Pseudomontella as well as fungicide and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pseudomontella as well as a microbial agent and application thereof. The invention provides pseudomonas montella, which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC NO.30836. The pseudomonas montella has the advantages that the preservation number is CGMCC NO.30836; the pseudomontella has relatively strong functions of fixing nitrogen and dissolving inorganic phosphorus, and has the capability of secreting auxin. The fertilizer can be applied to the fields of soil nitrogen fixation and plant growth promotion, has the advantages of reducing the use amount of chemical fertilizers and promoting plant growth in agricultural production, can promote green development of agriculture, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to Pseudomonas meningitidis and a bacterial agent and application thereof. Background Art

[0002] The continuous cropping of Chinese medicinal materials for many years and the excessive use of chemical fertilizers have led to soil degradation, a decline in the yield and quality of Chinese medicinal materials, and have seriously restricted the development of the Chinese medicinal materials industry. In addition, the large-scale use of chemical fertilizers has seriously damaged the ecological environment and is not conducive to the sustainable development of agriculture. It is urgent to find alternatives to chemical fertilizers.

[0003] Nitrogen and phosphorus are nutrients required for plant growth and development. 80% of nitrogen in the air cannot be directly used by plants. Phosphorus in the soil mostly exists in the form of aluminum phosphate and iron phosphate, which is also difficult for plants to directly use. Plant growth-promoting bacteria (PGPB) can promote plant absorption of nutrients through mechanisms such as nitrogen fixation and phosphorus solubilization, and can produce plant growth hormones to regulate plant growth.

[0004] Pseudomonas meningitidis is also used in plant growth promotion as a plant growth-promoting bacteria. CN118726192A discloses a synthetic bacterial community and its application for improving the quality and yield of simulated wild astragalus. Its synthetic bacterial community is composed of 3 nitrogen-fixing bacteria, 2 phosphate-dissolving bacteria and 1 potassium-dissolving bacteria. Among them, Pseudomonas meningitidis is used as a phosphate-dissolving bacteria. The synthetic bacterial community significantly improves the yield and quality of astragalus and improves the microbial community structure of astragalus planting soil. CN118146977A discloses a Pseudomonas meningitidis, which has a strong ability to produce indoleacetic acid, can be applied to low-yield fields and field grain crops in saline-alkali land, and has the effects of promoting emergence, increasing crop yield, and activating rhizosphere soil. At present, there is no literature report on Pseudomonas meningitidis with nitrogen fixation effect, and there is no report on Pseudomonas meningitidis with nitrogen fixation effect and soluble phosphorus at the same time, secreting indoleacetic acid (IAA) as a plant hormone.

[0005] Therefore, providing a Pseudomonas meningitidis strain that has significant nitrogen fixation, phosphorus solubilization, and IAA secretion as a plant growth-promoting bacterium can provide a new solution for weight loss and efficiency enhancement, sustainable agricultural development, and the construction of traditional Chinese medicine ecological agriculture. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a Pseudomonas meningitidis and a bacterial agent and application thereof.

[0007] On the one hand, the present invention provides a Pseudomonas meningitidis that can fix nitrogen, dissolve inorganic phosphorus, secrete auxin, effectively improve soil fertility, reduce the use of chemical fertilizers, improve plant nutrient supply, improve soil microecological environment, and promote plant growth. The deposit information of the Pseudomonas meningitidis FD-2 is as follows:

[0008] Taxonomic nomenclature: Pseudomonas mandelii;

[0009] Deposit time: June 3, 2024;

[0010] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0011] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0012] Deposit number: CGMCC NO.30836.

[0013] Furthermore, the 16S rRNA sequence of Pseudomonas mandelii FD-2 is shown in SEQ ID NO: 1.

[0014] Furthermore, the Pseudomonas mandelii FD-2 can secrete indoleacetic acid to promote plant growth.

[0015] Furthermore, the Pseudomonas mandelii FD-2 can form a phosphate-dissolving zone on the NBRIP inorganic phosphorus culture medium and can dissolve inorganic phosphorus; it can form a transparent zone on the Ashby nitrogen-free culture medium and has the ability to fix nitrogen.

[0016] In another aspect, the present invention provides a method for preparing Pseudomonas meningitidis, the method for preparing Pseudomonas meningitidis comprising the following steps:

[0017] Step 1: Seed activation: inoculate the cryopreserved Pseudomonas meningitidis on LB solid culture medium at 28-32°C for 24 hours to obtain activated seeds;

[0018] Step 2, liquid seed culture: from the activated seeds obtained in step 1, a single colony is picked and inoculated into LB liquid culture medium, and cultured at 28-32°C, pH 7.0-7.2, and 180 rpm for 24 hours to obtain seed liquid;

[0019] Step 3, fermentation culture: inoculate the seed solution obtained in step 2 into beef extract peptone culture medium at an inoculum of 1%, culture at 28-32° C., pH 7.0-7.2, and 180-220 rpm for 24-72 hours.

[0020] In another aspect, the present invention provides a bacterial agent containing the Pseudomonas mandelii FD-2.

[0021] Furthermore, the bacterial agent of Pseudomonas mandelii FD-2 contains Pseudomonas mandelii cells or Pseudomonas mandelii fermentation broth or a mixture of Pseudomonas mandelii cells and Pseudomonas mandelii fermentation broth.

[0022] According to the actual application needs, the bacterial agent of Pseudomonas mandelii FD-2 provided by the present invention can be prepared into a preparation dosage form that is convenient for agricultural application according to methods known to those skilled in the art, such as one of the dosage forms such as freeze-dried powder, suspension, wettable powder, water-dispersible granules, etc. It is easy for those skilled in the art to know that in addition to containing active ingredients, the preparation system also contains auxiliary agents required for preparation. The auxiliary agent can be selected from one or more of a solvent, an emulsifier, a wetting agent, a stabilizer, a dispersant, a thickener, a pH regulator, a defoamer, an antifreeze agent, a filler, etc. The auxiliary agents described in the present invention can all be known substances, such as various auxiliary agents commonly used in bacterial agents, which can be changed according to different situations and are not particularly limited.

[0023] Furthermore, the bacterial agent of Pseudomonas mandelii FD-2 contains Pseudomonas mandelii with a preservation number of CGMCC NO.30836.

[0024] Furthermore, the bacterial agent of the Pseudomonas mandelii or the Pseudomonas mandelii FD-2 is used to promote plant growth or soil nitrogen fixation.

[0025] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0026] The Pseudomonas mandelii FD-2 of the present invention is isolated by the inventor from the soil in the wolfberry field in Ningxia, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC NO. 30836. It is verified by experiments that the Pseudomonas mandelii FD-2 has significant characteristics of nitrogen fixation, inorganic phosphorus dissolution, and auxin secretion, can effectively improve soil fertility, improve soil microecological environment, promote plant growth, effectively reduce the use of chemical fertilizers, promote the green development of agriculture, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the phylogenetic tree of Pseudomonas mandelii FD-2.

[0028] Figure 2 This is a diagram showing the verification results of the nitrogen fixation effect of Pseudomonas mandelii FD-2.

[0029] Figure 3 This is a diagram showing the verification results of the inorganic phosphorus dissolution effect of Pseudomonas mandelii FD-2.

[0030] Figure 4 The results of qualitative determination of IAA production capacity of Pseudomonas mandelii FD-2F. CK+ represents the positive control, CK- represents the negative control, and from left to right represent the positive control group, negative control group, and FD-2 treatment group.

[0031] Figure 5 is the IAA standard curve. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is described below in conjunction with embodiments; however, the present invention is not limited to the following embodiments.

[0033] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0034] The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0035] Example 1

[0036] This example is the isolation and identification of Pseudomonas mandelii FD-2.

[0037] Isolation of Pseudomonas mandelii FD-2:

[0038] Take 1g of soil collected from the wolfberry field in Ningxia, put it into a sterile centrifuge tube, add 9mL of sterile water, put the centrifuge tube into a shaker at 28℃ and 180rpm for 30min, mix well and let it stand, take 1mL of the supernatant into a test tube, and then add 9mL of sterile water. -1 , 10 -2 , 10 -3 , 10 -4100 μL of soil dilutions of different concentrations were taken and spread on beef extract peptone medium for culture. When the bacteria just grew out, the bacteria were picked and repeatedly streaked to obtain a single strain, namely Pseudomonas mandelii FD-2.

[0039] Identification of Pseudomonas mandelii FD-2:

[0040] The Pseudomonas mandelii FD-2 isolated above was identified according to the following steps.

[0041] 1) Inoculate Pseudomonas mandelii FD-2 strain into beef extract peptone medium and culture on a shaking platform at 28°C and 180 rpm for 24 to 48 hours.

[0042] 2) After centrifugation, a small amount of Pseudomonas mandelii FD-2 bacteria was taken, and genomic DNA of Pseudomonas mandelii FD-2 bacteria was extracted using a bacterial genomic DNA extraction kit. The extraction steps were referred to the instruction manual of the bacterial genomic DNA extraction kit.

[0043] 3) Using the genomic DNA of Pseudomonas mandelii FD-2 as a template, PCR amplification was performed using universal primers 27F and 1492R. The primer sequences are as follows:

[0044] 27F: 5'-AGAGTTTGATCCTGGTCAGAACGAACGCT-3';

[0045] 1492R: 5'-TACGGCTACCTTGTTACGACTTCACCC-3'.

[0046] PCR amplification reaction system (50 μL): 5× buffer 10 μL; dNTP 4 μL; upstream primer 27F (10 μM) 1 μL; downstream primer 1492R (10 μM) 1 μL; Prime STAR 0.5 μL; template (1 μM) 1 μL; ddH2O 32.5 μL.

[0047] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min, for 30 cycles; and extension at 72°C for 5 min.

[0048] 4) The amplified product was sequenced (Beijing Qingke Biotechnology Co., Ltd.), and the nucleotide sequence of the 16S rRNA gene of Pseudomonas meningitidis is shown in SEQ ID NO: 1. The specific sequence is as follows:

[0049]

[0050] 5) The obtained sequences were compared with the NCBI nucleic acid database using BLAST for homology analysis, and some strains with high similarity were selected for phylogenetic analysis with Pseudomonas mandelii FD-2 strain abbreviation (FD-2). The phylogenetic tree was constructed using the neighbor-joining method in MEGA X software. The phylogenetic tree is shown in Figure 1 According to the homology and phylogenetic tree information, FD-2 was identified as Pseudomonas mandelii.

[0051] Example 2

[0052] This example is a test of the nitrogen fixation ability of Pseudomonas mandelii FD-2.

[0053] Medium preparation:

[0054] Ashby nitrogen-free medium: mannitol 10.0 g, potassium dihydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, sodium chloride 0.2 g, calcium sulfate dihydrate 0.1 g, calcium carbonate 5.0 g, agar 18-20 g, 0.5% Congo red solution 10 mL, distilled water 1000 mL, pH adjusted to 7.0, and sterilized by high-pressure steam at 121°C for 20 min.

[0055] LB liquid culture medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, 1000 mL of distilled water, pH 7.0-7.2, sterilized by high-pressure steam at 121°C for 20 min.

[0056] LB solid medium (FD-2 solid medium): 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, 20.0 g of agar, 1000 mL of distilled water, pH 7.0-7.2, sterilize with high-pressure steam at 121°C for 20 min, pour the medium into a plate before it cools down to prepare LB solid medium.

[0057] The nitrogen fixation ability of Pseudomonas mandelii FD-2 was detected by agar block confrontation method.

[0058] Use a sterilized bamboo stick to scrape the bacterial lawn of the test strain Pseudomonas mandelii FD-2 and inoculate it into the Ashby nitrogen-free medium. Then put it upside down in a 28℃ constant temperature incubator for 3 to 7 days to observe whether there is a nitrogen-fixing transparent circle at the inoculation site. After the transparent circle is formed, the nitrogen-fixing ability is tested by agar block confrontation. Use the end of a sterilized 1 ml pipette tip to punch holes on the solid medium of FD-2 to make agar truffle cakes of equal diameters, and then inoculate them into the Ashby nitrogen-free medium, with 4 replicates for each medium. Cultivate in a 28℃ constant temperature incubator for 3 to 7 days, record the diameter D of the transparent circle, and record the diameter d of the agar truffle cake. The diameter measurement method uses the cross method, and the calculation method is: D / d.

[0059] The test results are as follows Figure 2 As shown: strain FD-2 forms a transparent zone on Ashby nitrogen-free medium, indicating that it has the ability to fix nitrogen, and the D / d of strain FD-2 is 1.27±0.046, indicating that Pseudomonas mandelii FD-2 has a strong nitrogen fixation ability.

[0060] Example 3

[0061] This example is a test for detecting the ability of Pseudomonas mandelii FD-2 to dissolve inorganic phosphorus.

[0062] Medium preparation:

[0063] NBRIP inorganic phosphorus medium: sucrose (glucose) 10.0 g, calcium phosphate 5.0 g, magnesium chloride 5.0 g, magnesium sulfate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, agar 18-20 g, distilled water 1000 mL, pH adjusted to 7.0, and sterilized by high-pressure steam at 115°C for 30 min.

[0064] The agar block confrontation method was used to test the ability of Pseudomonas mandelii FD-2 to solubilize inorganic phosphorus.

[0065] Use a sterilized bamboo stick to scrape the bacterial lawn of the test strain Pseudomonas mandelii FD-2 (abbreviated as FD-2) and inoculate it in NBRIP inorganic phosphorus medium, then put it upside down in a 28°C constant temperature incubator for 3 to 7 days to observe whether there is a phosphorus dissolving circle at the inoculation site. After the transparent circle is formed, the inorganic phosphorus dissolving ability is tested by agar block confrontation. Use the sterilized 1 ml gun tip end to punch holes on the solid medium of FD-2 (using the same solid medium of FD-2 as in Example 2) to make agar truffle cakes with equal diameters, and then inoculate them in NBRIP inorganic phosphorus medium, with 4 replicates for each medium. Cultivate in a 28°C constant temperature incubator for 3 to 7 days, record the diameter D of the transparent circle, record the diameter d of the agar truffle cake, and use the cross method to measure the diameter to calculate D / d.

[0066] Test results such as Figure 3 As shown: strain FD-2 can form a phosphate-dissolving circle on NBRIP inorganic phosphorus medium, indicating that it can dissolve inorganic phosphorus, and the D / d of strain FD-2 is 1.22±0.05, indicating that Pseudomonas mandeliiF-D-2 can dissolve inorganic phosphorus.

[0067] Example 4

[0068] This example is a test for detecting the secretion performance of IAA by Pseudomonas mandelii FD-2.

[0069] Preparation of Salkowski reagent colorimetric solution and culture medium:

[0070] Salkowski reagent colorimetric solution: 150mL H2SO4, 250mL ddH2O, 7.5mL 0.5M FeCl3.

[0071] Kings medium: 20.0 g of tryptone, 1.50 g of dipotassium hydrogen phosphate, 1.50 g of magnesium sulfate, 1000 mL of distilled water, pH adjusted to 7.2, and sterilized by high-pressure steam at 121°C for 20 min.

[0072] Qualitative test of IAA secretion by Pseudomonas mandelii FD-2.

[0073] (1) Take the Pseudomonas mandelii FD-2 (FD-2) strain frozen in a -80°C glycerol tube and inoculate it into 20 mL LB liquid medium at a 1wt% inoculum. Grow the culture medium in a shaker at 180 rpm and 28°C until the OD value of the culture medium reaches 0. 600 It is about 0.8.

[0074] (2) The activated bacterial solution was inoculated into Kings medium containing tryptophan at a final concentration of 0.2 g / L at a 1 wt % inoculum. Three replicates were made for each strain and cultured in a shaker at 180 rpm and 28° C. for 3 days.

[0075] (3) After the culture was completed, the fermentation broth was centrifuged at 12000 rpm for 10 min. 800 μL of the supernatant after centrifugation was placed on a white porcelain plate, and an equal volume of Salkowski reagent colorimetric solution was added. The plate was covered with tin foil and allowed to react in the dark for 30 min. The positive control was 800 μL of 100 mg / L IAA standard solution and an equal volume of Salkowski reagent colorimetric solution. The negative control was 800 μL of Kings medium and an equal volume of Salkowski reagent colorimetric solution.

[0076] (4) Result determination: a positive result is displayed as pink, indicating that the strain can secrete IAA. The darker the pink color, the stronger the ability of the strain to secrete IAA. A negative result is displayed in the same color as the control, which is the color of the culture medium, indicating that the strain does not have the ability to secrete IAA.

[0077] The test results are as follows Figure 4 As shown, the results showed that Pseudomonas mandelii FD-2 had a strong IAA secretion ability.

[0078] IAA standard was purchased from Beijing Solebow Technology Co., Ltd., CAS#: 87-51-4.

[0079] Quantitative test of the ability of Pseudomonas mandelii FD-2 to secrete IAA.

[0080] (1) Prepare an IAA standard curve: Accurately weigh 0.005 g of IAA, dissolve it in a small amount of ethanol, and dilute to 50 mL with deionized water to prepare an IAA standard stock solution with a concentration of 100 mg / L. Accurately measure a certain amount of the standard stock solution into a 10 mL volumetric flask, dilute to the scale with deionized water, and prepare IAA standard solutions with concentrations of 0, 5, 10, 20, 30, and 40 mg / L, respectively, and store in a dark place. Take 2 mL of the standard solution of each concentration, add an equal volume of Salkowski reagent colorimetric solution, and protect from light for 30 minutes. Use a UV-visible spectrophotometer to detect the absorbance at 530 nm, adjust the value to zero with the 0 mg / L standard solution, and use the IAA standard concentration as the horizontal axis and the absorbance value as the vertical axis to draw the IAA standard curve, as shown below: Figure 5 shown.

[0081] (2) Pseudomonas mandelii FD-2 was inoculated into Kings liquid medium containing tryptophan and cultured in triplicate in a shaking incubator at 180 rpm and 28°C for 3 days.

[0082] (3) After 3 days of cultivation, the fermentation broth was centrifuged at 12,000 rpm for 10 min. 2 mL of the supernatant was placed in a glass test tube, 2 mL of Salkowski reagent was added, and the tube was covered with tin foil to avoid light and allowed to react for 30 min.

[0083] (4) After the reaction, the absorbance at 530 nm was measured using an ultraviolet spectrophotometer, and the IAA concentration in the supernatant of Pseudomonas mandelii FD-2 was calculated based on the IAA standard curve. The quantitative determination result of IAA production by Pseudomonas mandelii FD-2 was 31.67±1.34 mg / L.

[0084] In summary, the results of qualitative and quantitative tests showed that Pseudomonas mandelii FD-2 had a strong IAA secretion ability.

[0085] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A Pseudomonas meningitidis, characterized in that The Pseudomonas meningitidis is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC NO.30836.

2. The Pseudomonas meningitidis according to claim 1, characterized in that The 16S rRNA sequence of Pseudomonas meningitidis is shown in SEQ ID NO:

1.

3. The Pseudomonas meningitidis according to claim 1, characterized in that The Pseudomonas meningitidis can secrete indoleacetic acid.

4. The Pseudomonas meningitidis according to claim 1, characterized in that The Pseudomonas meningitidis can fix nitrogen and dissolve inorganic phosphorus.

5. The method for preparing Pseudomonas meningitidis according to claim 1, characterized in that: The steps include: Step 1: Seed activation: inoculate the cryopreserved Pseudomonas meningitidis on LB solid culture medium at 28-32°C for 24 hours to obtain activated seeds; Step 2, liquid seed culture: from the activated seeds obtained in step 1, a single colony is picked and inoculated into LB liquid culture medium, and cultured at 28-32°C, pH 7.0-7.2, and 180 rpm for 24 hours to obtain seed liquid; Step 3, fermentation culture: the seed liquid obtained in step 2 is inoculated into beef extract peptone medium at an inoculum amount of 1 wt %, and cultured at 28-32° C., pH 7.0-7.2, and 180-220 rpm for 24-72 hours.

6. A bacterial agent containing the Pseudomonas meningitidis according to claim 1, characterized in that: The bacterial agent contains Pseudomonas meningitii, Pseudomonas meningitii fermentation liquid, or a mixture of Pseudomonas meningitii and Pseudomonas meningitii fermentation liquid.

7. The bacterial agent according to claim 6, characterized in that The deposit number of the Pseudomonas meningitidis is CGMCCNO.30836.

8. The bacterial agent according to any one of claims 6 to 7, characterized in that The bacterial agent can be prepared into any industrially acceptable dosage form.

9. Use of the Pseudomonas meningitidis according to any one of claims 1 to 4 or the bacterial agent according to any one of claims 6 to 7 in promoting plant growth.

10. Use of the Pseudomonas meningitidis according to any one of claims 1 to 4 or the bacterial agent according to any one of claims 6 to 7 in soil nitrogen fixation.

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