Pseudomonas W5-1, microbial agent and application thereof

Abstract

The invention discloses pseudomonas W5-1, a microbial agent and application of the microbial agent, and belongs to the technical field of environmental microorganisms. In order to solve the technical problems of nitrogen loss and reduction of the fertilizer efficiency of the organic fertilizer caused by the fact that nitrogen in the organic fertilizer is formed by nitrate in compost fertilization and enters a denitrification path in soil in the prior art, the invention provides the pseudomonas W5-1, the microbial agent and the application of the pseudomonas W5-1, the microbial agent and the application of the pseudomonas W5-1, and the preservation number of the strain of the pseudomonas W5-1 is CGMCC (China General Microbiological Culture Collection Center) NO.31820; the pseudomonas W5-1 provided by the invention has a function of remarkably promoting nrfA gene expression, and further promotes generation of a DNRA process, so that NO2 <-> in compost fertility soil is promoted to be converted into NH4 < + >, and decomposition of soil organic matters and conversion of nitrogen are promoted; the pseudomonas W5-1 or the microbial agent provided by the invention can be applied to nitrogen fixation, nitrogen conservation and improvement of the nitrogen content of compost fertility improvement soil.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and specifically relates to a pseudomonas, a microbial agent and applications thereof. Background Art

[0002] Scientific and reasonable soil management and fertilization can not only effectively improve the physical and chemical properties of the soil, but also efficiently utilize soil resources and promote the sustainable use of agriculture. The methods for fertilizing planting soil include: chemical fertilizers, pesticides and organic compost materials (organic fertilizers); the large-scale use of chemical fertilizers and pesticides will lead to pesticide residues, causing pollution to the soil, water and air, and have low safety; while organic compost materials have a good improvement effect on the soil, which not only reduces economic consumption, but also protects the environment. In agricultural soil, crops mainly use ammonium nitrogen (NH4 + -N and nitrate nitrogen NO3 - -N), but during farming the soil nitrogen content often cannot meet the demand, and the greenhouse effect needs to be considered.

[0003] Organic fertilizer is an environmentally friendly fertilizer rich in various nutrients, but NO3 - -N is easily leached, and the N2O generated by the traditional denitrification pathway is the main source of greenhouse gases, and the final product N2 easily causes nitrogen loss; however, NO3 - -N can be reduced to another more bioavailable NH4 + -N, NH4 produced + Regenerate NO2 through nitrification - and NO3 - This nitrogen conservation mechanism is the dissimilatory nitrate reduction to ammonium (DNRA) process; therefore, those skilled in the art are eager to develop a method to increase the DNRA rate in composting, promote the formation of ammonium nitrogen, and have a good nitrogen conservation effect in composting soil. Summary of the invention

[0004] The present invention aims to solve the technical problem in the prior art that nitrogen in organic fertilizer is lost and the fertilizer efficiency of organic fertilizer is reduced due to the formation of nitrate in composting and soil and the entry into denitrification pathway. A Pseudomonas W5-1, a microbial agent and the application thereof are provided.

[0005] One of the objects of the present invention is to provide a Pseudomonas W5-1, wherein the Pseudomonas W5-1 strain deposit number is CGMCC NO.31820, and the classification name is Pseudomonas sp., which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is September 2, 2024.

[0006] A second object of the present invention is to provide a microbial agent, which contains the above-mentioned Pseudomonas W5-1.

[0007] In a preferred embodiment of the present invention, the effective concentration of Pseudomonas W5-1 in the microbial agent is 3×10 8 CFU / mL.

[0008] The third object of the present invention is to provide the use of the above-mentioned Pseudomonas W5-1 or microbial agent in nitrogen fixation, nitrogen conservation, and increasing the nitrogen content of composting soil.

[0009] In a preferred embodiment of the present invention, the nitrogen content includes one or more of total nitrogen content, nitrate nitrogen content, nitrite nitrogen content, and inorganic nitrogen content.

[0010] Beneficial effects of the present invention:

[0011] Dissimilatory reduction of nitrate to ammonium (DNRA) is different from denitrification. DNRA is the process of converting NO3 - , reduced to NH4 + Instead of N2, NH4 is produced + , regenerates NO2 through nitrification - and NO3 - , and the DNRA process is performed again. The present invention provides a Pseudomonas W5-1, a microbial agent and an application thereof, wherein the effective concentration of Pseudomonas W5-1 in the microbial agent is 3×10 8 The present invention proves that the Pseudomonas W5-1 has the function of significantly promoting the expression of nrfA functional gene, thereby promoting the occurrence of DNRA process, thereby promoting NO2 - Converted to NH4 + .

[0012] The present invention greatly reduces nitrate leaching through the DNRA process, reduces the denitrification rate and N2O emissions, improves plant nitrogen utilization and alleviates nitrogen pollution, reduces nitrogen loss, increases the nitrogen content of compost fertilizing soil, and increases the utilization rate of organic fertilizers and soil.

[0013] Before tilling the soil in spring, organic fertilizer is used to fertilize the soil, and then the Pseudomonas W5-1 microbial fertilizer provided by the present invention is evenly sprayed on the field. Since the Pseudomonas W5-1 provided by the present invention has the function of increasing the expression of the nrfA functional gene, thereby promoting the occurrence of the DNRA process, the decomposition of soil organic matter and the conversion of nitrogen can be promoted. The Pseudomonas W5-1 or microbial agent provided by the present invention can be used for nitrogen fixation, nitrogen conservation, and increasing the nitrogen content of compost fertilization soil.

[0014] [Biological Deposit Information]: The deposit number of Pseudomonas W5-1 is CGMCC NO.31820, and the classification name is Pseudomonas sp. It is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 2, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The 16S rRNA sequence alignment result diagram in Example 1;

[0016] Figure 2 It is a growth curve diagram of the mixed bacterial flora in Example 1;

[0017] Figure 3 This is an agarose gel electrophoresis diagram of nrfA gene amplification in Example 1;

[0018] Figure 4 The graph is a graph of nitrate nitrogen, nitrite nitrogen and ammonium nitrogen contents after different culture times of the DNRA bacterial solution in Example 1; the ordinate Inorganic nitrogen content is the inorganic nitrogen content, and the abscissa incubation time is the incubation time;

[0019] Figure 5 The figure is a graph showing the expression of the nrfA functional gene in different black soil treatment groups in Example 3; the abscissa Relativeabundance of nrfA in black soil is the relative abundance of nrfA in black soil;

[0020] Figure 6 This is a diagram showing the expression of the nrfA functional gene in different red soil treatment groups in Example 3. The abscissa Relative abundance of nrfA in black soil is the relative abundance of nrfA in red soil. DETAILED DESCRIPTION

[0021] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0022] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0023] The Giltay liquid culture medium used in the following examples consists of liquid A and liquid B, wherein the composition of liquid A is: 1.0g KNO3, 5.0mL 1% BTB alcohol solution and 500mL deionized water; the composition of liquid B is: 8.5g sodium citrate, 1.0g KH2PO4, 1.0g MgSO4.7H2O, 0.05g FeCl3.6H20, 0.2g CaCl2.2H20, 500mL deionized water; after liquid A and liquid B are evenly mixed, the pH value is adjusted to 7.0 using 1mol / L NaOH, and sterilized at 121°C for 20min; the Giltay solid culture medium is based on the Giltay liquid culture medium and 1.5% (w / v) agar is added.

[0024] Example 1: Screening and identification of Pseudomonas sp. W5-1

[0025] In this embodiment, 1 g / 1 mL of sludge or sewage was placed in a Giltay liquid culture medium without adding BTB alcohol solution, and cultured in a shaker at 140 rpm and 30° C. for 72 hours to obtain an enriched bacterial solution; 1 mL of the above-mentioned enriched bacterial solution was placed in a newly configured Giltay liquid culture medium, and cultured in a shaker at 140 rpm and 30° C. for 72 hours. As the bacteria grew, the color of the culture medium changed from light green to blue. After repeated acclimation, the bacteria adapted to the culture conditions to obtain Pseudomonas W5-1 bacterial solution; the Pseudomonas W5-1 bacterial solution obtained above was passaged and purified for multiple times to obtain Pseudomonas W5-1 with the function of nitrate dissimilatory reduction to ammonium, and the strain was named W5-1.

[0026] In this example, 16S rRNA sequencing was performed on the Pseudomonas W5-1 isolated above, and homology detection and comparison were performed with the Pseudomonas in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi); the results are as follows: Figure 1 As shown, the Pseudomonas W5-1 16S rRNA sequence isolated and obtained in this example has a similarity of 99.69% with the first similar sequence alignment result, and the Pseudomonas W5-1 16S rRNA sequence information is uploaded to the NCBI database (GenBank: PP396893.1).

[0027] Effect experiment:

[0028] (1) Driven by microorganisms, it is converted into different nitrogen compounds through a series of redox reactions, including nitrification, denitrification, anaerobic ammonium oxidation, and nitrate reduction. However, NO3 generated after organic nitrogen mineralization - It is easy to be leached. The N2O generated by the traditional denitrification pathway is the main source of greenhouse gases, and the final product N2 is easy to cause nitrogen loss. Among them, the dissimilatory reduction of nitrate to ammonium (DNRA) is different from denitrification. DNRA converts NO3 - , reduced to NH4 + Instead of N2, NH4 is produced + , regenerates NO2 through nitrification - and NO3 - , and is utilized again by denitrification, DNRA and anaerobic ammonium oxidizing bacteria, indicating that DNRA is an important internal connection process of the nitrogen cycle.

[0029] The DNRA bacterial solution obtained in this embodiment was used for streaking and plate coating. The DNRA bacterial solution can grow on Giltay plates using nitrate as a nitrogen source and sodium citrate as a carbon source, and the nitrate is reduced to solid ammonium. The above-mentioned plate was stored at a low temperature of 4°C for ≤15 days and observed.

[0030] As a result, crystals were observed to precipitate on the plate. The crystals were needle-shaped in three-dimensional space and gathered in the middle of the agar layer, looking like "snowflakes". When Nessler's reagent was dripped onto the crystals, the crystals turned yellow, and it was preliminarily judged that the precipitated crystals were ammonium salts, indicating that the DNRA mixed bacterial solution obtained in this example had the ability to perform dissimilatory reduction of nitrate to ammonium (DNRA), which is different from denitrification. The growth curve of the mixed bacterial community obtained in this example is shown in FIG. Figure 2 As shown in the figure, it can be seen that the mixed bacterial community grows rapidly, reaching the logarithmic phase at 12 hours, reaching the maximum value at 48 hours and entering the decline phase.

[0031] (2) Functional identification of DNRA bacterial solution

[0032] The first step of the DNRA process and the denitrification process is catalyzed by nitrate reductase (Nar) to convert NO3 - Reduction to NO2 - The functional genes encoding Nar are narG and napA genes; the DNRA process and the second step of the denitrification process are different in the following ways: the second step of the denitrification process is the conversion of NO2 - Finally, it is reduced to NO, which is then reduced to N2O by nitrous oxide reductase (Nor), and finally reduced to N2 by nitrous oxide reductase (Nos); the second step of the DNRA process is that Nir converts NO2 - Direct reduction to NH4 + It can be seen that the role of Nir in the denitrification process is completely different from that in the DNRA process. The functional genes encoding Nir in the denitrification process are nirS and nirK, and the functional genes encoding Nir in the DNRA process are nirB, nirC and cysG; the Nir in the fermentation DNRA process is cytochrome C nitrite reductase (pentahemecytoclrome C nitrite reductase, nrfA), so the nrfA gene is considered to be involved in the conversion of NO2 - Converted to NH4 + marker genes.

[0033] Therefore, the DNRA bacterial liquid DNA obtained in this example was extracted as a template to perform PCR amplification on the nrfA gene. In order to enhance the versatility of the primers, degenerate primers were designed in this example. The upstream primer nucleotide sequence of the primers was shown in SEQ ID NO.1: CARTGYCAYGTBGARTA, and the downstream primer nucleotide sequence was shown in SEQ ID NO.2: TWNGGCATRTGRCARTC, wherein in the nucleotide sequence, R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, W=A / T, H=A / C / T, B=C / G / T, V=A / C / G, D=A / G / T, and N=A / C / G / T; the PCR amplification system was: 2xTaq PCR Mix 5.0μL, DNA template 1.0μL, total reaction volume 10.0μL; primer usage 0.3-0.5μL; amplification program: 5min pre-denaturation -95℃, 30s denaturation -95℃, 30s annealing -49℃, 30s extension -72℃, 72℃-7min.

[0034] The results are as follows Figure 3 As shown, this example successfully amplified and obtained a 250 bp nrfA gene amplification product.

[0035] (3) DNRA process verification

[0036] The DNRA bacterial solution obtained in this example was inoculated into the Giltay liquid medium without the addition of BTB alcohol solution, and the NO3 in the Giltay liquid medium after culturing for 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 96 h was measured. - 、NO2 - NH4 + The content was measured, and the DNRA bacterial solution was sampled regularly after culture. 25 mL of the bacterial solution was measured and placed in a centrifuge tube. The tube was centrifuged at 10,000 rpm for 1 min to obtain the DNRA bacterial solution supernatant.

[0037] The nitrate nitrogen was measured by ultraviolet spectrophotometry (HJ / T 346-2007), the nitrite nitrogen was determined by N-(1-naphthyl)-ethylenediamine photometry (GB 7493-87), and the ammonium nitrogen content was determined by Nessler's reagent photometry (HJ 535-2009).

[0038] The specific steps include:

[0039] S1: Add 250 μL of zinc sulfate solution (10% zinc sulfate solution: weigh 10 g ZnSO4, add deionized water to dissolve, and dilute to 100 mL) to the supernatant of the DNRA bacterial culture after culturing for 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 96 h, respectively, and shake well; use 5M NaOH (5 mol / L NaOH: weigh 20 g NaOH, add deionized water while stirring to dissolve, and dilute to 100 mL) to adjust the pH to 7; after standing, take 2 mL of the supernatant into a 50 mL colorimetric tube, add water to the mark, and add 1 mL of 1.0 mol / L HCI solution (1.0 mol / L HCI: weigh 8.3 mL HCl, add it to deionized water, and then dilute to 100mL) and 0.1mL0.8% sulfamic acid solution (0.8% sulfamic acid solution: weigh 0.8g sulfamic acid, add deionized water to dissolve, dilute to 100mL, and store in a 4°C refrigerator away from light) are added to a colorimetric tube; a quartz cuvette with an optical path of 10mm is used to perform dual-wavelength measurement at 220nm and 275nm, and 50mL of deionized water plus 1mL of 1.0mol / L hydrochloric acid solution is used as a reference, and nitrate nitrogen is determined by ultraviolet spectrophotometry, and the nitrate nitrogen content is:

[0040] A 校 =A 220 -2A 275 ;

[0041] S2: Determine nitrite nitrogen by N-(1-naphthyl)-ethylenediamine spectrophotometry. If the pH of the supernatant of the DNRA bacterial solution after culturing for 0h, 12h, 24h, 36h, 48h, 60h, 72h, and 96h is ≥11, add 1 drop of phenolphthalein indicator and add (1+9) phosphoric acid solution while stirring until the red color disappears. If there is color and suspended matter in the supernatant, add 2mL of alumina suspension to every 100mL of DNRA bacterial solution supernatant, stir, let stand, filter and discard 25mL of the primary filtrate; take 5mL of the DNRA bacterial solution supernatant after the primary filtration in a 50mL colorimetric tube, add water to the mark, and add 1mL of color developer (color developer: 250mL of deionized water and 50mL 1.7g / mL phosphoric acid is mixed evenly, then 20g of p-aminobenzenesulfonamide is added, and then 1g of N-(1-naphthyl)-ethylenediamine hydrochloride is dissolved in the above mixed solution, the volume is fixed to 500mL, and stored in a brown bottle at 2-4℃. The solution is toxic). After standing for 20 minutes, it is placed at 540nm for colorimetry, and deionized water is used instead of water sample for full blank determination as a reference;

[0042] S3: Determine ammonium nitrogen by Nessler's reagent spectrophotometry. Add 250 μL of zinc sulfate solution (10%) to the supernatant of DNRA bacterial solution after culture for 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 96 h, respectively, and shake well. Use 5 mol / L NaOH to adjust the pH to 10.5. After standing, take 200 μL of the supernatant into a 50 mL colorimetric tube, add deionized water to the mark, add 1 mL of potassium sodium tartrate solution (potassium sodium tartrate: 50 g dissolved in 100 mL, heated and boiled to remove ammonia, and fixed to 100 mL after cooling) and mix well. Add 1.5 mL of Nessler's reagent (Nessler's reagent: 16 g NaOH dissolved in 50 mL water, cooled to room temperature; 7 g KI, 10 g HgI was dissolved in 20-30 mL of water, and the above-prepared NaOH was added to make the volume 100 mL), and mixed; after standing for 10 minutes, the solution was placed at 420 nm for colorimetry, and deionized water was used instead of the water sample as a blank for the entire measurement.

[0043] The results are as follows Figure 4 As shown in the figure, the nitrate nitrogen (NO3 - ) concentration is always accompanied by the decrease in the intermediate product nitrite nitrogen (NO2 - ) accumulation and ammonium nitrogen (NH4 + ) concentration increased; at 96h of culture, the concentration of ammonium nitrogen was close to 40mg / L, while the concentration of nitrate nitrogen was close to 0mg / L; NO3 - There is an upward trend at 48h, and the product NH4 +There was also a downward trend for a period of time after 12 hours, which suggests that humans, due to the complexity of the bacterial community system in the DNRA bacterial solution, have adopted a mutually beneficial symbiotic model in order to maintain the nutritional balance in this environment.

[0044] Example 2: Preparation of Pseudomonas W5-1 microbial agent

[0045] Pseudomonas W5-1 was placed in Giltay liquid medium without BTB alcohol solution for two passages, and then cultured in a shaker at 140 rpm and 30°C for 72 hours to obtain an enriched bacterial solution; 1 mL of the enriched bacterial solution was placed in a newly configured Giltay liquid medium, and cultured in a shaker at 140 rpm and 30°C for 72 hours. As the bacteria grew, the color of the culture medium changed from light green to blue. After repeated acclimation, a Pseudomonas W5-1 microbial inoculant was obtained, and the effective concentration of Pseudomonas W5-1 in the microbial inoculant was 3×10 8 CFU / mL.

[0046] Example 3: Application of Pseudomonas W5-1 microbial agent in nitrogen fixation, nitrogen conservation, and increasing nitrogen content in composting soil.

[0047] Functional microorganisms promote the expression of nrfA functional gene under simulated fertilization conditions:

[0048] In this embodiment, 2.3 kg of compost (pig manure + straw decomposed fertilizer) and soil mixture were put into a cylindrical plastic (diameter 15 cm and length 20 cm), and the upper and lower ends of the cylinder were respectively designed with air inlets and air outlets and then sealed, and the soil was replenished with water according to the optimal water holding rate during the whole process; during the culturing process, samples were taken at 0d, 3d, 10d, 20d, 28d, 35d, 42d, and 50d of culturing, and the materials were evenly mixed before each sampling, and the quartering method was used for sampling. A total of 12 different fertilization simulation treatments were set, and the added Pseudomonas W5-1 microbial agent was prepared in Example 2, as shown in Table 1.

[0049] Table 1

[0050] Serial number name Processing Type 1. BS-CK+CM Black soil + medium without bacterial agent 2. BS-CK Black soil 3. BS-F10% Black soil + 10% compost 4. BS-F30% Black soil + 30% compost 5. BS-F10%+B Black soil + 10% compost + microbial agent 6. BS-F30%+B Black soil + 30% compost + microbial agent 7. RS-CK+CM Red soil + medium without bacterial agent 8. RS-CK Red soil 9. RS-F10% Red soil + 10% compost 10. RS-F30% Red soil + 30% compost 11. RS-F10%+B Red soil + 10% compost + microbial agent 12. RS-F30%+B Red soil + 30% compost + microbial agent

[0051] In this example, the expression of nrfA functional gene was detected in the samples of the above-mentioned different treatment groups, and the results are as follows: Figure 5As shown, compared with the black soil + compost material treatment group without adding microbial agents, the expression of nrfA functional gene in the treatment group with the microbial agent prepared in Example 2 was significantly increased. When the mass proportion of compost material was 30%, the expression of nrfA functional gene was the highest at 28 days of culture, and the expression of nrfA functional gene decreased significantly with the increase of culture time; when the mass proportion of compost material was 10%, the expression of nrfA functional gene was high at 10 days and 50 days of culture, and the expression of nrfA functional gene decreased at 28 days of culture.

[0052] like Figure 6 As shown, compared with the red soil + compost material treatment group without the addition of microbial agents, in the red soil + 10% compost material treatment group with the addition of the microbial agent prepared in Example 2, the expression level of the nrfA functional gene was significantly increased after 28 days of cultivation, and the expression level of the nrfA functional gene was significantly reduced with the increase of the cultivation time.

[0053] It can be seen that the expression of nrfA functional gene is affected by a variety of environmental factors. The expression of nrfA functional gene is best in the red soil + 10% compost material and black soil + 10% compost material, as well as the treatment group with the microbial agent prepared in Example 2. The nrfA functional gene promotes the conversion of NO2 into - Converted to NH4 + The occurrence of nitrogen can be achieved, thereby achieving the purpose of nitrogen fixation, nitrogen conservation, and increasing the nitrogen content of the compost fertilization soil.

[0054] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A Pseudomonas sp. W5-1, characterized in that The Pseudomonas W5-1 strain has a deposit number of CGMCCNO.31820 and is classified as Pseudomonas sp. It is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 2, 2024.

2. A microbial agent, characterized in that: The microbial agent contains the Pseudomonas W5-1 described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The effective concentration of Pseudomonas W5-1 in the microbial agent is 3×10 8 CFU / mL.

4. Use of the Pseudomonas W5-1 described in claim 1 or the microbial agent described in any one of claims 2 to 3 in nitrogen fixation, nitrogen conservation, and increasing the nitrogen content of composting soil.

5. The use according to claim 4, characterized in that: The nitrogen content includes one or more of total nitrogen content, nitrate nitrogen content, nitrite nitrogen content, and inorganic nitrogen content.

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