deposit number CGMCC No. 33691 as claimed in claim 1 or a fermentation liquid containing the same or the microbial agent as claimed in claim 2 in increasing the nitrogenous organic matter content of corn seedlings.
6. The use according to any one of claims 3, 4 or 5, characterized in that The Pseudomonas rhodesiae or fermentation liquid containing the same is obtained by inoculating Pseudomonas rhodesiae, whose Latin name is Pseudomonas rhodesiae and whose preservation number is CGMCC No. 33691, into a sterilized culture medium containing a carbon source, a nitrogen source and an inorganic salt, and then culturing the culture medium with shaking.
7. The use according to claim 6, characterized in that In the culture medium, sucrose is selected as the carbon source, soybean powder is selected as the nitrogen source, and calcium nitrate is selected as the inorganic salt.
8. The use according to claim 7, characterized in that The culture medium is composed of the following raw material components: Sucrose 7.5 g / L; soybean powder 10.0 g / L; calcium nitrate 12.5 g / L; the balance is water; the initial pH of the culture medium = 7.
0.
9. The use according to claim 6, characterized in that The shaking culture conditions are as follows: The culture medium filling volume is 20%, the capacity of the shaking flask containing the culture medium is 50mL-250mL, the inoculation volume is 15.0%, and the shaking culture is carried out at a temperature of 30°C and a rotation speed of 170 rpm for 36 hours.