A biological organic fertilizer suitable for saline-alkali soil, a preparation method and application thereof

Abstract

The application provides a biological organic fertilizer suitable for saline-alkali soil and a preparation method and application thereof, and belongs to the field of microbial fertilizers.The biological organic fertilizer is prepared from halomonas sp ZH-1, including organic acid fermentation liquor, perlite, activated carbon, distilled water, halomonas sp, pancreatin and yeast extract.The biological organic fertilizer can convert insoluble phosphate into soluble phosphate which can be absorbed by plants, can synthesize indole acetic acid by using tryptophan, and has the ability of synthesizing iron carrier.The biological organic fertilizer can improve the chemotaxis distance of halomonas sp, enhance the activity of halomonas sp in soil, improve the colonization ability of the strain in the rhizosphere, improve the stress resistance of wheat under salt stress, promote the growth of wheat, improve the thousand-grain weight of wheat, and finally improve the yield of wheat.

Description

Technical Field

[0001] This invention relates to the field of microbial fertilizers, specifically to a bio-organic fertilizer suitable for saline-alkali land, its preparation method, and its application. Background Technology

[0002] In my country, approximately 9.91 × 10⁻⁶ m² of land is saline-alkali. 7 km 2 Saline soils account for 1.03% of China's land area. Xinjiang Uygur Autonomous Region is the largest saline soil area in my country, with saline arable land accounting for 37.72% of the total irrigated arable land area, thus earning it the nickname "treasure trove" of saline-alkali land. High salt and alkalinity environments inhibit crop growth, leading to the death of many crops and reduced yields, severely limiting the types and yields of crops in saline-alkali areas. Extensive research has discovered a type of microorganism that can tolerate extreme saline-alkali environments. When these microorganisms are extracted from the soil and applied to salt-sensitive crops, their growth in saline-alkali environments is significantly improved, enhancing their salt tolerance. We call these bacteria, which colonize the plant rhizosphere and promote plant growth, PGPRs. PGPRs are very common among all beneficial microorganisms, can colonize almost all plants, and are easily developed into products applicable to agricultural production without causing environmental pollution or pesticide residues. Therefore, microbial fertilizers made from rhizosphere growth-promoting bacteria have become a new direction for the fertilizer market.

[0003] Currently, microbial fertilizers are categorized into liquid, powder, and solid forms. Solid fertilizers have a longer shelf life but are more difficult to produce and suffer from problems such as weak colonization of beneficial microorganisms, easy loss, and weak competition with other microorganisms, thus failing to exert their true effects. Commercially available microbial liquid fertilizers primarily focus on supplementing nutrients and promoting crop growth. There are currently few products specifically designed to improve plant tolerance to saline-alkali soils. This product utilizes salt-tolerant and growth-promoting bacteria isolated from the rhizosphere of salt-tolerant plants growing in saline-alkali areas of Xinjiang Uygur Autonomous Region. Combined with auxiliary materials such as factory fermentation waste liquid, this creates a highly efficient bio-organic fertilizer suitable for promoting crop growth in saline-alkali soils. It exhibits excellent microbial colonization, significantly increasing the survival rate of beneficial microorganisms and improving crop yields in saline-alkali lands, thus providing a new approach for the development and utilization of saline-alkali lands. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a bio-organic fertilizer suitable for saline-alkali land, its preparation method and application. The bio-organic fertilizer can convert insoluble phosphate into soluble phosphorus that can be absorbed by plants, can also synthesize indoleacetic acid using tryptophan, and has the ability to synthesize siderophores, thereby improving the colonization ability of Haloxylon ammodendron; and can also increase wheat yield under salt stress conditions.

[0005] To achieve the above objectives, the present invention provides a bio-organic fertilizer suitable for saline-alkali land, comprising the following raw materials in parts by weight:

[0006]

[0007] The halometabolite strain is ZH-1, with the strain preservation number CCTCCNO: M 2021333.

[0008] Preferably, the preparation method of the organic acid fermentation broth is as follows: corn is used as raw material, crushed and pulped, and mixed into fermentation raw materials. The raw materials are continuously sterilized and cooled in a fermentation tank. The fermentation broth is heated and filtered to obtain the organic acid fermentation tail liquid.

[0009] Preferably, the organic acid fermentation broth has an organic matter content of 150-250 g / L, an amino acid content of 0.8-1.1 g / L, a total sugar content of 75-85 g / L, a total nitrogen content of 10-12 g / L, a total potassium content of 16-20 g / L, and a pH of 3.5-4.5.

[0010] Preferably, the perlite has an organic matter content of 3 wt%; the activated carbon has an organic matter content of 5 wt%. The activated carbon and perlite used in this invention are filter residues containing organic matter used in the organic acid fermentation process.

[0011] Preferably, the perlite has a particle size of 0.1-0.5 mm.

[0012] Preferably, the number of *Haloxymonas* cells is 0.2 × 10⁻⁶. 8 -1×10 8 cfu / g.

[0013] In this invention, tryptone and yeast extract provide basic nutrients for microbial proliferation; the organic acid fermentation broth ensures sufficient organic matter content in the product, and the abundant small-molecule active substances can reduce salinity and alkalinity, providing ample nutrients for plants, while simultaneously enabling microbial proliferation to occupy favorable ecological niches, resisting the invasion of pathogenic microorganisms, improving soil structure, and increasing crop yield; activated carbon, as an adsorbent material, provides sufficient attachment space for the microorganisms; perlite plays a retaining role, protecting the internal microorganisms and ensuring the stability of the product. Furthermore, the combination of activated carbon and organic acid fermentation broth in this application can increase the chemotactic distance of *Halomonas*, enhance its activity in saline-alkali soil, and make it easier for it to act on plant roots.

[0014] This invention also provides a method for preparing the above-described bio-organic fertilizer, comprising the following steps:

[0015] (1) Weigh the raw materials according to the proportion and adjust the pH value of the organic acid fermentation broth to 6.5-6.8 for later use;

[0016] (2) The pH-adjusted organic acid fermentation broth, the weighed activated carbon, halometa bacteria broth, tryptone, and yeast paste are mixed together, and an appropriate amount of water is added and stirred to obtain a granular mixture.

[0017] (3) Mix the granular mixture described in step (2) with the weighed perlite, dry and sieve to obtain a bio-organic fertilizer suitable for saline-alkali land.

[0018] Preferably, the pH value of the organic acid fermentation broth is adjusted using ammonia water.

[0019] This invention first adjusts the pH of the organic acid fermentation broth to 6.5-6.8, which can ensure the normal survival of the bacteria. By using ammonia to adjust the pH, no excess base ions are added, thus preventing the aggravation of salt and alkali damage.

[0020] Preferably, the method for culturing the Haloxylon ammodendron is as follows: a halophilic rhizosphere growth-promoting bacterium ZH-1 with growth-promoting effects is screened from the rhizosphere of saline-alkali vegetation in Xinjiang Uygur Autonomous Region. The strain ZH-1 is inoculated into liquid LB medium and cultured in a shake flask at 30°C for 7 days. The supernatant is centrifuged and the bacterial cells are enriched and stored in a refrigerator at 4°C for later use.

[0021] Preferably, the liquid LB culture medium consists of: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, and 1 L distilled water.

[0022] Preferably, the diameter of the bio-organic fertilizer is 1-2 mm.

[0023] Preferably, the bio-organic fertilizer has a moisture content of 20-25% and an organic matter content of 75-85%.

[0024] The present invention also provides the application of the above-mentioned bio-organic fertilizer suitable for saline-alkali land in increasing wheat yield, wherein the application rate of the bio-organic fertilizer is 200 kg / mu.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The *Haloxylon ammodendron* ZH-1 described in this invention possesses the ability to tolerate salt and alkaline environments, and can convert insoluble phosphates into soluble phosphorus that can be absorbed by plants, with a conversion capacity of 0.74 mg·L⁻¹ for organic phosphorus. -1 ·2d -1The conversion rate of inorganic phosphorus was 1.05 mg·L⁻¹. -1 ·2d -1 It can also synthesize indoleacetic acid (IAA) from tryptophan; after 48 hours of cultivation, the IAA content in the fermentation broth can reach 21.24 mg·L⁻¹. -1 It also has the ability to synthesize siderophores, with siderophore activity as high as 82%.

[0027] The bio-organic fertilizer prepared by this invention can increase the chemotactic distance of Halomonas bacteria and enhance their activity in the soil. The number of live bacteria colonizing wheat roots is significantly higher than that of individual bacteria, and it has a clear advantage in improving the colonization of growth-promoting bacteria in the rhizosphere.

[0028] The bio-organic fertilizer prepared by this invention promotes the fresh weight of wheat under salt stress conditions, while enhancing the activity of antioxidant enzymes in wheat roots and reducing malondialdehyde content. Under salt stress, Halomonas ZH-1 promotes plant growth and improves plant stress resistance by slowing down membrane lipid peroxidation. Under normal growth conditions, it can effectively promote the fresh weight of wheat aboveground parts, dry weight of aboveground parts, fresh weight of underground parts, and root length, thus promoting plant growth.

[0029] It also promotes the quality of both above-ground and below-ground wheat materials and increases the thousand-grain weight of wheat. Under normal growth conditions, it can also effectively promote the quality of above-ground and below-ground wheat materials. Therefore, this product can promote wheat yield under moderately saline soil conditions. Attached Figure Description

[0030] Figure 1 Bar chart showing the effect of bio-organic fertilizer on wheat aboveground dry matter.

[0031] Figure 2 Bar chart showing the effect of bio-organic fertilizer on wheat underground dry matter.

[0032] Figure 3 Bar chart showing the effect of bio-organic fertilizer on wheat plant height

[0033] Figure 4 and Figure 5 Bar chart showing the effect of bio-organic fertilizer on the thousand-grain weight of wheat Detailed Implementation

[0034] 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. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0035] The Halomonas spp. ZH-1 used in the following examples was deposited on April 6, 2021, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Wuchang, with accession number CCTCCNO: M 2021333.

[0036] The preparation method of Halomonas is as follows:

[0037] Rhizosphere samples were taken from halophytes in the saline-alkali areas of the Manas River Basin in Xinjiang Uygur Autonomous Region. Well-grown and robust plants were selected. The roots were carefully dug out from around the roots using a shovel, the loose soil on the root surface was shaken off, and the spherical soil clumps adhering to the roots were carefully broken up and placed into sterilized self-sealing bags. The bags were then immediately placed in an insulated box with ice packs for cold storage.

[0038] Prepare the following instruments and materials: a spreading rod, a 200μL pipette, a 1mL pipette, a 15mL test tube (with a stopper), a 500mL Erlenmeyer flask, a glass petri dish (10cm in diameter), and disposable sterilizable pipette tips. Place all items in a high-temperature sterilization bag and sterilize in a high-temperature autoclave at 120℃ for 20 minutes. Then, place the sterilized items in a clean bench under a UV lamp.

[0039] LB medium: 5g yeast extract, 10g tryptone, 10g NaCl, 1L double-distilled water, using 1mol·L⁻¹ -1 Adjust the pH of the culture medium to 8.5-9 with NaOH solution (for solid culture medium, add agar to 1-2%).

[0040] The collected soil samples were processed within one week. 10g of the well-mixed soil was weighed and dissolved in sterile double-distilled water, diluted to 100mL, and sealed with sealing film in a sterile Erlenmeyer flask. The solution was then refrigerated at 180 rpm. -1 Shake for 30 minutes, let stand for a while, then dilute. Take 1 mL of 10 -1 The bacterial suspension was injected into a test tube containing 9 mL of sterile double-distilled water, and the suspension was evenly dispersed, avoiding contact between the pipette tip and the test tube wall. This yielded 10... -2 Bacterial suspension, and so on, dilute the bacterial suspension to 10. -3 10 -4 10 -5 10 -6 (Replace the pipette tip with a new one after each dilution). After dilution, take 10 μL of each solution. -3 10 -4 10 -5 10 -6Add 200 μL of bacterial suspension to LB solid nutrient medium and spread evenly using a sterile spreader (the spreader should be replaced after each application). Seal the medium and incubate upside down at 30°C for 3-7 days (all steps are performed under aseptic conditions). Repeat each concentration three times. Observe colony growth daily. After 7 days of incubation, use a modified photocopying method to transfer the entire colony from the LB solid medium to LB solid medium with NaCl concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% for salt tolerance screening. Incubate upside down at 30°C for 3-7 days, observing colony growth daily. Select colonies that grow well at each concentration for isolation and purification using an inoculation needle. Perform 3-4 isolation and purification cycles on salt-tolerant strains until a single, clean colony appears on the medium. Inoculate the colony onto LB slant agar and store at 4°C after bacterial growth.

[0041] Screening and growth-promoting performance testing of Halomonas ZH-1:

[0042] Based on high colonization strength and salt tolerance, salt-tolerant growth-promoting bacteria with various growth-promoting functions were screened out. Specific functions include phosphorus solubilization (organic and inorganic phosphorus), nitrogen fixation, iron carrier production, and IAA production.

[0043] The various properties of Halomonas ZH-1 were determined using different culture media.

[0044] Ashube nitrogen-free medium: mannitol 10.0g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaCO3 5.0g, CaSO4·2H2O 0.1g, peptone 20.0g, deionized water 1000mL.

[0045] Mongkina Organic Phosphorus Medium: Glucose 10.0g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, NaCl 0.3g, KCl 0.3g, FeSO4·7H2O 0.03g, MnSO4·H2O 0.03g, lecithin 0.2g, CaCO3 1.0g, yeast extract 0.5g, deionized water 1000mL.

[0046] PKO Inorganic Phosphorus Medium: Glucose 10.0g, MgSO4·7H2O 0.3g, MnSO4·H2O 0.03g, KCl 0.3g, (NH4)2SO4 0.5g, NaCl 0.3g, Ca3(PO4)2 2.0g, FeSO4·7H2O 0.036g, Deionized Water 1000ml.

[0047] MKB medium: 5.0 g casein amino acids, 15.0 mL glycerol, 2.5 g K2HPO4, 0.2 g MgSO4·7H2O, 1000 mL deionized water, pH 7.0.

[0048] Preparation of CAS colorimetric solution: Dissolve 0.0605g of chromeazurol S (CAS) in 50mL of deionized water, add 0.0027g of FeCl3 and stir well, label this solution A; then weigh 0.0729g of hexadecyltrimethylammonium bromide and dissolve it in 40mL of deionized water, label this solution B; finally, slowly pour solution A into solution B and stir well.

[0049] Nitrogen fixation capacity determination: Salt-tolerant strains were inoculated into nitrogen-free solid medium of Assumption and incubated upside down at 28°C for 3 days. The growth of colonies was checked and recorded regularly. Those that could grow well on nitrogen-free medium and form uniform colonies after 3 days were nitrogen-fixing bacteria.

[0050] Phosphate solubilization capacity assay: Each strain was activated in LB medium, and 1 mL of culture solution was incubated at 6000 r·min. -1 Centrifuge for 5 minutes, discard the supernatant, wash the bacterial cells three times with sterile water, resuspend, and adjust the bacterial concentration to 10. 8 CFU·mL -1 The bacteria were inoculated onto NBRIP and PKO solid media using the dropwise method and incubated at 28°C for 48 hours. The ability of the bacteria to dissolve organic / inorganic phosphorus was determined by the ratio of the phosphate-solubilizing zone diameter to the colony diameter (HD / CD). Phosphate-solubilizing strains were then inoculated into NBRIP and PKO liquid media, with no inoculation serving as a blank control. The culture was incubated at 28°C and 180 rpm. -1 The cells were cultured under shaking conditions for 7 days, and the solubility of organic / inorganic phosphorus was quantitatively determined by the molybdenum antimony colorimetric method.

[0051] IAA production capacity assay: The test strain was prepared in Landy medium (with 0.5 g·L⁻¹ added). -1 L-tryptophan, approximately 2.5 mmol·L⁻¹ -1 25℃, 140 r·min -1 After culturing under shaking conditions for 72 hours, take 1 mL of culture medium and incubate at 12000 rpm. -1 Centrifuge for 5 min, take 500 μL of supernatant and add an equal volume of Salkowski reagent (10.8 mol·L⁻¹). -1 Sulfuric acid containing 4.5 g of ferric chloride was used for color development at room temperature in the dark for 30 min. The optical density was measured at 530 nm. A blank culture medium was used as a control, and a standard curve was plotted using the optical density corresponding to pure IAA. The yield of IAA (mg·L⁻¹) was calculated. -1 )

[0052] Siderophore production capacity determination: Each strain to be tested was spotted onto LB agar plates and incubated at 28°C for 24 h. Then, CAS agar (containing CAS blue detection solution, 30.24 g·L⁻¹) was used. -1 Pour PIPES (0.9% agarose) onto LB agar plates and observe for pale yellow or pale red halos around the cells after 15-30 minutes to preliminarily determine their siderophore-producing ability. Inoculate salt-tolerant, siderophore-producing bacteria into MKB liquid medium at 28°C and 160 rpm. -1 Shaking culture for 3 days, then stirring the culture medium at 1000 rpm. -1 Centrifuge 5 ml, discard the supernatant, wash 2-3 times with sterile double-distilled water, and adjust to OD. 600 =1 bacterial suspension, take 2ml of bacterial suspension and add an equal volume of 2ml CAS detection solution, and measure OD. 630 The value is recorded as Ar. Take 1 ml of the control MKB culture medium without bacterial inoculation, and record the OD value as As in the same procedure as above. The concentration of siderophores is expressed in siderophore units (SU), SU=[(Ar-As) / Ar]×100%. The measurement is repeated 3 times, and the average value is used for comparison and analysis.

[0053] The experimental results are shown in Table 1:

[0054] Table 1. Growth-promoting properties of strains

[0055]

[0056] The results show that strain ZH-1 has the strongest overall growth-promoting ability and better performance. Therefore, strain ZH-1 of this invention was selected as the strain used in subsequent experiments.

[0057] Simultaneous testing revealed that the ZH-1 strain in this invention converted 0.74 mg·L⁻¹ of organophosphates. -1 ·2d -1 The conversion rate of inorganic phosphorus was 1.05 mg·L⁻¹. -1 ·2d -1 It has a very significant promoting effect on the conversion of insoluble phosphorus into soluble phosphorus.

[0058] Research on the growth-promoting effects of ZH-1 on wheat:

[0059] Preparation of bacterial suspension: The bacterial strain was inoculated into LB liquid medium and cultured with shaking for 72 h, then incubated at 10000 r·min. -1 Centrifuge for 5 minutes to collect bacterial cells, discard the supernatant, wash three times with sterile double-distilled water, resuspend, and adjust the bacterial concentration to 10⁻⁶. 8 CFU·mL -1 .

[0060] "Xinchun No. 5" wheat seeds, disinfected with 75% alcohol for 20 minutes, were rinsed five times with sterile water, then soaked for 8 hours. The soaked seeds were then placed in germination boxes lined with filter paper and germinated at room temperature in the dark for 24 hours. Afterward, the germinated seeds were sown in germination boxes containing sterilized sand, treated as CK, 150mM NaCl, ZH-1, and 150mM NaCl+ZH-1, respectively. Each wheat plant was inoculated with 10ml of bacterial solution. Samples were taken for testing after 7 days, and the results are shown in Table 2.

[0061] Table 2. Effects of strain ZH-1 on wheat biomass under salt stress

[0062]

[0063] The table above shows that under normal conditions, adding ZH-1 significantly increases the fresh weight of both the aboveground and underground parts of wheat, with the whole plant fresh weight increasing by approximately 76.8%. Under salt stress conditions, adding ZH-1 also significantly increases the whole plant fresh weight of wheat, by 43.7% compared to salt stress.

[0064] When wheat is subjected to salt stress, it will reduce the damage by adjusting its own osmotic substance content and antioxidant enzyme activity. Therefore, the osmotic substance content and antioxidant enzyme activity can indirectly reflect the effect of the strain on improving the salt tolerance of wheat.

[0065] Determination and methods of permeate substances:

[0066] Proline content in wheat leaves: The proline content was determined using the sulfosalicylic acid method. 0.1 g of leaves were weighed and placed in a test tube. 3 mL of 5% sulfosalicylic acid was added. The mixture was extracted in a boiling water bath for 10 min. After cooling, it was filtered into another clean test tube; the filtrate was the proline extract. 2 mL of the extract + 2 mL of glacial acetic acid + 2 mL of acidic ninhydrin reagent were transferred to a covered test tube. The tube was incubated in a boiling water bath for 30 min. After cooling, 4 mL of toluene was added, the mixture was shaken for 30 s, and allowed to stand for a moment. The supernatant was gently pipetted into a cuvette and the absorbance was measured at 520 nm. The proline content X (μg·2mL) in 2 mL of the test solution was calculated using a regression equation. -1 Then, the proline content in the sample was calculated. Proline content (μg·g) -1 ) = (X·5 / 2) × sample mass (g). The test results are shown in Table 3.

[0067] Table 3. Effects of strain ZH-1 on osmotic regulatory substances in wheat under salt stress

[0068]

[0069] Plants exhibit physiological water deficiency under salt stress, leading to a decrease in leaf osmotic pressure, cell dehydration and shrinkage, and in severe cases, death. Therefore, under external stress, plants secrete osmotic regulators to balance cell osmotic pressure and alleviate damage. Table 3 shows that treatment with strain ZH-1 significantly increased the content of proline, an osmotic substance in wheat leaves, by approximately 58%, thereby increasing leaf osmotic pressure, alleviating leaf water loss, reducing the damage of salt stress to wheat, and improving wheat's salt tolerance.

[0070] The activity of antioxidant enzyme system in wheat leaves was detected, as shown in Table 4.

[0071] Table 4. Effects of strain ZH-1 on the activity of wheat antioxidant enzyme system under salt stress

[0072]

[0073] Under adverse conditions, when reactive oxygen species (ROS) accumulate to a certain concentration in plant organs, chlorophyll is damaged. Simultaneously, the plant's antioxidant mechanisms activate, increasing the activity of antioxidant enzymes to scavenge ROS. When these mechanisms fail to maintain a balance between ROS production and scavenging, excessive ROS can lead to peroxidation of tissue or organ membrane lipids. Malondialdehyde (MDA) is one of the products of membrane lipid peroxidation under adverse conditions, thus its content reflects the degree of damage to plant organs. After adding strain ZH-1 of this invention, the antioxidant enzyme activities in wheat organs were significantly enhanced. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased by 35%, 153%, and 238%, respectively, while the corresponding content of the harmful substance MDA decreased by 28%. Therefore, strain ZH-1 of this invention enhances wheat's ability to resist salt stress and improves its salt tolerance.

[0074] In summary, the *Haloxylon ammodendron* ZH-1 provided by this invention can tolerate salt stress, convert insoluble phosphorus in the soil into phosphorus that can be absorbed by plants, and simultaneously synthesize indoleacetic acid and siderophores. The *Haloxylon ammodendron* described in this invention can also enhance plant stress resistance and promote plant growth.

[0075] The preparation method of organic acid fermentation broth is as follows: Corn is used as raw material, crushed and pulped, and mixed into fermentation raw materials. The mixture is then continuously sterilized and cooled in a fermentation tank. The fermentation broth is heated and filtered to obtain the organic acid fermentation tail liquid. Since the composition of the tail liquid varies between different batches, tail liquids with different compositions are shown in the examples.

[0076] Example 1

[0077] 1) Weigh out 400 mL of organic acid fermentation broth (organic matter content 200 g / L, amino acid content 1.0 g / L, total sugar content 80 g / L, total nitrogen content 11 g / L, total potassium content 18 g / L, pH 4), 200 g of perlite (3 wt% organic matter content), 200 g of activated carbon (5 wt% organic matter content), 119 mL of distilled water, and 0.2 × 10⁻⁶ salinomyces ZH-1. 8 -1.0×10 8 7g of cfu / g, 2g of tryptone and 1g of yeast extract;

[0078] 2) Adjust the pH of the organic acid fermentation broth to 6.6 using ammonia water;

[0079] 3) Mix the pH-adjusted organic acid fermentation broth, activated carbon, Halomonas bacteria, tryptone, yeast extract, and distilled water to obtain a granular mixture;

[0080] 4) After mixing the obtained granular mixture with perlite, dry it to a moisture content of 20% and pass it through a 2mm sieve to obtain a bio-organic fertilizer suitable for saline-alkali land. The organic matter content of the obtained bio-organic fertilizer is 80%.

[0081] Example 2

[0082] 1) Weigh out 300 mL of organic acid fermentation broth (organic matter content 150 g / L, amino acid content 0.8 g / L, total sugar content 75 g / L, total nitrogen content 10 g / L, total potassium content 16 g / L, pH 3.5), 100 g of perlite (3 wt% organic matter content), 100 g of activated carbon (5 wt% organic matter content), 209 mL of distilled water, and 0.2 × 10⁻⁶ seromonas ZH-1. 8 -1.0×10 8 5g of cfu / g, 1g of tryptone and 2g of yeast extract;

[0083] 2) Adjust the pH of the organic acid fermentation broth to 6.5 using ammonia water;

[0084] 3) Mix the pH-adjusted organic acid fermentation broth, activated carbon, Halomonas bacteria, tryptone, yeast extract, and distilled water to obtain a granular mixture;

[0085] 4) After mixing the obtained granular mixture with perlite, dry it to a moisture content of 25% and pass it through a 1.5mm sieve to obtain a bio-organic fertilizer suitable for saline-alkali land. The organic matter content of the obtained bio-organic fertilizer is 75%.

[0086] Example 3

[0087] 1) Weigh 500 mL of organic acid fermentation broth (the organic acid fermentation broth has an organic matter content of 250 g / L, an amino acid content of 1.1 g / L, a total sugar content of 85 g / L, a total nitrogen content of 12 g / L, a total potassium content of 20 g / L, and a pH of 4.5), 300 g of perlite (3 wt% organic matter content), 300 g of activated carbon (5 wt% organic matter content), 289 mL of distilled water, and 0.2 × 10⁻⁶ spores of Haloxylon ammodendron ZH-1. 8 -1.0×10 8 8g of cfu / g, 1g of tryptone and 3g of yeast extract;

[0088] 2) Adjust the pH of the organic acid fermentation broth to 6.8 using ammonia water;

[0089] 3) Mix the pH-adjusted organic acid fermentation broth, activated carbon, Halomonas bacteria, tryptone, yeast extract, and distilled water to obtain a granular mixture;

[0090] 4) After mixing the obtained granular mixture with perlite, dry it to a moisture content of 25% and pass it through a 1mm sieve to obtain a bio-organic fertilizer suitable for saline-alkali land. The organic matter content of the obtained bio-organic fertilizer is 85%.

[0091] Comparative Example 1

[0092] Same as Example 1, except that the raw materials do not contain Halomonas ZH-1.

[0093] Comparative Example 2

[0094] Same as Example 1, except that the raw materials do not contain activated carbon.

[0095] Comparative Example 3

[0096] Same as Example 1, except that the raw materials do not contain organic acid fermentation liquid.

[0097] Comparative Example 4

[0098] Same as Example 1, except that the raw materials do not contain activated carbon and organic acid fermentation liquid.

[0099] Experimental Example 1

[0100] Chemotaxis of ZH-1 by major fertilizer components:

[0101] A chemotactic medium with an agar content of 0.15% was prepared to investigate the effect of activated carbon on improving ZH-1 colonization. The medium composition was: 10 g / L tryptone, 5 g / L yeast extract, 0.15% agar, and 10 g / L NaCl.

[0102] Centrifuge the ZH-1 bacterial culture that has been cultured in liquid LB medium for 48 hours at 10000 r / min for 4 min, discard the supernatant, rinse the solid bacteria twice with sterile distilled water, resuspend and adjust the OD value to 0.8-1.0 for later use.

[0103] The treatments were sterile water (CK), activated carbon (S1), organic acid fermentation broth (S2), and activated carbon + organic acid fermentation broth (S3).

[0104] (1) Chemotaxis of activated carbon on ZH-1

[0105] Activated carbon was ground and sieved. 1g of activated carbon was dissolved in 100ml of sterile distilled water and thoroughly mixed by ultrasonication. The mixture was then filtered through a 0.22μm disposable sterile filter to obtain a 1% activated carbon solution. 10μL of bacterial culture was pipetted into the center of the culture medium, and 10μL of the 1% activated carbon solution was inoculated at a distance of 4cm from the bacterial culture. The culture was incubated at 30℃ for 48h, and the chemotactic distance of the bacterial zone was measured.

[0106] (2) Chemotaxis of ZH-1 by organic acid fermentation broth

[0107] Dissolve 1 ml of organic acid fermentation broth in 100 ml of sterile distilled water, mix thoroughly using an ultrasonic mixer, and then filter the mixture through a 0.22 μm disposable sterile filter membrane to obtain a 1% organic acid fermentation broth. Pipette 10 μL of the bacterial culture into the center of the culture medium, and inoculate another 10 μL of the 1% organic acid fermentation broth at a distance of 4 cm from the bacterial culture. Incubate at 30°C for 48 h and measure the chemotactic distance of the bacterial zone.

[0108] (3) Chelogenic effect of activated carbon and organic acid components on ZH-1

[0109] 10 μL of bacterial culture was inoculated into the center of the culture medium using a pipette. 5 μL of 1% organic acid fermentation broth and 5 μL of 1% activated carbon solution were inoculated at a distance of 4 cm from the bacterial culture. The culture was incubated at 30 °C for 48 h and the chemotaxis distance of the bacterial zone was measured.

[0110] The chemotactic distance detection results are shown in Table 5.

[0111] Table 5. Effects of different treatments on the chemotaxis of strain ZH-1

[0112]

[0113] The chemotaxis and colonization strength of rhizosphere growth-promoting bacteria determine the strength of their growth-promoting effects. Therefore, enhancing the chemotaxis of bacterial strains is an important prerequisite for improving the fertilizer efficiency of microbial fertilizers.

[0114] As shown in Table 5, both activated carbon and organic acid fermentation broth showed significant chemotaxis towards strain ZH-1. However, the organic acid fermentation broth with added activated carbon showed a more significant effect compared to the fermentation broth with activated carbon or organic acid added alone, indicating that activated carbon significantly improved the chemotaxis towards ZH-1.

[0115] Experimental Example 2

[0116] Research on the growth-promoting effects of bio-organic fertilizer on wheat:

[0117] Verification of the growth-promoting effect of bio-organic fertilizer

[0118] The “Xinchun No. 5” wheat seeds were disinfected with alcohol, rinsed with sterile water, and then sown in seedling trays filled with nutrient soil for 7 days. Then, seedlings with uniform growth were selected and transplanted into flower pots (top diameter 23cm, bottom diameter 20cm, height 23cm) filled with 4.5kg of sand and soil (sand: soil = 3:1).

[0119] Configuration and processing:

[0120] (1) Treatment 1 (SK): 150mM NaCl

[0121] (2) Treatment 2 (JF-Y): 150mM NaCl + the bio-organic fertilizer obtained in Example 1

[0122] (3) Treatment 3 (F0-Y): 150 mM NaCl + Bio-organic Fertilizer obtained from Comparative Example 1

[0123] (4) Treatment 4 (CK): Distilled water

[0124] (5) Treatment 5 (JF): Distilled water + bio-organic fertilizer obtained in Example 1

[0125] (6) Treatment 6 (F0): Distilled water + bio-organic fertilizer obtained from Comparative Example 1

[0126] (7) Treatment 7(F1-Y): 150mM NaCl + Bio-organic Fertilizer obtained from Comparative Example 2

[0127] (8) Treatment 8(F2-Y): 150mM NaCl + Bio-organic Fertilizer obtained from Comparative Example 3

[0128] (9) Treatment 9(F3-Y): 150mM NaCl + Bio-organic Fertilizer obtained from Comparative Example 4

[0129] Each treatment was replicated in triplicate, with 6 seedlings per pot. Before transplanting, the treatment chemicals and fertilizers were evenly mixed into the sand. The fertilizer application rate was calculated using the field application formula of 200 kg / mu, resulting in 112.5 g per pot. After the wheat matured, the roots were rinsed with deionized water, the moisture was blotted dry with filter paper, and the average biomass, plant height, and yield per wheat plant were measured.

[0130] Depend on Figure 1-4 It was found that the bio-organic fertilizer with ZH-1 added significantly increased the aboveground and underground biomass of wheat, and also increased the thousand-grain weight of wheat. Although the fertilizer without ZH-1 added also had a promoting effect on wheat, the promoting effect was not obvious, especially for wheat growth under salt stress.

[0131] Depend on Figure 4-5 It can be seen that, under salt stress conditions, the bio-organic fertilizer with added activated carbon and organic acid fermentation tail liquid significantly increased the thousand-grain weight of wheat compared with the treatment with single application of microbial agent; and the treatment with added activated carbon also significantly increased the aboveground fresh weight and thousand-grain weight of wheat compared with the treatment without activated carbon.

[0132] In summary, applying this product to moderately saline soil conditions can increase the thousand-grain weight of wheat to the level of normal soil conditions, and applying this product to normal soil conditions can increase the thousand-grain weight of wheat by approximately 40%.

[0133] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A bio-organic fertilizer suitable for saline-alkali land, characterized in that, Including the following parts by weight of raw materials: 300-500 parts organic acid fermentation broth, 100-300 parts perlite, 100-300 parts activated carbon, 5-8 parts Halomonas, 1-2 parts tryptone, and 1-3 parts yeast extract; The Halomonas strain is ZH-1, with the strain preservation number CCTCC NO: M 2021333; The preparation method of the organic acid fermentation broth is as follows: corn is used as raw material, crushed and pulped, and mixed into fermentation raw materials. The raw materials are continuously sterilized and cooled in a fermentation tank. The fermentation broth is heated and filtered to obtain organic acid fermentation tail liquid.

2. The bio-organic fertilizer according to claim 1, characterized in that, The organic acid fermentation broth has an organic matter content of 150-250 g / L, an amino acid content of 0.8-1.1 g / L, a total sugar content of 75-85 g / L, a total nitrogen content of 10-12 g / L, a total potassium content of 16-20 g / L, and a pH of 3.5-4.

5.

3. The bio-organic fertilizer according to claim 1, characterized in that, The perlite has an organic matter content of 3 wt%; the activated carbon has an organic matter content of 5 wt%.

4. The bio-organic fertilizer suitable for saline-alkali land according to claim 2, characterized in that, The perlite has a particle size of 0.1-0.5 mm.

5. The bio-organic fertilizer according to claim 1, characterized in that, The bacterial count of the *Haloxymonas* was 0.2 × 10⁻⁶. 8 -1×10 8 cfu / g.

6. The method for preparing the bio-organic fertilizer according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Weigh the raw materials according to the proportion and adjust the pH value of the organic acid fermentation broth to 6.5-6.8 for later use; (2) The pH-adjusted organic acid fermentation broth, the weighed activated carbon, halometa bacteria broth, tryptone, and yeast paste are mixed together, and an appropriate amount of water is added and stirred to obtain a granular mixture. (3) Mix the granular mixture described in step (2) with the weighed perlite, dry and sieve to obtain a bio-organic fertilizer suitable for saline-alkali land.

7. The preparation method according to claim 6, characterized in that, The diameter of the bio-organic fertilizer is 1-2 mm.

8. The preparation method according to claim 6, characterized in that, The bio-organic fertilizer has a moisture content of 20-25% and an organic matter content of 75-85%.

9. The application of the bio-organic fertilizer suitable for saline-alkali land according to any one of claims 1 to 5 in increasing wheat yield, characterized in that, The application rate of the bio-organic fertilizer is 200 kg / mu.

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