Pseudomonas koreensis WF3 and saline-alkali soil microbial fertilizer, and preparation method and application thereof

Abstract

The application provides a pseudomonas koreensis WF3 and a saline-alkali soil microbial fertilizer, a preparation method and application thereof, and belongs to the technical field of biological composting.The preparation method of the saline-alkali soil microbial fertilizer provided by the application comprises the following steps: (1) configuring a fertilizer carrier comprising matured organic fertilizer, humic acid, attapulgite powder and polyglutamic acid; (2) inoculating the pseudomonas koreensis WF3 into the fertilizer carrier, and carrying out sealed fermentation to obtain the saline-alkali soil microbial fertilizer.The pseudomonas koreensis WF3 has the abilities of producing extracellular polysaccharides and organic acids, and can survive in a strong saline-alkali environment; the fertilizer carrier is fermented by the pseudomonas koreensis WF3; the prepared microbial organic fertilizer can effectively improve the soil organic carbon, total nitrogen and water-stable large aggregate (>2.5mm) content, increase the corn yield, reduce the soil pH and total salt content, and has a remarkable improvement effect on the saline-alkali soil.

Description

Technical Field

[0001] This invention relates to the field of biocomposting technology, and in particular to a Pseudomonas krusei WF3 microbial fertilizer for saline-alkali land, its preparation method and application. Background Technology

[0002] High salinity and alkalinity soils inhibit crop growth. A small number of crops can survive in these soils by secreting organic acids, but the ability of crops to secrete organic acids varies; not all crops can secrete organic acids to combat saline-alkali environments. Microbial strains that produce organic acids can neutralize alkaline compounds in the soil, lowering soil pH and improving soil physicochemical properties. Some of the organic acids secreted by microorganisms can also serve as nutrients for the growth and metabolism of crops or other microorganisms, increasing the richness of the soil microbial community, improving the rate of soil nutrient metabolism, promoting crop growth, and increasing crop yield. Microbial secreted exopolysaccharides (EPS) are large molecules containing numerous negatively charged functional groups. They can promote the formation of water-stable aggregates in the soil through various physical forces and cementation processes, improving soil physical structure and enhancing the soil's water retention capacity. EPS can also enrich soil nutrient types, improve crop resistance, and promote crop growth. Currently, commonly used EPS-producing microorganisms in agricultural production mainly include Bacillus and Pseudomonas. Pseudomonas ), genus *Cymbidium* ( Ensifer ), nitrogen-fixing bacteria ( Azotobacter Rhizobium ( ) Rhizobium )wait.

[0003] Microbial fertilizers prepared through microbial fermentation are a safer and more environmentally friendly option for improving saline-alkali land, benefiting both the environment and agricultural practices. Microbial fertilizers containing functional bacteria that produce extracellular polysaccharides and organic acids can minimize the impact of salt stress on crop growth, improve soil fertility, increase crop yields, and reduce chemical fertilizer application, playing a crucial role in achieving sustainable agricultural development. Therefore, developing microbial fertilizers with functional bacteria producing extracellular polysaccharides and organic acids is an effective way to improve saline-alkali land.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing microbial fertilizer for saline-alkali land, comprising the following steps:

[0007] (1) The fertilizer carrier consists of decomposed organic fertilizer, humic acid, attapulgite powder and polyglutamic acid;

[0008] (2) Inoculate the fertilizer carrier with Pseudomonas krusei ( Pseudomonas knackmussii WF3, sealed fermentation, yields microbial fertilizer for saline-alkali land;

[0009] The Pseudomonas kirchii ( Pseudomonas knackmussii WF3 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:65562, and deposited on November 28, 2024.

[0010] Preferably, the fertilizer carrier comprises the following raw materials in parts by weight: 700-800 parts of well-rotted organic fertilizer, 100-150 parts of humic acid, 100-145 parts of attapulgite powder, and 3-5 parts of polyglutamic acid; the moisture content of the fertilizer carrier is ≤25%.

[0011] Preferably, the decomposed organic fertilizer has a particle size of 120-200 mesh, a moisture content of <25 wt.%, and an organic matter content of ≥45%; the humic acid has a particle size of 100-120 mesh, a moisture content of <25 wt.%, and an organic matter content of ≥65%; the attapulgite has a particle size of 100-120 mesh, a moisture content of <25 wt.%; and, on a dry basis, the polyglutamic acid has an effective content of ≥20%, a moisture content of <6 wt.%, and a molecular weight of 100-500 kDa.

[0012] Preferably, the inoculum amount of *Pseudomonas krusei* WF3 in the fertilizer carrier is 8-10% by mass, and the bacterial concentration of *Pseudomonas krusei* WF3 is (5-10) × 10⁻⁶. 9 cfu / mL.

[0013] Preferably, the fermentation is carried out at 25~30℃ for 96~120 hours.

[0014] The present invention also provides a microbial fertilizer for saline-alkali land prepared by the preparation method described above.

[0015] Preferably, the effective viable count of *Pseudomonas krusei* WF3 in the microbial fertilizer for saline-alkali land is ≥1.0 × 10⁻⁶. 8 cfu / g, organic matter content ≥50%, pH 6.5~7.5.

[0016] This invention also provides a *Pseudomonas kuribsiella* strain used in the preparation method described above. Pseudomonas knackmussiiThe *Pseudomonas kirkinensis* WF3 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:65562, and deposited on November 28, 2024.

[0017] The present invention also provides the application of the aforementioned microbial fertilizer for saline-alkali land or the aforementioned Pseudomonas kirchwitz WF3 in improving the resistance of crops in saline-alkali land, promoting the growth of crops in saline-alkali land, and improving saline-alkali land soil.

[0018] Preferably, the crop in saline-alkali land is corn, and improving the resistance of the crop in saline-alkali land involves increasing the content of soluble sugars, proline, and malondialdehyde in corn seedlings under saline-alkali stress; promoting the growth of the crop in saline-alkali land involves increasing the germination rate of corn seeds, the fresh weight, dry weight, total root length, average root diameter, and number of root tips of corn seedlings, as well as increasing corn yield; improving the saline-alkali soil involves increasing the content of organic carbon, total nitrogen, and water-stable macroaggregates in the saline-alkali soil, and reducing the soil pH and total salt content.

[0019] This invention uses *Pseudomonas kuribsiella* WF3, which has the ability to produce extracellular polysaccharides and organic acids and can survive in strongly saline-alkali environments, to ferment fertilizer carriers. The resulting microbial organic fertilizer can effectively increase the content of soil organic carbon, total nitrogen, and water-stable macroaggregates (>2.5 mm), increase corn yield, and reduce soil pH and total salt content, showing significant improvement effects on saline-alkali land.

[0020] In addition, the *Pseudomonas krusei* WF3 provided by this invention can grow normally in a culture medium with pH ≥ 8.5 and sodium chloride content ≥ 10%. Experiments show that it can significantly improve the germination rate of maize seeds, fresh weight, dry weight, total root length, average root diameter and root tip number of maize seedlings under salt-alkali stress, and reduce the content of soluble sugar, proline and malondialdehyde in salt-stressed maize seedlings. Soluble sugar, proline and malondialdehyde are osmotic regulators. *Pseudomonas krusei* WF3 can help crops adapt to the salt-alkali stress environment and promote crop growth under salt stress by affecting the content of osmotic regulators in crops under salt-alkali stress. Attached Figure Description

[0021] Figure 1 The growth morphology of Pseudomonas kuribsiella pneumoniae WF3;

[0022] Figure 2 Microscopic morphology of Pseudomonas kuribda WF3 (scale bar in figure is 5 μm).

[0023] Figure 3 Phylogenetic tree of WF3;

[0024] Figure 4 The morphology of strain WF3 on aniline blue solid medium;

[0025] Figure 5 The clear zone of acid production by strain WF3 on Congo red medium;

[0026] Figure 6 The effects of strain WF3 on germination rate, fresh weight and dry weight of maize;

[0027] Figure 7 The effects of strain WF3 on root length, average root diameter, and number of root tips in maize seedlings;

[0028] Figure 8 The effect of strain WF3 on the soluble sugar content of maize seedlings;

[0029] Figure 9 The effect of strain WF3 on proline content in maize seedlings;

[0030] Figure 10 The effect of strain WF3 on malondialdehyde content in maize seedlings;

[0031] Figure 11 The impact of microbial fertilizers on soil organic carbon content;

[0032] Figure 12 The impact of microbial fertilizers on soil total nitrogen content;

[0033] Figure 13 The effect of microbial fertilizer on the content of large soil aggregates (>2.5 mm);

[0034] Figure 14 The effect of microbial fertilizers on soil pH;

[0035] Figure 15 The effect of microbial fertilizers on soil total salt content;

[0036] Figure 16 The impact of microbial fertilizers on corn yield.

[0037] Preservation Instructions

[0038] Pseudomonas kuribda ( Pseudomonas knackmussii WF3 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:65562, and deposited on November 28, 2024. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] The *Pseudomonas kuribsiella* provided by this invention ( Pseudomonas knackmussii WF3, derived from yak dung samples from Minlian Town, Minle County, Zhangye City, Gansu Province, is a Gram-negative bacillus. Its morphology and microstructure are as follows: Figure 1 , 2 As shown. Incubated at 30°C for 2 days on NA (nutrient agar) plates, the colonies were flat, opaque, with irregular edges, and grayish-white with a slight yellow tinge. Figure 1 Under a microscope, the bacteria appear rod-shaped, without spores or capsules, and are Gram-negative. Figure 2 ).

[0042] The physicochemical characteristics of Pseudomonas kuribda WF3 are shown in Table 1 below. Pseudomonas kuribda WF3 can produce catalase and oxidase, and can utilize glucose and fructose, but cannot utilize arabinose, mannitol, galactose, sucrose, maltose, xylose, trehalose and sorbitol. The optimal growth temperature is 41℃, and it cannot grow at low temperatures of 4℃ and high temperatures of 47℃.

[0043] Table 1 Physicochemical characteristics of Pseudomonas kurstii WF3

[0044]

[0045] Note: ("+" indicates a positive reaction, "-" indicates a negative reaction).

[0046] Pseudomonas kuribda WF3 ( Pseudomonas knackmussii Molecular identification results: The 16S rRNA gene sequence of *Pseudomonas kuriformis* WF3 was 1319 bp. Nucleotide homology comparison with already registered sequences in Genebank was performed using the Blast program. The 16S rRNA gene sequence of this bacterium... Pseudomonas knackmussii The homology with (Pseudomonas krusei) is 100%.

[0047] The 16S rRNA gene sequencing sequence (SEQ ID NO: 1) of *Pseudomonas kuriformis* WF3 is as follows:

[0048]

[0049] Using MEGA software, a phylogenetic tree was constructed based on the 16S rRNA gene sequence using the neighbor-joining method, such as... Figure 3 As shown. After 1000 repeated calculations, the node displays a Bootstrap value greater than 50%, and the superscript "T" indicates the type strain.

[0050] Aniline blue is a basic dye that can specifically bind to hydroxyl groups and other groups in polysaccharide molecules to form a blue complex. When a strain that produces extracellular polysaccharides grows on a plate containing aniline blue, the EPS it secretes will bind to the dye, forming a distinct blue halo or blue precipitate ring around the colony.

[0051] Plate tests using Congo red and aniline blue agar plates showed that *Pseudomonas kuribda* WF3 ( Pseudomonas knackmussii It has the ability to produce extracellular polysaccharides and organic acids, such as Figure 4 , Figure 5 As shown.

[0052] The total protein content in the cell extract was determined colorimetrically, and a standard curve of bovine serum albumin was plotted using bovine serum albumin as a standard. ACC deaminase activity was expressed as the amount of ACC deaminase catalyzed by each milligram of bacterial protease per hour to produce α-butanone, expressed in μmol / (mg·h). A blank control was included. The isolated and purified strain was inoculated into LB liquid medium containing L-tryptophan and cultured on a shaker for 24 h. 50 μL of the bacterial suspension was dropped onto a white ceramic plate, and an equal volume of Salkowski chromogenic solution was added for color development. An equal volume of IAA standard solution was added as a positive control. The plate was incubated at room temperature in the dark for 0.5 h; a red color indicated the production of IAA. The results showed that the *Pseudomonas kuribda* WF3 ( Pseudomonas knackmussii It can secrete ACC deaminase and IAA (indoleacetic acid) to promote crop growth, thus possessing growth-promoting abilities. Furthermore, experimental results also indicate that *Pseudomonas kurii* WF3 also has nitrogen-fixing capabilities. (See Table 2.)

[0053] Table 2. Evaluation of the growth-promoting function of strain WF3

[0054]

[0055] Note: NPA represents a culture medium for screening inorganic phosphorus bacteria; OPA represents a culture medium for screening organic phosphorus bacteria; NFb represents a culture medium for screening nitrogen-fixing bacteria. "+" indicates positive, and "-" indicates negative.

[0056] Example 2

[0057] The *Pseudomonas krusei* WF3 isolated and purified in Example 1 was inoculated into liquid culture medium until the effective viable count was ≥5 × 10⁻⁶. 9 When the cfu / mL concentration is 100 mL, centrifuge 10000 r / min, discard the supernatant, and add 20 mL of sterile water to obtain a concentrated bacterial solution (effective viable count ≥ 3 × 10⁻⁶). 10 (cfu / mL), for later use. The liquid culture medium consisted of 0.3% beef extract, 1% peptone, 11% sodium chloride, and a pH of 9.0. The culture conditions in the liquid culture medium were 30°C for 36 hours.

[0058] (1) Effects of Pseudomonas krusei WF3 on germination rate, fresh weight and dry weight of maize seeds

[0059] Take an appropriate amount of corn seeds and sterilize them in a beaker, then rinse them three times with sterile water. Prepare a 9cm sterile petri dish and lay defatted cotton balls at the bottom. Prepare an 11% sodium chloride solution, sterilize it, and pour it into the petri dish, ensuring the cotton balls are submerged. Place the corn seeds in distilled water, the 11% sodium chloride solution, and WF3 bacterial suspension (5mL / dish, effective bacterial concentration 5×10⁻⁶). 9 Ten seeds were placed in each petri dish containing 10 CFU / mL corn seeds, 11% sodium chloride solution, 5 mL / dish of culture medium (sterile-free), 11% sodium chloride solution, and 5 mL / dish of WF3 bacterial suspension. Ten replicates were made per dish (for the bacterial suspension treatment group, pre-soaked cleaned corn seeds in WF3 fermentation broth for 3 hours). The petri dishes were then incubated at 25°C for 48 hours. After 48 hours, the germination rate (%) and the fresh and dry weight (mg) of the germinated corn seeds were recorded. Statistical results are as follows: Figure 6 As shown, WF3 bacterial solution can increase the germination rate of corn seeds under stress with 11% NaCl solution by 272.87%, increase fresh weight by 148.78%, and increase dry weight by 288.84%.

[0060] (2) Effects of Pseudomonas krusei WF3 on maize seedling roots and salt-alkali stress resistance indicators

[0061] A potting mix was prepared by mixing nutrient soil and coconut coir (volume ratio 4:1), then sterilized at 121℃ for 1 hour. After cooling, the mixture was filled into seedling trays (10cm x 10cm x 10cm), with 200g of corn seeds per tray. Five control groups (distilled water), treatments with 11% sodium chloride solution, WF3 bacterial suspension + 11% sodium chloride solution, culture medium + 11% sodium chloride solution, and Pseudomonas kurstii WF3 bacterial suspension were set up, with five replicates per group. After corn seedling emergence, 10mL of the solution was applied to each treatment once a week, with sterile water applied at other times. After emergence, one seedling was retained per tray. After two weeks of cultivation, growth indicators such as total root length, average root diameter, and number of root tips, as well as physiological indicators such as soluble sugar, proline, and malondialdehyde content, were measured. Statistical results are shown below. Figures 7-10 As shown, *Pseudomonas krusei* WF3 bacterial suspension increased the total root length, average root diameter, and number of root tips in maize seedlings under salt stress by 169.57%, 116.67%, and 178.19%, respectively. It also decreased the contents of soluble sugars, proline, and malondialdehyde in maize seedlings under salt stress by 33.12%, 29.16%, and 38.98%, respectively.

[0062] Example 3

[0063] Well-rotted organic fertilizer (organic matter content ≥45%), humic acid (organic matter content ≥65%), attapulgite powder, and polyglutamic acid (on a dry basis, effective polyglutamic acid content ≥20%, molecular weight 100~500kDa) were purchased from Guangzhou Zhenwei Microbial Technology Co., Ltd. The well-rotted organic fertilizer was sieved through a 200-mesh sieve and the moisture content was adjusted to <25wt.%, and it was kept for later use. The humic acid was sieved through a 120-mesh sieve and the moisture content was adjusted to <25wt.%, and it was kept for later use. The attapulgite powder was sieved through a 120-mesh sieve and the moisture content was adjusted to <25wt.%, and it was kept for later use. The polyglutamic acid had a moisture content of <6wt.%, and it was kept for later use.

[0064] Preparation of fertilizer carrier (parts by weight): Mix 750 parts of well-rotted organic fertilizer, 120 parts of humic acid, 120 parts of attapulgite powder and 4 parts of polyglutamic acid evenly, and adjust the moisture content to 25 wt.% to obtain fertilizer carrier.

[0065] Viable bacteria count ≥3×10 10 A concentrated bacterial suspension of *Pseudomonas kurstani* WF3 (cfu / mL) was added to a fertilizer carrier at an inoculum size of 8 wt.%, stirred thoroughly, sealed, and fermented in a 25°C incubator for 120 hours to obtain microbial fertilizer. The microbial fertilizer was found to have an organic matter content ≥50%, a pH between 6.5 and 7.5, and an effective viable count ≥1.0 × 10⁻⁶. 8 cfu / g.

[0066] The prepared microbial fertilizer was applied as a base fertilizer at a rate of 150 kg / mu to cornfields in saline-alkali soil in Ganzhou District, Zhangye City, Gansu Province. The saline-alkali soil had a pH of 9.2 and a salt content of 0.32%. Simultaneously, a no-fertilizer setting was implemented; WF3 microbial agent (with a viable count ≥3×10⁻⁶) was applied at a rate of 1.2 kg / mu. 10 Concentrated solution (cfu / mL); 150 kg / mu of fertilizer carrier; conventional fertilization group (20 kg / mu of nitrogen, 8 kg / mu of phosphorus, and 4 kg / mu of potassium), 30% basal fertilizer, 20% topdressing at the jointing stage, 20% topdressing at the large trumpet stage, and 20% topdressing at the tasseling stage. Figure 11 As shown, the microbial organic fertilizer provided by the present invention can effectively increase the soil organic carbon content, and the effect is better than that of the conventional fertilization group, with the organic carbon content increasing by 9.22% compared with the no-fertilization treatment.

[0067] like Figure 12 As shown, the microbial organic fertilizer provided by this invention can effectively increase the total nitrogen content of the soil by 7.38% compared with the no-fertilizer treatment.

[0068] like Figure 13 As shown, the microbial organic fertilizer provided by the present invention can effectively increase the content of soil water-stable large aggregates (>2.5mm), and the effect is better than that of the conventional fertilization group, increasing by 36.17% compared with the no-fertilization treatment.

[0069] like Figure 14 , 15 As shown, the microbial organic fertilizer provided by the present invention can reduce soil pH and total salt, and the effect is better than that of the conventional fertilization group. Compared with the no-fertilization treatment, the pH decreased by 2.06% and the total salt decreased by 9.5%.

[0070] like Figure 16 As shown, the microbial organic fertilizer provided by the present invention can increase corn yield by 13.04% compared with the no-fertilizer treatment.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a microbial fertilizer for saline-alkali land, characterized in that, Includes the following steps: (1) The fertilizer carrier consists of well-rotted organic fertilizer, humic acid, attapulgite powder and polyglutamic acid; (2) Inoculate the fertilizer carrier with Pseudomonas krusei ( Pseudomonas knackmussii WF3, sealed fermentation, yields microbial fertilizer for saline-alkali land; The Pseudomonas kirchii ( Pseudomonas knackmussii WF3 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:65562, and deposited on November 28, 2024.

2. The preparation method according to claim 1, characterized in that, The fertilizer carrier comprises the following raw materials in parts by weight: 700-800 parts of well-rotted organic fertilizer, 100-150 parts of humic acid, 100-145 parts of attapulgite powder, and 3-5 parts of polyglutamic acid; the moisture content of the fertilizer carrier is ≤25%.

3. The preparation method according to claim 2, characterized in that, The decomposed organic fertilizer has a particle size of 120-200 mesh, a moisture content of <25 wt.%, and an organic matter content of ≥45%; the humic acid has a particle size of 100-120 mesh, a moisture content of <25 wt.%, and an organic matter content of ≥65%; the attapulgite has a particle size of 100-120 mesh, a moisture content of <25 wt.%; and, on a dry basis, the polyglutamic acid has an effective content of ≥20%, a moisture content of <6 wt.%, and a molecular weight of 100-500 kDa.

4. The preparation method according to claim 1, characterized in that, The amount of *Pseudomonas krusei* WF3 inoculated into the fertilizer carrier is 8-10% by weight, and the bacterial concentration of *Pseudomonas krusei* WF3 is (5-10) × 10⁻⁶. 9 cfu / mL.

5. The preparation method according to claim 1, characterized in that, The fermentation is carried out at 25~30℃ for 96~120 hours.

6. A microbial fertilizer for saline-alkali land prepared by the preparation method according to any one of claims 1 to 5.

7. The microbial fertilizer for saline-alkali land as described in claim 6, characterized in that, The effective viable count of *Pseudomonas krusei* WF3 in the microbial fertilizer for saline-alkali land is ≥1.0 × 10⁻⁶. 8 cfu / g, organic matter content ≥50%, pH 6.5~7.

5.

8. A *Pseudomonas kuribsiella* strain used in the preparation method according to any one of claims 1 to 5. Pseudomonas knackmussii WF3, characterized in that, The *Pseudomonas kuribda* WF3 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:65562, and deposited on November 28, 2024.

9. The application of the microbial fertilizer for saline-alkali land as described in claim 6 or 7, or the *Pseudomonas kirchiii* WF3 as described in claim 8, in improving the resistance of crops in saline-alkali land, promoting the growth of crops in saline-alkali land, and improving saline-alkali soil.

10. The application as described in claim 9, characterized in that, The crop in the saline-alkali land is corn. Improving the resistance of the crop in the saline-alkali land involves increasing the content of soluble sugars, proline, and malondialdehyde in corn seedlings under saline-alkali stress. Promoting the growth of the crop in the saline-alkali land involves increasing the germination rate of corn seeds, the fresh weight, dry weight, total root length, average root diameter, and number of root tips of corn seedlings, as well as increasing corn yield. Improving the saline-alkali soil involves increasing the content of organic carbon, total nitrogen, and water-stable macroaggregates in the saline-alkali soil, and reducing the soil pH and total salt content.

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