A method for preparing a long-acting bio-fertilizer comprising a dse strain

Abstract

The application belongs to the field of microbial fertilizers, and particularly relates to a preparation method of a long-acting biofertilizer containing a DSE strain. The DSE strain has a preservation number of CGMCC No. 42008, a preservation date of May 19, 2025, a classification name of Paraphoma pye, and a name of HQ-21. The long-acting biofertilizer comprises a core layer and a shell layer. The core layer comprises trehalose, a slow-release carrier and the DSE strain. The slow-release carrier is a carboxymethyl cellulose-xanthan gum composite matrix, and the slow-release carrier is loaded with calcium ions and magnesium ions. The shell layer comprises a pH-responsive chitosan / calcium pectate film, PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles. The long-acting biofertilizer solves the technical problem that the current DSE is susceptible to soil pH, water, ultraviolet rays and the like, is difficult to survive, and cannot long-term colonize to exert the growth-promoting effect in the application of soil improvement, degraded soil repair and crop growth promotion.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizers, and specifically relates to a method for preparing a long-acting bio-fertilizer containing DSE strains. Background Technology

[0002] Dark seperate endophytes (DSEs) are a diverse group of ascomycetes or deuteromycetes that colonize plant roots and are either non-spore-producing or produce asexual spores. DSEs are found within the epidermis, cortex, and even vascular tissues of healthy plant roots, forming dark-septate hyphae and microsclerotia within the root cells or intercellular spaces. However, they do not contribute to the pathological features caused by pathogens within the root tissue. DSEs belong to the fungal kingdom and are characterized by septate hyphae and melanin-containing cell walls. They are widely colonized within the cortical cells or intercellular spaces of plant roots. DSEs promote plant growth by secreting growth hormones (such as IAA), lysozymes, and siderophores, thus promoting nutrient absorption (especially phosphorus and iron). They can also chelate heavy metals to reduce their toxicity and accumulation in plants. Furthermore, DSEs can alleviate soil-borne diseases (such as Fusarium wilt) by secreting antibiotics (such as chitinase). Currently, DSE is mainly used for marginal soil improvement (such as mine reclamation), ecological agriculture (reducing reliance on chemical fertilizers), and medicinal plant cultivation (such as increasing the content of flavonoid secondary metabolites). Studies have found that DSE is widely present in the roots of various genera of plants, such as oats and astragalus, and that DSE has a significant impact on promoting plant growth and stress resistance. The growth-promoting effects of DSE are documented in relevant patents such as CN118480449A, CN118620745B, CN107083335B, and CN118272236A.

[0003] However, the use of DSE faces several limitations: the growth-promoting effect of DSE is greatly affected by soil pH and moisture, and it is easily competed for by native microorganisms in the soil, and may even become a weak parasite under high stress. In addition, under the stress of ultraviolet radiation, competing bacteria, high salinity and alkalinity, DSE applied to the soil is also prone to failure to survive, and cannot establish itself for a long time to play its role in promoting growth and inhibiting soil-borne pathogens. Summary of the Invention

[0004] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for preparing a long-acting bio-fertilizer containing DSE strains, which solves the technical problems of DSE in soil improvement, degraded soil remediation and crop growth promotion applications, which are greatly affected by soil pH and moisture, have difficulty surviving, cannot be established for a long time, and crops are susceptible to soil-borne root rot. It also reduces the heavy metal content of high-value crops (such as Chinese herbal medicines) and improves safety.

[0005] (II) Technical Solution In a first aspect, the present invention provides a DSE strain, the DSE strain having the accession number CGMCC No.42008, the accession date being May 19, 2025, the classification name being Paraphoma pye, the name being HQ-21, and the accession address being No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The DSE strain possesses functions such as inorganic phosphorus decomposition, IAA production, nitrogen fixation, siderophore production, and excellent resistance to pathogens.

[0007] Secondly, the present invention provides a long-acting bio-fertilizer containing DSE strains, characterized in that it comprises a core layer and a shell layer; the core layer comprises trehalose, a slow-release carrier and the aforementioned DSE strains; the slow-release carrier is a carboxymethyl cellulose-xanthan gum composite matrix, and calcium ions and magnesium ions are loaded on the slow-release carrier; The shell comprises a pH-responsive chitosan / calcium pectate membrane, PLA-coated ammonium dihydrogen phosphate microspheres, and humic acid nanoparticles.

[0008] According to a preferred embodiment of the present invention, the molar ratio of calcium ions to magnesium ions is 1:2 to 2:1.

[0009] According to a preferred embodiment of the present invention, the pH-responsive chitosan / calcium pectate membrane degrades at pH ≤ 6.5 and has a thickness of 50-100 μm; wherein the calcium pectate is derived from agricultural waste (such as citrus peel residue).

[0010] According to a preferred embodiment of the present invention, the DSE strain content in the long-acting bio-fertilizer is 1×10⁻⁶. 6 -1×10 8 The concentration of CFU / g, trehalose content is 5-10wt%, carboxymethyl cellulose content is 3-6wt%, xanthan gum content is 1.5-3wt%, calcium ion content is 0.1-0.3mol / kg, and magnesium ion content is 0.2-0.6mol / kg.

[0011] According to a preferred embodiment of the present invention, the long-acting bio-fertilizer contains 10-15 wt% chitosan / calcium pectate film, 20-30 wt% PLA-coated ammonium dihydrogen phosphate microspheres, and 5-8 wt% humic acid nanoparticles.

[0012] Thirdly, the present invention provides a method for preparing a long-acting bio-fertilizer containing DSE strains, comprising the following steps: S1, Core layer preparation The aforementioned DSE strain was cultured to the logarithmic growth phase. After adding PEG-400, the cells were centrifuged at 4℃ and 4000-6000 rpm for 5-8 min to collect the DSE cells. The cells were then suspended in phosphate buffer containing 4-6% trehalose to obtain a bacterial suspension. CMC and xanthan gum were dissolved in hot water at 60-70℃. CaCl2 and MgCl2 were added, and the mixture was stirred to form a homogeneous gel. The homogeneous gel was then cooled to 25℃ and mixed with the bacterial suspension. The mixture was then spray-dried, with the inlet temperature controlled at 75-80℃ and the outlet temperature at 35-40℃ to obtain the core particles. S2, Shell Preparation Chitosan with a degree of deacetylation ≥ 85% was dissolved in 1% acetic acid solution, and calcium pectate was dissolved in deionized water. They were mixed in a 1:1 volume ratio and the pH was adjusted to 5.5 to obtain a chitosan / pectic acid weak gel system. Ammonium dihydrogen phosphate solution was added to an industrial-grade ethyl acetate solution of PLA with a Mw between 40-55 kDa. After emulsification, the solvent was evaporated to obtain PLA-coated ammonium dihydrogen phosphate microspheres. The PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles were then dispersed together in water to form a homogeneous colloidal solution. S3, Composite Coating The core particles are placed in a fluidized bed, and a chitosan / pectinic acid weak gel system and a homogeneous colloidal solution are sprayed on them. After drying, the long-lasting bio-fertilizer is obtained.

[0013] According to a preferred embodiment of the present invention, in S1, when the bacterial cells contain two types, the bacterial cells are cultured separately to the logarithmic phase, and then the wet bacterial cells are collected by centrifugation. The two or more wet bacterial cells are then mixed according to a predetermined ratio.

[0014] According to a preferred embodiment of the present invention, in S1, CMC and xanthan gum are dissolved in hot water at 60°C at a mass ratio of 2:1, and then CaCl2 and MgCl2 at a molar ratio of 1:2 are added to the solution and stirred to form a homogeneous gel.

[0015] According to a preferred embodiment of the present invention, in S2, the concentration of ammonium dihydrogen phosphate solution is 20-25 wt%; the ammonium dihydrogen phosphate solution is added to an industrial-grade ethyl acetate solution of PLA with a Mw between 50 kDa, and after emulsification, the solvent is evaporated to obtain PLA-encapsulated ammonium dihydrogen phosphate microspheres.

[0016] According to a preferred embodiment of the present invention, in S2, PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles are dispersed together in water containing 0.1-0.5 wt% Tween 80 and ultrasonically dispersed together for 15-30 min to obtain a homogeneous colloidal solution.

[0017] According to a preferred embodiment of the present invention, in S3, the spraying speed is 2 mL / min and the air pressure is 0.3 MPa.

[0018] Fourthly, the present invention provides the application of the above-mentioned long-acting bio-fertilizer containing DSEDSE in promoting the growth of oats or astragalus.

[0019] (III) Beneficial Effects To reduce the limitations of DSE applications, this invention first screened a DSE strain with highly efficient inorganic phosphorus solubilization, IAA production, nitrogen fixation, siderophore production, and pathogen resistance, thus promoting growth. This DSE strain was then encapsulated with trehalose, a slow-release carrier, calcium ions, and magnesium ions to produce a long-acting bio-fertilizer containing DSE bacteria. The encapsulation material protects DSE from environmental stresses, resisting ultraviolet radiation and drought to extend the colonization cycle and improve the survival rate and stability of the DSE bacteria. This long-acting bio-fertilizer is suitable for crop growth promotion and stress mitigation in marginal soils (such as saline-alkali land and heavy metal-contaminated farmland), solving the technical problems of low field survival rate of DSE bacteria, asynchronous nutrient release with plant needs, and susceptibility to soil-borne root rot in crops.

[0020] Specifically, the long-acting bio-fertilizer prepared by this invention has the following synergistic effects among its components: (1) Trehalose provides attachment sites and nutrient signals for DSE, inducing its biofilm formation. Fungi themselves also secrete extracellular polysaccharides, which can constitute the matrix of biofilms, encapsulating fungal cells and promoting biofilm formation and stability. Trehalose replaces traditional glycerol, avoiding the osmotic stress caused by traditional glycerol and improving the survival rate of fungi under drought conditions.

[0021] (2) The carboxymethyl cellulose-xanthan gum composite matrix forms a three-dimensional network structure. The calcium and magnesium ions loaded on it promote the secretion of organic acids (such as oxalic acid) by DSE mycelia, which is beneficial to the efficiency of phosphorus solubilization during fermentation. Calcium and magnesium ions are fixed by the carboxymethyl cellulose-xanthan gum composite matrix and can be slowly released, which may promote the formation of fungal biofilm. Calcium ions can participate in the signal transduction process within fungal cells, affecting the physiological activities and gene expression of fungi, and thus affecting the formation of biofilm. Magnesium ions are activators of many enzymes and promote the metabolic activities of fungi. Calcium and magnesium ions work synergistically to indirectly affect the formation of biofilm by affecting the growth and metabolism of fungi. The core layer continuously releases calcium and magnesium ions and DSE metabolites, maintaining the rhizosphere growth-promoting microenvironment.

[0022] (3) The chitosan / calcium pectate membrane is pH responsive and can encapsulate and protect DSE bacteria inside under highly alkaline conditions, reducing their release. When the rhizosphere pH is ≤6.5 (such as when plants secrete organic acids), calcium pectate dissociates, triggering membrane degradation and releasing more DSE bacteria. Calcium pectate can degrade to produce oligosaccharides, which can also promote the growth of DSE bacteria.

[0023] (4) PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles have the dual function of slow nutrient release and heavy metal passivation, reducing phosphorus loss. PLA slowly degrades and releases phosphorus, matching phosphorus supply with the plant's nutrient requirement cycle and promoting plant growth. The surface of humic acid nanoparticles (20-100nm) is rich in carboxyl or hydroxyl groups, which can chelate harmful heavy metals such as Cd / Pb and also delay the explosive release of calcium and magnesium ions.

[0024] This invention's long-acting bio-fertilizer promotes plant root health and growth in multiple ways, leveraging the role of DSE in IAA production, phosphorus solubilization, and iron carrier function. It also promotes nutrient absorption, reduces heavy metal toxicity, and mitigates soil-borne diseases (such as Fusarium oxysporum). Experiments have shown that after 90 days of application, the abundance of DSE in the plant rhizosphere increased more than three times compared to direct application. Verification using the chlorophyll fluorescence parameter Fv / Fm confirmed a significant increase in leaf photosynthetic rate, promoting nutrient accumulation in oat forage. Furthermore, it can reduce the heavy metal content in high-value crops (such as traditional Chinese medicine), thereby improving the safety of these herbs. Attached Figure Description

[0025] Figure 1 A standard curve for testing the soluble sugar content in crop leaves.

[0026] Figure 2 A standard curve for testing the soluble protein content in crop leaves. Detailed Implementation

[0027] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to specific embodiments. Example 1

[0028] The strain used in this embodiment is DSE, which the inventors isolated from the roots of Astragalus membranaceus in Inner Mongolia. It was identified as *Paraphoma pye*, and deposited on May 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42008 and name HQ-21. This strain has been shown to possess functions such as inorganic phosphorus solubilization, IAA production, nitrogen fixation, siderophore production, inhibition of plant pathogenic fungi, and in vitro disease resistance. The following are the performance verification experiments of this strain.

[0029] 1. Determination of inorganic phosphorus solubility DSE fungal cakes (diameter = 5 mm) were placed in Monkina inorganic phosphorus medium and cultured at 28°C for 7-10 days. During the growth process, inorganic phosphorus-dissolving fungi secreted some substances and diffused them to the surrounding area, causing the Ca3(PO4)2 in the medium to dissolve and become transparent.

[0030] 2. IAA production capacity determination The Salkowski colorimetric method was used to determine the growth factor (IAA) of DSE fungi. Preparation of DSE fungal liquid culture: Solid culture medium with good growth and mycelium was broken into pieces and transferred to 100 mg / L L-Try PDB liquid medium and PDB liquid medium without L-Try. PDB medium containing sterile water was used as a control. The cultures were incubated at 28°C and 180 rpm / min for 7 days. The fermented culture was then centrifuged at 4°C and 10,000 rpm / min for 20 minutes. The fungal liquid culture was mixed with Salkowski reagent at a volume ratio of 1:2. The mixture was incubated at room temperature in the dark for 30 minutes, and the color development (pink or pinkish-red) was observed. Color development indicated that the strain could secrete indole-like substances. The maximum absorption wavelength of the pinkish substance was 530 nm. The OD value at 530 nm was measured using a UV spectrophotometer to quantitatively analyze the IAA produced by the endophytic fungi.

[0031] 3. Determination of nitrogen fixation capacity of DSE strains Place DSE bacterial cakes (diameter = 5 mm) on Assumption nitrogen-fixing medium and incubate at 28°C for 7-10 days. Observe the growth status and morphological characteristics of the strain. If the strain produces a clear zone, it indicates that the DSE strain has nitrogen-fixing ability.

[0032] 4. Determination of the siderophore production capacity of DSE strains Different DSE strains were inoculated on ferrophilic assay medium (CAS) and cultured at 28°C for 7-10 days. The growth status and morphological characteristics of the strains were observed. If the strain produced an orange-red reaction, it indicated that the DSE strain had the ability to produce siderophores.

[0033] 5. In vitro disease resistance test of DSE strain The tested pathogenic fungal strain was Fusarium oxysporum, which is a pathogenic fungus for a variety of plants, including important economic crops such as oats and astragalus.

[0034] (1) Inhibition rate of DSE strain against F. oxysporum A two-point confrontation method was used, inoculating identical-sized *F. oxysporum* and *DSE* mycelial discs at 4 cm intervals at both ends of a petri dish containing PDA medium. A plate inoculated solely with *F. oxysporum* served as a control group. The dishes were incubated in the dark at 28°C, with three replicates. Once the *Fusarium oxysporum* fungus had essentially covered the petri dishes in the control group, the colony radius of the blueberry root rot fungus was measured in both the control and treatment groups.

[0035] (2) Inhibitory effect of DSE volatile products on F. oxysporum DSE mycelial cakes (5 mm in diameter) were placed in the center of PDA agar plates and cultured for 10 days. Then, they were incubated separately on top of each other with another PDA plate inoculated with *F. oxysporum* mycelial cakes. A control group was prepared by inverting a PDA plate inoculated with *F. oxysporum* onto a sterile PDA plate. The inverted areas of both plates were sealed with sealing film to prevent gas evaporation. The plates were then incubated in the dark at 28°C, with three replicates. When the *F. oxysporum* colonies in the control group nearly completely covered the plates, the colony diameter of *F. oxysporum* in the treatment group was measured.

[0036] (3) Inhibitory effect of DSE fermentation filtrate on F. oxysporum One DSE bacterial cake (diameter = 5 mm) was transferred into an Erlenmeyer flask containing PDB culture medium. The Erlenmeyer flask was placed in a constant temperature shaking incubator at 28℃ and 170 rpm for 10 days. The culture medium was first filtered through 8 layers of sterile gauze, centrifuged at 12000 rpm for 15 min, and the supernatant was first filtered through a 0.45 μm microporous membrane for preliminary filtration, and then through a 0.22 μm microporous membrane for later use. PDA (15 mL) that had been melted and cooled to a certain temperature was mixed with the collected DSE strain fermentation filtrate (5 mL). The F. oxysporum bacterial cake (diameter = 5 mm) was transferred into the prepared plate. The DSE strain fermentation filtrate concentration was 0 as a control group, and each group was repeated 3 times. The plates were incubated at 28℃ in the dark until the control group F. oxysporum almost completely covered the petri dish, and the colony diameter was measured.

[0037] The test results for items 1-4 above are shown in Table 1, and the in vitro disease resistance test results for the above DSE strains are shown in Table 2.

[0038] Table 1: Table 2: Example 2

[0039] In this embodiment, the DSE strain screened in Example 1 was used to prepare a long-acting bio-fertilizer according to the following method, wherein the composition of the long-acting growth promoter is as follows: Core layer: Trehalose, DSE strain, slow-release carrier, calcium ions, and magnesium ions. The slow-release carrier is a composite matrix composed of carboxymethyl cellulose and xanthan gum in a 2:1 mass ratio. Calcium and magnesium ions are provided by calcium chloride and magnesium chloride, respectively, and are loaded onto the slow-release carrier. Shell layer: pH-responsive chitosan / calcium pectate membrane, PLA-coated ammonium dihydrogen phosphate microspheres, and humic acid nanoparticles. The chitosan / calcium pectate membrane degrades at pH ≤ 6.5 and has a thickness of 50-100 μm; the calcium pectate is derived from citrus peel residue.

[0040] The DSE strain content in the long-acting bio-fertilizer was 1×10⁻⁶. 8 The composition includes CFU / g, trehalose (6wt%), carboxymethyl cellulose (6wt%), xanthan gum (3wt%), calcium ions (0.1mol / kg), and magnesium ions (0.2mol / kg). The chitosan / calcium pectate film contains 15wt%, PLA-coated ammonium dihydrogen phosphate microspheres contain 20wt%, and humic acid nanoparticles contain 5wt%. All of these materials are biodegradable. The remaining components are deionized water and necessary process additives, specifically magnesium stearate to improve the fluidity of the shell material during fluidized bed coating, an anti-caking agent (nano-silica) to prevent particle adhesion after spray drying, PEG-400, and phosphate buffer, etc.

[0041] The preparation method of the long-acting bio-fertilizer is as follows: (1) Weigh each material and auxiliary material according to the above ingredients.

[0042] (2) The DSE strain was cultured to the logarithmic phase, and 1.5% PEG-400 was added. The DSE cells were collected by centrifugation at 4℃ and 6000rpm for 6min. The cells were then suspended in phosphate buffer containing 5% trehalose to obtain a bacterial suspension.

[0043] (3) CMC and xanthan gum were dissolved in hot water at 60°C. CaCl2 and MgCl2 were added and stirred to form a homogeneous gel. The homogeneous gel was then cooled to 25°C and mixed with the bacterial suspension. The mixture was then spray-dried, with the inlet temperature controlled at 80°C and the outlet temperature at 40°C, to obtain the core layer particles. 0.2% nano silica (gas phase method) was used during the spray drying process to prevent the core layer particles from clumping.

[0044] (4) Chitosan with a degree of deacetylation ≥ 85% was dissolved in 1% acetic acid solution, and calcium pectate was dissolved in deionized water. They were mixed in a 1:1 volume ratio and the pH was adjusted to 5.5 to obtain a chitosan / pectic acid weak gel system.

[0045] (5) Add 20wt% ammonium dihydrogen phosphate solution to an industrial grade ethyl acetate solution of PLA with Mw=50kDa, emulsify and evaporate the solvent to obtain PLA-coated ammonium dihydrogen phosphate microspheres.

[0046] (6) Disperse PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles (20-100nm) together in water containing 0.5wt% Tween 80 and ultrasonically disperse them together for 20min (or use a homogenizer at 50MPa) to form a homogeneous colloidal solution.

[0047] (7) After premixing the core layer particles and magnesium stearate, place them in a fluidized bed. Set the spraying speed to 2 mL / min and the air pressure to 0.3 MPa. Spray the chitosan / pectinic acid weak gel system and homogeneous colloidal solution onto the surface of the core layer particles in sequence. After drying, the long-acting bio-fertilizer is obtained. Drying conditions: inlet temperature 40℃ (to prevent heat damage from DSE bacteria), outlet humidity 15%. Example 3

[0048] In this embodiment, the composition of the long-acting bio-fertilizer is adjusted as follows: the content of DSE strains is 1×10 8 The composition includes: CFU / g, trehalose content 8wt%, carboxymethyl cellulose content 5wt%, xanthan gum content 2.5wt%, calcium ion content 0.2mol / kg, and magnesium ion content 0.4mol / kg. The chitosan / calcium pectate membrane content is 15wt%, PLA-coated ammonium dihydrogen phosphate microspheres content is 24wt%, and humic acid nanoparticles content is 8wt%. All of the above materials are biodegradable. The remaining components are deionized water and necessary process additives. Example 4

[0049] The test investigated the growth-promoting effects of the long-acting bio-fertilizer from Examples 2-3 and the application of DSE strains alone (equal application rates per unit area based on bacterial count) to the rhizosphere of potted plants (oats and astragalus). The experiments in this example included the following test items: I. Test Plants Test plant 1: Due to the short growth cycle of oats, oats were selected as the test plant. Ten days after sowing, DSE microbial cells and slow-release bio-fertilizer were applied to compare the growth-promoting effects of the two forms of growth promoters in the plant.

[0050] Test plant 2: Astragalus membranaceus, seeds from Darhan Muminggan United Banner. Samples were taken 30 days after application of DSE inoculum or slow-release bio-fertilizer for index determination. Several root segments were taken for colonization rate determination and morphological observation. Five plants were randomly selected from each treatment for growth index determination. A suitable amount of leaves were taken for physiological index determination.

[0051] Both of the tested plants were potted plants.

[0052] II. Application Method Application method: Inject the DSE bacterial solution into the soil around the seedling roots using a syringe, with an inoculation amount of 10 mL per seedling. Prepare a suspension of the long-acting bio-fertilizer and inject it into the soil around the seedling roots using a syringe, ensuring that the long-acting bio-fertilizer comes into direct contact with the main root of the crop.

[0053] Preparation method of DSE bacterial culture: The pure dry matter content of each milliliter of bacterial culture is 32 mg. The strain is inoculated into PDA culture medium and placed in an environment at 25℃, and fermented at 120 rpm for 15 days to allow the fungi to grow and reproduce in the culture medium. The cultured mycelium is filtered through a 230-mesh sieve (i.e., sieve opening size of 63 μm) to remove larger particles and incompletely broken mycelium in order to obtain a relatively pure fermentation broth. Then, the mycelium on the sieve is rinsed three times with sterile water to ensure that all attached mycelium is washed off. Finally, the washed mycelium is placed in a mixer, 500 mL of sterile water is added, and the mixture is stirred and broken for 40 seconds to prepare the bacterial culture.

[0054] III. Sampling and Measurement Samples were taken 80 days after inoculation for index determination. Several root segments were taken for colonization rate determination and morphological structure observation. Five plants were randomly selected from each treatment for growth index determination. An appropriate amount of leaves were taken for physiological index determination.

[0055] IV. DSE colony rate determination The root DSE staining method was used, as follows: ① Transparent: Rinse the roots soaked in FAA fixative, cut them into small sections of about 1cm, place them in a test tube containing a certain amount of 10% KOH, heat in a 90℃ water bath for 20 minutes, then discard the KOH, gently rinse the root sample several times with tap water and drain the water.

[0056] ② Decolorization: After decolorizing with hydrogen peroxide for 3 minutes, rinse the root sample several times with tap water and then drain the water.

[0057] ③ Acidification: Soak the root samples in a 5% lactic acid solution for 5 minutes to acidify them. After acidification, gently rinse the root samples several times with tap water and then drain them.

[0058] ④ Staining: Soak the root segments in 0.05% tribenzyl blue staining solution and stain at room temperature for 24 hours.

[0059] ⑤ Decolorization: After dyeing, recover the dye solution, rinse gently with tap water several times, soak for 12 hours, and decolorize with lactic acid glycerin solution.

[0060] ⑥ Slide preparation: Randomly select 60 root segments, arrange them neatly horizontally on a glass slide, soak them in polyethylene-lactic acid-glycerin, and place a coverslip on top.

[0061] The infection status of each root segment was observed under a microscope, and the DSE colonization rate (i.e., infection rate) of the sample was calculated according to the formula: In the formula: 10%, 20%, etc., refer to the grading weights of the root segment infection degree, representing the percentage of the root segment infected by DSE (infection intensity). 0%: The root segment is completely uninfected by DSE (level 0); 10%: Approximately 10% of the root segment is infected by DSE hyphae / microsclerotia (level 1); 20%: Approximately 20% of the root segment is infected by DSE (level 2); 100%: 100% of the root segment is infected by DSE (highest level).

[0062] V. Measurement of growth indicators All the Astragalus membranaceus in the pot was removed for growth index testing.

[0063] (1) Determination of plant height, root length, and longest lateral root length: Using a ruler, the distance from the base of the plant to the highest point is the plant height, and the distance from the root to the end of the main root is the root length. Among all lateral roots, select the longest one and measure it, which is the longest lateral root length.

[0064] (2) Measurement of stem and root diameter: Use vernier calipers to measure at a uniform location on the stem and root of the plant.

[0065] (3) Biomass determination: The plants were carefully washed with clean water to remove surface dirt, dust, and other impurities to ensure accurate measurement results. After washing, the surface moisture of the plants was gently absorbed with absorbent paper to prevent excess moisture from interfering with subsequent fresh and dry weight measurements. After the fresh weight was measured, the plants were packaged into resealable bags and numbered. They were then placed in an oven at 105℃ for blanching. After 10 minutes, the temperature was reduced to 65℃ and the drying continued until the weight of the plants no longer changed. The dry weight was then obtained by weighing the plants again using an electronic scale.

[0066] VI. Measurement of Physiological Indicators Physiological indicators reflect a plant's growth status, health condition, and stress resistance. Chlorophyll, an essential component of photosynthesis, directly reflects the plant's photosynthetic capacity. MDA (methyl hydroxylamine) is an oxidation product produced by plants under abiotic stress; its content reflects the degree of lipid peroxidation in plant cell membranes. Higher MDA content indicates stronger abiotic stress and more severe cell damage in Astragalus membranaceus. Soluble sugars and soluble proteins play a role in maintaining osmotic pressure balance inside and outside cells. Their content is closely related to a plant's stress resistance.

[0067] (1) Chlorophyll content Chlorophyll content was determined using the ethanol extraction colorimetric method. Several leaves were selected, chopped, mixed thoroughly, and 0.5 g was quickly weighed and placed in a 50 mL graduated test tube. 20 mL of 95% ethanol was added, and the tube was sealed. The tube was placed in a dark room at room temperature, and the fading of the leaves was observed after 24 hours until all leaves had completely turned green, thus obtaining accurate chlorophyll extraction. The absorbance was then measured using a UV spectrophotometer at wavelengths of 665 nm and 649 nm.

[0068] Calculated according to the formula: Ca = 13.95 × A665 - 6.88 × A649 Cb = 24.96 × A649 - 7.32 × A665 C = [(Ca + Cb) × V] / (m × 1000) In the formula: Ca and Cb are the contents of chlorophyll a and chlorophyll b, respectively (mg / L); C is the chloroplast pigment content (mg / g); A665 and A649 are the absorbance values ​​at wavelengths of 665nm and 649nm, respectively; V is the volume of the extract (mL); and m is the sample mass (g).

[0069] (2) Malondialdehyde (MDA) content The thiobarbituric acid (TBA) colorimetric method was used for determination. Prepare 10% trichloroacetic acid (TCA) solution and 0.6% TBA solution in advance. Weigh 0.3g of leaf material and place it in a 2mL centrifuge tube. After a period of time, remove the tube and grind it into powder using a sample grinder. Add 3mL of 10% chloroacetic acid solution and grind until homogenized. Transfer the homogenate to a centrifuge tube and centrifuge (4000 rpm) for 10 minutes. Take 2mL of the supernatant and add 2mL of 0.6% TBA solution. Mix well and heat in a 95℃ water bath for 30 minutes. After cooling, centrifuge and collect the supernatant. Using distilled water as a blank control, measure the absorbance at 450nm, 532nm, and 600nm.

[0070] Calculated according to the formula: MDA concentration (μmol / L) = 6.45 × (A532 - A600) - 0.56 × A450 MDA content (μmol / g) = C × W / V In the formula: C is the MDA concentration (μmol / L) calculated by substituting into the formula; A532, A600 and A450 are the absorbance values ​​at wavelengths of 532nm, 600nm and 450nm, respectively; V is the volume of the extract (mL); W is the fresh weight of the sample (g).

[0071] (3) Determination of soluble sugar content The anthrone colorimetric method was used for determination. 0.1 g of leaf material was weighed and placed in a centrifuge tube in liquid nitrogen. After a period of time, the material was removed and ground into powder using a grinder. The powder was then transferred to a volumetric flask with distilled water and brought to a final volume of 50 mL. After shaking for 30 min, the extract was filtered. 0.4 mL of the extract was taken and brought to a final volume of 1 mL with distilled water. 5 mL of anthrone reagent was immediately added, and the mixture was shaken well and placed in a boiling water bath for 10 min. After cooling, the absorbance at 620 nm was measured using a spectrophotometer.

[0072] Measure the absorbance of glucose standard solutions of different mass concentrations and plot the glucose standard curve (e.g.) Figure 1 (As shown). The standard curve equation is y = 0.0017x + 0.0046, R0 2 =0.9985. Where: y is the absorbance value; x is the mass concentration, μg / mL.

[0073] Calculate the soluble sugar content by referring to the standard curve. Calculate using the formula: Soluble sugar content (%) = 100 × (C × V × dilution factor) / (W × 10) 6 ) In the formula: C is the glucose content per tube obtained from the standard curve (μg / mL); V is the total volume of the extract (mL); W is the sample mass (g).

[0074] (4) Determination of soluble protein content The soluble protein content was determined using the Coomassie Brilliant Blue G-250 staining method. 0.1 g of Astragalus membranaceus leaves were weighed and placed in a 2 mL centrifuge tube in liquid nitrogen. After a period of time, the tube was removed and ground into powder using a grinder. Distilled water was added to form a homogenate, which was then transferred to a centrifuge tube and brought to a final volume of 5 mL. After shaking, the tube was centrifuged (4000 rpm) for 10 min. 0.5 mL of the supernatant was collected in a 10 mL test tube, and 0.5 mL of distilled water and 5 mL of Coomassie Brilliant Blue G-250 were added. After shaking and standing for 3 min, the absorbance at 595 nm was measured using a spectrophotometer. The corresponding soluble protein content was calculated using a standard curve.

[0075] Measure the absorbance of protein standard solutions of different mass concentrations and plot the protein standard curve (see [reference]). Figure 2 (As shown). The standard curve equation is y = 0.0054x + 0.0043, R0 2 =0.9987. Where: y is the absorbance value; x is the mass concentration, μg / mL.

[0076] Calculate the soluble protein content by referring to the standard curve, using the formula: Soluble protein content (μg / g) = C × V × dilution factor / W In the formula: C is the protein concentration per tube obtained from the standard curve (μg / mL); V is the total volume of the extract (mL); W is the sample mass (g).

[0077] VII. Measurement Results 1. The experimental results of the effects of applying growth promoters or DSE bacterial solution on the growth indicators of potted oats are shown in Table 3, and the experimental results of the effects on the physiological indicators of potted oats are shown in Table 4.

[0078] Table 3: Table 4: Note: Free proline was determined according to the "acidic ninhydrin colorimetric method" in GB_T 30987-2020 "Determination of Free Amino Acids in Plants".

[0079] As shown in Table 3, compared with the blank control group, both the long-acting bio-fertilizer prepared in this embodiment of the invention and the DSE bacterial solution applied alone significantly promoted the growth of oat seedlings (Table 3). The plant height, stem diameter, root length, fresh weight, and dry weight of the oats in the long-acting bio-fertilizer group were significantly higher than those in the DSE bacterial solution group. Oat root staining and microscopic observation revealed that DSE fungi could colonize the oat roots, with a colonization rate exceeding 43%. Furthermore, root samples from inoculated oat seedlings yielded only strains identical to those with the applied bacteria, further demonstrating that the growth advantage exhibited by the inoculated oat seedlings was caused by DSE colonization in the oat roots.

[0080] As shown in Table 4, compared with the blank control group, the long-acting bio-fertilizer prepared in the present invention and the DSE bacterial solution applied alone both significantly promoted various physiological and biochemical indicators of oat seedlings. The chlorophyll content, soluble protein content, soluble sugar content and free proline content of oats in the long-acting bio-fertilizer group were significantly higher than those in the DSE bacterial solution group applied alone, while the malondialdehyde content was significantly lower than that in the DSE bacterial solution group applied alone.

[0081] In summary, the long-acting bio-fertilizer prepared according to the embodiments of the present invention has a better growth-promoting effect compared with the bacterial solution applied alone.

[0082] 2. The experimental results of the effects of applying growth promoters or DSE bacterial solution on the growth indicators of potted Astragalus membranaceus are shown in Table 5, the experimental results of the effects on the biomass of potted Astragalus membranaceus are shown in Table 6, and the experimental results of the effects on the physiological indicators of potted Astragalus membranaceus are shown in Table 7.

[0083] Table 5: Table 6: Table 7: Note: POD refers to peroxidase, determined according to GB / T 32131-2015 "Horseradish Peroxidase Activity Detection Method - Colorimetric Method" using a commercially available kit. Table 5 shows that applying the long-acting growth promoter or DSE bacterial solution prepared in this invention significantly improved various growth indicators of Astragalus membranaceus seedlings (Table 5), including increased plant height, stem diameter, root length, root diameter, number of lateral roots, and the longest lateral root length; Astragalus membranaceus biomass also increased (Table 6), with significant increases in aboveground fresh weight, underground fresh weight, total fresh weight, aboveground dry weight, underground dry weight, and total dry weight. Therefore, applying the growth promoter or DSE bacterial solution can promote the growth of Astragalus membranaceus seedlings and the accumulation of biomass in both the aboveground and underground parts. Furthermore, compared to applying DSE bacterial solution alone, the increase in various growth indicators and biomass of Astragalus membranaceus seedlings after applying the long-acting growth promoter prepared in this invention was more significant. As shown in Table 7, after applying the long-acting growth promoter or DSE bacterial solution prepared in this invention, the chlorophyll, soluble protein, soluble sugar, and POD contents of Astragalus membranaceus seedlings were increased, while the malondialdehyde (MDA) content was reduced (Table 7). Compared with the DSE bacterial solution applied alone, the long-acting growth promoter showed better results in increasing the chlorophyll, soluble protein, soluble sugar, and POD contents of Astragalus membranaceus seedlings and reducing the MDA content. Therefore, the long-acting bio-fertilizer of this invention can better improve the stress resistance of Astragalus membranaceus plants and the nutrient content in the leaves. Example 5

[0084] This embodiment further compares the growth-promoting effects of the long-acting growth promoter of Example 2 and DSE bacterial solution applied alone on Astragalus membranaceus in the field. The experiment in this embodiment includes the following parts: I. Information on tested plants and sample plots The tested plant was *Astragalus mongholicus*, which was an annual seedling. The field experiment was conducted in the Hailiutu Science and Technology Park, Tuozuo Banner, Hohhot, Inner Mongolia.

[0085] II. For the application method, please refer to Example 4.

[0086] III. Sampling and Measurement Astragalus membranaceus samples were taken 6 months after planting for index determination, yield measurement, and statistical analysis of root rot susceptibility (Fusarium oxysporum). Several root segments were taken for colonization rate determination and morphological observation. Five plants were randomly selected from each treatment for growth index determination. A suitable amount of roots were taken for physiological index determination.

[0087] IV. For the determination of DSE colonization rate, growth indicators and physiological indicators, please refer to Example 4.

[0088] V. Measurement Results The experimental results of the effects of applying growth promoters or DSE bacterial solution on the growth indicators of Astragalus membranaceus in the field are shown in Table 8; the experimental results of the effects on the physiological indicators of Astragalus membranaceus in the field are shown in Table 9; and the experimental results of the effects on the root colonization rate of Astragalus membranaceus in the field are shown in Table 10.

[0089] Table 8: Table 9: Table 10: As shown in Table 8, the application of the long-acting growth promoter or DSE bacterial solution prepared in this invention can effectively promote the growth and biomass accumulation of two-year-old Astragalus membranaceus, increase the root length, root diameter, fresh weight and dry weight of two-year-old Astragalus membranaceus, increase the yield per mu of Astragalus membranaceus, and reduce the incidence of root rot. Among them, after the application of the long-acting growth promoter, the incidence of root rot of Astragalus membranaceus decreased to 39%, indicating that the long-acting growth promoter has a very good biocontrol effect on root rot of Astragalus membranaceus, and its biocontrol effect is better than that of the bacterial solution applied alone.

[0090] Table 9 shows that applying the long-acting growth promoter prepared in this invention or DSE bacterial solution effectively increased the content of soluble protein, soluble sugar, and free proline in the roots of two-year-old Astragalus membranaceus, while simultaneously reducing the malondialdehyde (MDA) content. Furthermore, compared to applying DSE bacterial solution alone, the long-acting growth promoter prepared in this invention showed a greater increase in the content of soluble protein, soluble sugar, and free proline in Astragalus membranaceus roots, and a greater reduction in MDA content than applying DSE bacterial solution alone. Therefore, the long-acting growth promoter of this invention can significantly improve the stress resistance of Astragalus membranaceus plants in the field and the content of nutrients in the roots.

[0091] As shown in Table 10, on day 30 after applying the long-acting growth promoter or DSE bacterial solution prepared according to this invention, the DSE colonization rate was higher with the bacterial solution injected alone. However, by day 90, the DSE colonization rate of Astragalus membranaceus roots was higher with the bacterial solution applied in the form of the long-acting growth promoter. By day 120, the colonization rate of Astragalus membranaceus roots with DSE applied in the form of the long-acting growth promoter far exceeded that of the injected bacterial solution. This indicates that as Astragalus membranaceus matures, the DSE colonization rate of its roots decreases, but the colonization rate increases after treatment with the long-acting growth promoter. This demonstrates that the long-acting growth promoter of this invention can achieve long-term DSE colonization, thereby promoting plant growth.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A long-acting bio-fertilizer containing DSE strain, characterized in that, It includes a core layer and a shell layer; the core layer contains trehalose, a slow-release carrier, and a DSE strain; the slow-release carrier is a carboxymethyl cellulose-xanthan gum composite matrix, on which calcium and magnesium ions are loaded; the DSE strain has the accession number CGMCC No.42008, the accession date is May 19, 2025, and its classification name is Paraphoma pye, with the name HQ-21; The shell comprises a pH-responsive chitosan / calcium pectate membrane, PLA-coated ammonium dihydrogen phosphate microspheres, and humic acid nanoparticles.

2. The long-acting bio-fertilizer according to claim 1, characterized in that, The molar ratio of calcium ions to magnesium ions is 1:2 to 2:

1.

3. The long-acting bio-fertilizer according to claim 1, characterized in that, The pH-responsive chitosan / calcium pectate membrane degrades at pH ≤ 6.5 and has a thickness of 50-100 μm.

4. The long-acting bio-fertilizer according to claim 1, characterized in that, The long-acting bio-fertilizer contained 1×10⁻⁶ DSE strains. 6 -1×10 8 The composition includes CFU / g, trehalose content of 5-10wt%, carboxymethyl cellulose content of 3-6wt%, xanthan gum content of 1.5-3wt%, calcium ion content of 0.1-0.3mol / kg, magnesium ion content of 0.2-0.6mol / kg, chitosan / calcium pectate film content of 10-15wt%, PLA-coated ammonium dihydrogen phosphate microspheres content of 20-30wt%, and humic acid nanoparticles content of 5-8wt%.

5. A method for preparing a long-acting bio-fertilizer containing DSE strain, characterized in that, Includes the following steps: S1, Core layer preparation The DSE strain was cultured to the logarithmic growth phase, and then PEG-400 was added. Collect DSE cells by centrifugation at 4℃ and 4000-6000 rpm for 5-8 min; suspend the cells in phosphate buffer containing 4-6% trehalose to obtain a bacterial suspension; dissolve CMC and xanthan gum in hot water at 60-70℃, add CaCl2 and MgCl2, stir to form a homogeneous gel, cool the homogenate to 25℃, mix it with the bacterial suspension, spray dry, control the inlet temperature at 75-80℃ and the outlet temperature at 35-40℃ to obtain core particles; the DSE strain has the preservation number CGMCC No.42008, the preservation date is May 19, 2025, and the classification name is Paraphoma pye, with the name HQ-21; S2, Shell Preparation Chitosan with a degree of deacetylation ≥ 85% was dissolved in 1% acetic acid solution, and calcium pectate was dissolved in deionized water. They were mixed in a 1:1 volume ratio and the pH was adjusted to 5.5 to obtain a chitosan / pectic acid weak gel system. Ammonium dihydrogen phosphate solution was added to an industrial-grade ethyl acetate solution of PLA with a Mw between 40-55 kDa. After emulsification, the solvent was evaporated to obtain PLA-coated ammonium dihydrogen phosphate microspheres. The PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles were then dispersed together in water to form a homogeneous colloidal solution. S3, Composite Coating The core particles are placed in a fluidized bed, and a chitosan / pectinic acid weak gel system and a homogeneous colloidal solution are sprayed on them. After drying, the long-lasting bio-fertilizer is obtained.

6. The preparation method according to claim 5, characterized in that, In S1, when the bacterial cells contain two types, the bacterial cells are cultured separately to the logarithmic phase, and then the wet bacterial cells are collected by centrifugation. The two or more wet bacterial cells are then mixed according to a predetermined ratio.

7. The preparation method according to claim 5, characterized in that, In S1, CMC and xanthan gum are dissolved in hot water at 60°C at a mass ratio of 2:1, and then CaCl2 and MgCl2 at a molar ratio of 1:2 are added and stirred to form a homogeneous gel.

8. The preparation method according to claim 5, characterized in that, In S2, the concentration of ammonium dihydrogen phosphate solution is 20-25 wt%. The ammonium dihydrogen phosphate solution is added to an industrial-grade ethyl acetate solution of PLA with a Mw between 50 kDa. After emulsification, the solvent is evaporated to obtain PLA-coated ammonium dihydrogen phosphate microspheres. Then, the PLA-coated ammonium dihydrogen phosphate microspheres and humic acid nanoparticles are co-dispersed in water containing 0.1-0.5 wt% Tween 80 and ultrasonically dispersed for 15-30 min to obtain a homogeneous colloidal solution.

9. The preparation method according to claim 5, characterized in that, In S3, the spraying speed is 2 mL / min and the air pressure is 0.3 MPa.

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