A strain of nils from lemna and a hydrogel bacterial agent prepared therefrom and applications thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a leguminosus from lemna and a hydrogel bacterial agent prepared from the leguminosus and application. The present application provides a new leguminosus with the abilities of phosphorus solubilization and nitrogen fixation, and the leguminosus can secrete IAA and iron carrier and has the plant growth promoting effect. Based on the leguminosus GY23, the present application further provides a hydrogel. The hydrogel can improve the soil properties, enhance the activities of enzymes such as urease and phosphatase in the soil, increase the contents of available nitrogen and phosphorus in the soil, and can release the leguminosus GY23 slowly and promote the plant growth for a long time. The leguminosus and the bacterial agent provided by the present application can serve different crops at the same time, and the product has high practical value and strong market adaptability. Moreover, the preparation process of the bacterial agent provided by the present application is simple, the raw materials are easy to obtain, the conditions are mild, the operation is simple, and the preparation method is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of endophytic Niell bacillus derived from duckweed, a hydrogel bacterial agent prepared from it, and its application. Background Technology

[0002] The excessive use of chemical fertilizers and pesticides is a significant factor restricting sustainable agricultural development, easily leading to soil compaction, acidification, and a decline in organic matter. Developing bio-agents using beneficial microorganisms is an important way to achieve green and high-yield crops. Some microorganisms possess rhizosphere growth-promoting properties, promoting crop growth through mechanisms such as nitrogen fixation, phosphorus solubilization, and the secretion of plant growth hormones. However, existing microbial inoculants generally suffer from low cell survival rates, weak rhizosphere colonization ability, and poor tolerance to environmental stresses (such as ultraviolet radiation and drought) in field applications, resulting in unstable growth-promoting effects.

[0003] The existing technology still has the following shortcomings: 1. Existing reports do not adequately explore broad-spectrum and efficient strains that have multiple growth-promoting functions (such as nitrogen fixation, phosphorus solubilization, and hormone production) and can be adapted to both rice and leafy vegetable crops; 2. Existing research focuses on the direct growth-promoting effect of microbial agents, and does not pay enough attention to the comprehensive benefits of simultaneously improving soil microecology and enhancing the soil's sustainable fertilization capacity after the application of microbial agents.

[0004] Therefore, developing a microbial agent based on a new, highly efficient strain that combines the functions of promoting growth in multiple crops and improving soil is of great significance for achieving green agricultural production and sustainable development. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of endophytic Nyctalobacterium derived from duckweed, a hydrogel bacterial agent prepared therefrom, and its applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: a strain of Niallia taxi GY23, which was deposited on April 23, 2026 at the China General Microbiological Culture Collection Center, with the accession number CGMCC NO.38415.

[0007] Correspondingly, a bacterial preparation was prepared using the aforementioned Niger GY23.

[0008] Accordingly, a hydrogel is provided, wherein the hydrogel contains the *Nyctalobacterium* GY23 as described in claim 1. The hydrogel also comprises sodium alginate, carboxymethyl chitosan, and calcium chloride.

[0009] Accordingly, the preparation method of the hydrogel includes the following steps:

[0010] (1) Prepare sodium alginate solution and carboxymethyl chitosan solution respectively;

[0011] (2) Prepare a culture medium of Niger GY23, and collect the cells of Niger GY23 by centrifuging the culture medium;

[0012] (3) The cells of the bacterium Niger GY23 were mixed with the sodium alginate solution to prepare a cell-sodium alginate mixture;

[0013] (4) The bacterial cell-sodium alginate mixture is mixed with the carboxymethyl chitosan solution to obtain a bacterial cell-polymer mixture;

[0014] (5) The bacterial cell-polymer mixture is mixed with calcium chloride solution to obtain the hydrogel.

[0015] Accordingly, the use of the Niger bacteria GY23 or the bacterial preparation in the production of IAA and / or siderophores.

[0016] Accordingly, the application of *Nyctalobacterium* GY23, the bacterial preparation, the hydrogel, or the hydrogel prepared using the aforementioned method in promoting plant growth. The plants include rice and Shanghai bok choy.

[0017] Accordingly, the application of *Nyctalobacterium* GY23, the bacterial preparation, the hydrogel, or the hydrogel prepared by the method thereof in soil improvement. The application includes enhancing the activity of urease and phosphatase in the soil, and increasing the content of available nitrogen and phosphorus in the soil.

[0018] The present invention has the following beneficial effects:

[0019] This invention provides a novel, highly efficient growth-promoting strain: *Nyctalobacterium* GY23. This strain exhibits significant growth-promoting potential on a variety of crops, overcoming the problems of existing strains having limited functionality and application targets.

[0020] Based on the aforementioned Niger GY23, this invention also developed a highly stable form of inoculant: the strain is embedded and fixed in a sodium alginate-carboxymethyl chitosan-calcium chloride ion crosslinking hydrogel system, which greatly improves the survival ability of live bacteria under adverse environments and solves the problem of unstable field effects of microbial inoculants.

[0021] The microbial agent of this invention can not only effectively promote the growth and development of rice and Shanghai bok choy, but also synergistically improve the soil micro-ecological environment, increase soil enzyme activity and effective nutrient content, and has the potential to reduce fertilizer use and increase efficiency, thus solving the problem that existing technologies do not pay enough attention to the comprehensive benefits of soil improvement.

[0022] The microbial agent provided by this invention is multi-purpose, serving different crops simultaneously. It has high practical value and strong market adaptability. Furthermore, the preparation process of the microbial agent provided by this invention is simple, the raw materials are readily available, the conditions are mild, and the operation is straightforward, making it suitable for large-scale production. Attached Figure Description

[0023] Figure 1 A schematic diagram showing the comparison of different duckweed growth rates;

[0024] Figure 2 A schematic diagram showing the growth rate of duckweed 7 days after different strains were reintroduced;

[0025] Figure 3 This is a colony morphology diagram of strain GY23;

[0026] Figure 4 Gram staining image of strain GY23;

[0027] Figure 5 A phylogenetic tree constructed based on the 16S rRNA gene sequence;

[0028] Figure 6 A phylogenetic tree constructed based on 31 housekeeping genes;

[0029] Figure 7 Structural characterization of PMH and GY23-PMH;

[0030] Figure 8 The activity control diagram of strain GY23 with added PMH at 2, 4, 6 and 8 h;

[0031] Figure 9 The activity control diagrams for PMH and GY23-PMH at weeks 1, 3, and 5 are shown.

[0032] Figure 10 A control diagram showing the effect of hydrogel bacterial agents on the growth of hydroponic rice seedlings;

[0033] Figure 11 Figure showing the effect of hydrogel bacterial agent on the growth of hydroponic rice seedlings;

[0034] Figure 12 A control diagram showing the effect of hydrogel bacterial agents on the growth of potted rice;

[0035] Figure 13 A control diagram showing the effect of hydrogel bacterial agents on rice yield;

[0036] Figure 14 The diagram shows the effect of hydrogel bacterial agents on rice grains and panicles.

[0037] Figure 15 A control diagram showing the effect of hydrogel bacterial agent on the growth of Shanghai bok choy;

[0038] Figure 16 The figure shows the effect of hydrogel bacterial agent on the growth of Shanghai bok choy. Detailed Implementation

[0039] This invention provides a novel endophytic Niallia taxi strain GY23 derived from duckweed, whose 16S rDNA sequence is shown in SEQ ID NO: 1. Niallia taxi GY23 was deposited on April 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 38415. Niallia taxi GY23 possesses phosphorus solubilization and nitrogen fixation capabilities, and can secrete IAA (indole-3-acetic acid) and siderophores, exhibiting plant growth-promoting effects.

[0040] Based on the aforementioned *Nyctalobacterium* GY23, this invention also provides a hydrogel. The hydrogel comprises *Nyctalobacterium* GY23, sodium alginate, carboxymethyl chitosan, and calcium chloride. Sodium alginate and carboxymethyl chitosan serve as the backbone materials, undergoing ionic cross-linking under the action of calcium chloride to form a three-dimensional network hydrogel encapsulating *Nyctalobacterium* GY23. The volume ratio of sodium alginate:carboxymethyl chitosan:calcium chloride is 1:1:0.2. An optional feature is that the OD of *Nyctalobacterium* GY23 in the hydrogel... 600 It ranges from 0.5 to 1.0.

[0041] The present invention also provides a method for preparing the hydrogel, comprising the following steps:

[0042] 1. Prepare a 14 g / L sodium alginate (SA) solution (0.15 mol / L NaCl) and a 6 g / L carboxymethyl chitosan (CMCS) solution (distilled water) respectively.

[0043] 2. Take an OD equal to the final volume of the target hydrogel. 600 =0.5-1.0 of Niger GY23 culture medium, centrifuge to collect bacterial cells, and mix the bacterial cells with 0.4-0.5 times the target final volume of SA solution to prepare bacterial cell-SA mixture;

[0044] 3. Add an equal volume of CMCS solution to the bacterial cell-SA mixture to obtain the bacterial cell-SA-CMCS mixture;

[0045] 4. The bacterial cell-SA-CMCS mixture is mixed with a 7.8 g / L calcium chloride solution to carry out an ionic cross-linking reaction, forming a hydrogel bacterial agent encapsulating the bacterial cells. The volume of the calcium chloride solution is 8% to 12% of the volume of the bacterial cell-SA-CMCS mixture.

[0046] The hydrogel can improve soil properties, enhance the activity of enzymes such as urease and phosphatase in the soil, increase the content of available nitrogen and phosphorus in the soil, and can slowly release the Niger bacteria GY23 to promote plant growth in a long-term manner.

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0048] The culture media and reagents used in the examples are as follows:

[0049] 1. Culture medium formula for bacterial isolation

[0050] (1) LB medium: 10g peptone, 5g yeast extract, 10g sodium chloride, 18g agar and 1000mL distilled water, pH 7.1-7.4, sterilized at 121℃ for 20min.

[0051] (2) Beef extract peptone medium: 3g beef extract, 10g peptone, 5g sodium chloride, 15g agar and 1000mL distilled water, pH 7.4-7.6, sterilized at 121℃ for 20min.

[0052] (3) Gao's No. 1 culture medium: 20g soluble starch, 1g potassium nitrate, 0.5g sodium chloride, 0.5g dipotassium hydrogen phosphate trihydrate, 0.5g magnesium sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate and 1000mL distilled water, pH 7.4-7.6, sterilized at 121℃ for 20min.

[0053] (4) Czapek's medium: 30g sucrose, 2g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 0.01g ferrous sulfate heptahydrate, 18g agar and 1000mL water, pH 7.0-7.2, sterilized at 121℃ for 20min.

[0054] (5) PDA culture medium: Peel 200g of potatoes and cut them into pieces about 2cm in size. 2 Add small pieces of the liquid to a 1500mL beaker and boil for 30 minutes, stirring gently with a glass rod to prevent burning. Then filter through double-layered gauze, add 20g of glucose and 13g of agar to the filtrate, and add distilled water to bring the total volume to 1000mL. Sterilize at 121℃ for 20 minutes.

[0055] 2. Culture medium formulation for in vitro plant growth-promoting characteristics determination

[0056] (1) Assumption nitrogen-free medium: 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 5g calcium carbonate, 0.1g calcium sulfate, 10g mannitol, 15g agar powder, add distilled water to 1000mL, pH 7.0~7.1, autoclave at 121℃ for 15min.

[0057] (2) PKO inorganic phosphorus medium: 10g glucose, 5g calcium phosphate, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g magnesium sulfate monohydrate, 0.03g ferrous sulfate heptahydrate, 0.5g ammonium sulfate, 0.2g lecithin, 0.5g yeast extract, 20.0g agar, add distilled water to 1000mL, pH 7.0~7.5, autoclave at 121℃ for 15min.

[0058] (3) Mongkina Organic Phosphorus Medium: 10g glucose, 5g calcium carbonate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate monohydrate, 0.03g ferrous sulfate heptahydrate, 0.5g ammonium sulfate, 0.2g lecithin, 0.5g yeast extract, 20g agar, add distilled water to 1000mL, pH 7.0~7.5, autoclave at 121℃ for 15min.

[0059] (4) CAS detection medium: 60.5 mg Crazin S (CAS), 72.9 mg hexadecyltrimethylammonium bromide (HDTMA), 2.645 mg ferric chloride hexahydrate, 1213.5 mg sodium dihydrogen phosphate dihydrate, 125 mg ammonium chloride, 37.5 mg potassium dihydrogen phosphate, 62.5 mg sodium chloride, 9000 mg agar and 1000 mL distilled water, pH 6.8-6.9, autoclaved at 116℃ for 30 min.

[0060] 3. Components of the culture medium used for duckweed cultivation

[0061] Hoagland culture medium: 20 mL of solution A (59 g / L calcium nitrate tetrahydrate, 15.76 g / L potassium nitrate and 34 g / L potassium dihydrogen phosphate), 1 mL of solution B (3 g / L tartaric acid), 1 mL of solution C (5.6 g / L ferrous sulfate heptahydrate), 1 mL of solution D (9 g / L ethylenediaminetetraacetic acid), 10 mL of solution E (50 g / L magnesium sulfate heptahydrate), 1 mL of solution F (2.86 g / L boric acid, 0.22 g / L zinc sulfate heptahydrate, 0.12 g / L sodium molybdate, 0.08 g / L copper sulfate pentahydrate and 3.62 g / L manganese chloride tetrahydrate), 15 g sucrose, and 966 mL distilled water, pH 5.0–5.5, sterilized at 121°C for 20 min.

[0062] 4. International Rice Research Institute (IRRI) nutrient solutions: Solution A 1 mL (calcium chloride dihydrate 147 g / L); Solution B 1 mL (ammonium chloride 106.98 g / L); Solution C 1 mL (potassium dihydrogen phosphate 40.83 g / L); Solution D 1 mL (potassium sulfate 60.9 g / L); Solution E 1 mL (sodium silicate nonahydrate 142.1 g / L); Solution F 1 mL (manganese chloride tetrahydrate 1.781 g / L, sodium molybdate dihydrate 0.0944 g / L, boric acid 1.2366 g / L, zinc sulfate heptahydrate 0.2214 g / L, copper sulfate hexahydrate 0.0799 g / L); Solution G 1 mL (ferrous sulfate heptahydrate 5.57 g / L and disodium ethylenediaminetetraacetate 7.45 g / L, complexed by heating in a constant temperature oven at 70℃ for 2 h); Solution H 5 mL (magnesium sulfate heptahydrate 98.6 g / L). Bring the volume to 1L with distilled water and adjust the pH to 5.0–5.5.

[0063] Example 1: Screening and Identification of Strains

[0064] 1. Screening of superior duckweed strains

[0065] Duckweed was collected from natural water bodies and the explants were disinfected in a clean bench. The duckweed was then sequentially immersed in centrifuge tubes containing 5% sodium hypochlorite and 75% ethanol for 30 seconds each, followed by rinsing with sterile water 3–5 times to obtain sterile duckweed. Sterile duckweed that had undergone at least five consecutive sterile subcultures was used as material for subsequent studies.

[0066] Sterile duckweed was transferred to fresh Hoagland solution containing 1.5% sucrose for cultivation. Subsequent experiments were conducted after its growth was deemed satisfactory. Fifty species of sterile duckweed (belonging to the genera *Lemna minor*, *Lemna polytropoides*, and *Lemna minor*) were randomly selected for the experiment. After expansion culture, excess water was absorbed using sterile filter paper, and 0.5g of fresh weight of each species was weighed and cultured in 250mL Erlenmeyer flasks containing 100mL of Hoagland culture medium. Each strain was divided into three replicates. Cultivation was carried out at 25℃ with a 16h:8h light-dark ratio. After 7 days of cultivation, excess water was absorbed, and the final fresh weight was measured. The 7-day growth rate of the duckweed was used as the screening index to obtain the duckweed strain with the fastest growth rate. The results are as follows: Figure 1 As shown, the growth rate of duckweed strain 0208 (Lemnaaequinoctialis) was significantly higher than that of all other strains.

[0067] 2. Isolation, purification, and screening of endophytic bacteria in duckweed

[0068] (1) Isolation and screening of growth-promoting endophytes

[0069] Sterile *L. aequinoctialis* 0208 in good growth condition was collected, surface-sterilized with 75% ethanol, and rinsed with sterile water. The final rinse solution was plated to verify thorough sterilization. Subsequently, physiological saline was added to homogenize *L. aequinoctialis* 0208, resulting in a tissue homogenate. After serial dilution, the homogenate was plated onto LB agar, beef extract peptone agar, Gao's 1 agar, and Czapek's agar, respectively. After standing for 30 minutes, the culture was incubated upside down at 28°C in the dark for 3–5 days. Based on differences in colony morphology, continuous streaking and purification were performed until pure cultures with consistent morphology were obtained. A total of 31 endophytic bacteria strains were obtained, numbered GY01–GY31.

[0070] (2) Effect of endophytic bacteria re-inoculation on the growth rate of duckweed

[0071] Thirty-one isolates were inoculated into LB liquid medium for activation, then transferred to LB liquid medium at a ratio of 1% (v / v), and cultured with shaking until the logarithmic growth phase. The isolates were then centrifuged, the supernatant discarded, and subsequently diluted with sterile Hoagland broth to OD0.05. 600 ≈0.3. At 25℃, a light-dark ratio of 16 h:8 h, and a light intensity of 5000 Lux, 0.5 g of *L. aequinoctialis* 0208 in good growth condition was co-cultured with the bacterial suspension for 24 hours (OD). 600 (≈0.3), and then the duckweed with attached bacteria was transferred to a 250mL Erlenmeyer flask containing 100mL of sterile Hoagland culture medium and cultured for 7 days. The growth rate after 7 days was used as an indicator to screen for growth-promoting strains.

[0072] The growth rate of duckweed after inoculation with 31 endophytic bacteria was used as an indicator for screening, such as... Figure 2 As shown in the figure. The results indicate that strain GY23 has the most significant effect on promoting the growth of duckweed.

[0073] 3. Identification of strain GY23

[0074] (1) Physiological and biochemical identification

[0075] The colony morphology of strain GY23 is as follows Figure 3 As shown, it is off-white in color, with irregular edges, a raised center, and a smooth, moist, and opaque surface. The Gram staining results of the strain are as follows. Figure 4 As shown, strain GY23 is a Gram-positive bacterium with rod-shaped cells. It has a relatively wide range of growth conditions: it can grow in the pH range of 5–9, and the optimal temperature range is 25–35℃.

[0076] (2) Molecular identification

[0077] DNA was extracted using a bacterial genome extraction kit, and PCR amplification was performed using universal primers 27F and 1492R. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were uploaded to EzBioCloud for homology analysis, and a phylogenetic tree was constructed using MEGA 6.0 software, as shown below. Figure 5 As shown.

[0078] Comparison of the 16S rRNA gene sequence (SEQ ID NO: 1) revealed that strain GY23 is highly similar to several strains of the genus *Niallia*, with similarities exceeding 99% with *Niallia circulans*, *Niallia taxi*, and *Niallia nealsonii*. Since the 16S rRNA gene alone is insufficient for species identification, GY23 is preliminarily identified as *Niallia* sp.

[0079] To further clarify its taxonomic position, a phylogenetic tree was constructed based on 31 housekeeping genes (e.g., Figure 6 (As shown in Table 1). The results showed that strain GY23 and Niallia taxi (GCF 004005475.1) clustered in the same branch. Species delineation was further performed using ANI and dDDH analyses.

[0080] Table 1. ANI and dDDH values ​​of strain GY23 and its closely related species.

[0081]

[0082] The results showed that GY23 and *Niallia taxi* had ANI values ​​exceeding 95% and dDDH values ​​exceeding 70%, both reaching the species definition thresholds for prokaryotic species (ANI ≥ 95%, dDDH ≥ 70%). In contrast, GY23 had ANI values ​​below 95% and dDDH values ​​below 70% compared to other *Niallia* strains. Based on the combined physiological and biochemical characteristics and molecular identification results, strain GY23 was identified as *Nialliataxi*.

[0083] Example 2: Demonstration of the growth-promoting effect of strain GY23

[0084] 1. Phosphorus solubilization ability

[0085] After activation, strain GY23 was inoculated onto PKO inorganic phosphorus medium and Monkina organic phosphorus medium, respectively, and incubated at 180 rpm. -1 The cells were cultured in a shaker at 28°C for 7 days. The results showed that a clear zone formed around the colonies, indicating that strain GY23 has phosphate-solubilizing activity.

[0086] Seven days later, the fermentation broth was centrifuged at 5000 r / min for 20 min to obtain the supernatant. The available phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method, and finally the available phosphorus content in the fermentation broth was calculated according to the formula.

[0087] Available phosphorus content in culture medium (μg / mL) = In the formula, ρ is the mass concentration of P (μg / mL) obtained from the working curve, V is the volume of water used for color development (mL), and T is the volume of water used for color development. s V0 is the volume (mL) of the fermentation broth being measured.

[0088] Each group was set up with three replicates and a blank control (all other conditions were the same, except that no strain was inoculated).

[0089] The results showed that after 7 days of culture, strain GY23 had a solubility of 4.07±0.94 mg / L for organic phosphorus and 138.23±24.44 mg / L for inorganic phosphorus, indicating that the strain has a strong phosphorus solubilizing ability.

[0090] 2. Ability to generate IAA

[0091] After activation, strain GY23 was inoculated into 50 mL of LB liquid medium containing L-tryptophan (200 mg / L). Simultaneously, 1 mL of indoleacetic acid standard (200 mg / L) and sterile water were used as two controls. After incubation at 28℃ and 180 rpm for 2 days with shaking, 5 mL of culture medium was taken and centrifuged at 4℃ and 10000 rpm for 10 min. Then, an equal volume of supernatant and colorimetric reagent was added to a white ceramic plate. Finally, the plate was incubated in the dark for 30 min, and the OD was measured using the blank control group as a reference. 530 value.

[0092] The pink color indicates that strain GY23 can produce IAA. Substitute the OD values ​​from the above sample... 530 The IAA production of strain GY23 was calculated based on the standard curve. The IAA yield after 2 days of fermentation was 32.43 ± 5.37 mg / L.

[0093] 3. Iron-producing capacity

[0094] After activation, single colonies of GY23 were picked and inoculated onto CAS detection medium and incubated at 30°C for 48 hours.

[0095] Observation revealed a yellow halo around the colony, indicating that strain GY23 can produce siderophores. Take 1 mL of OD... 600A GY23 bacterial suspension with a pH of 0.6 was inoculated into 10 mL of CAS liquid medium and cultured at 28 °C with shaking at 180 rpm for 48 h. 5 mL of the culture was centrifuged at 5000 x g for 20 min, with deionized water as a control for zeroing, and the absorbance at 630 nm was measured using a UV spectrophotometer. s Separately, 1 mL of LB medium and 10 mL of CAS liquid medium were mixed and used as a blank control. The absorbance at 630 nm was used as the reference value (A). r The concentration of siderophores produced by the strain is expressed in siderophore units (SU), and the SU value is calculated.

[0096]

[0097] The results showed that the siderophore activity (SU) of strain GY23 after 2 days of fermentation was 72.44±9.41%.

[0098] 4. Nitrogen fixation capacity

[0099] After activation, strain GY23 was inoculated onto Assumption nitrogen-free solid medium. Strain GY23 was able to grow normally on this medium, indicating that strain GY23 has nitrogen-fixing capabilities.

[0100] Example 3: Preparation and structural characterization of hydrogels

[0101] 1. Preparation and structural characterization of PMH and GY23-PMH

[0102] (1) Preparation of basic materials: Sodium alginate (SA) powder was dissolved in 0.15 mol / L sodium chloride solution to reach a concentration of 14 g / L. Carboxymethyl chitosan (CMCS) powder was dissolved in distilled water to reach a concentration of 6 g / L. A calcium chloride (CaCl2) solution of 7.8 g / L was prepared with distilled water. The strain GY23 was cultured in LB liquid medium until OD 600 The concentration is approximately 0.5 or 1.0. Centrifuge the bacterial fermentation broth, discard the supernatant, and obtain the bacterial cells.

[0103] (2) Preparation of PMH hydrogel: Sterilized SA solution and CMCS solution are mixed in a 1:1 volume ratio to form SA-CMCS polymer. Then, 10% volume of sterile CaCl2 solution of SA-CMCS polymer is added and mixed evenly to obtain a hydrogel (Polymer hydrogel, PMH) made of CMCS, SA and CaCl2.

[0104] (3) Preparation of GY23-PMH hydrogel: The bacterial cells were first mixed with SA solution, and then an equal volume of CMCS solution was added. Finally, sterile CaCl2 solution was added at 10% of the above solution volume (SA+CMCS) to prepare hydrogel bacterial agent (GY23-PMH).

[0105] (4) Electron microscopy analysis

[0106] Fresh samples (PMH, GY23-PMH) were sent to Tianjin Hengqu Technology Co., Ltd. for cryo-scanning electron microscopy (cryo-SEM) analysis. The specific operating procedure for cryo-SEM is as follows: First, a cold-resistant adhesive was evenly coated on the sample stage surface. Then, the sample was carefully adhered to the adhesive layer using tweezers. After adhesion, the sample stage was immediately immersed in liquid nitrogen snow for rapid freezing for 30 seconds. Next, the frozen sample was transferred to the preparation chamber under vacuum using a cryogenic preparation transport system. Inside the chamber, the sample was first sublimated at -70°C for 15 minutes, and then sputtered with gold at a current of 10 mA for 60 seconds. After gold plating, the treated sample was transferred to the SEM sample chamber, and the morphology of the sample was observed under the conditions of maintaining a cold stage temperature of -100°C and an accelerating voltage set to 1–30 kV.

[0107] like Figure 7 As shown, cryo-scanning electron microscopy (cryo-SEM) revealed that both PMH and GY23-PMH materials possess a three-dimensional porous network with continuously interconnected channels. PMH exhibits a uniformly distributed porous morphology with interconnected internal pores, resulting in a high specific surface area. This structural characteristic helps maintain internal humidity and gas exchange, thus providing a suitable microenvironment for the encapsulated microorganisms. Observation of GY23-PMH showed that strain GY23 was encapsulated within the three-dimensional network structure of PMH. This encapsulation method can mitigate the direct damage to the bacteria from external environmental stresses and enables the slow release of the strain.

[0108] 2. Changes in bacterial activity and survival rate in hydrogel bacterial agents

[0109] Treatment group (GY23-PMH): 1% PMH was added to LB liquid medium containing 1% GY23 bacterial suspension; Control group (GY23): 1% sterile water was added to LB liquid medium containing 1% GY23 bacterial suspension. Both groups were incubated in a shaking incubator for 2h, 4h, 6h, and 8h, respectively, and the OD was measured using a spectrophotometer. 600 The bacterial cell activity of GY23 in GY23-PMH was determined by the resazurin reduction method, with PMH as the control group, before storage (week 0) and after storage for 1, 3 and 5 weeks.

[0110] The results are as follows Figure 8 , 9 As shown in the figure. The results showed that, compared with the control group, the growth trend of strain GY23 after the addition of PMH was not significantly different in the short term, indicating that PMH itself had no obvious toxic side effects on strain GY23. During long-term storage, the survival rate of strain GY23 in the treated group was consistently significantly higher than that in the control group throughout the storage period. Even after 5 weeks of storage, the treated group still maintained a high survival rate, while the survival rate of the control group decreased significantly. This indicates that PMH can not only serve as a safe carrier for GY23, but also significantly enhance the stability of the strain during storage.

[0111] Example 4: Effects of hydrogel bacterial agents on plant growth

[0112] 1. The growth-promoting effect of hydrogel bacterial agents on rice

[0113] To evaluate the practical application effect of GY23-PMH, four treatments were set up: sterile water control (CK), PMH group, GY23 group, and GY23-PMH group, with three replicates for each group. The number of bacteria in the GY23 group was kept consistent with that in the GY23-PMH group. The GY23 group refers to the group where strain GY23 was cultured to the corresponding absorbance in LB liquid medium before application. The GY23-PMH group was prepared according to the method in Example 3; the GY23 group was prepared using the same volume of the original GY23 bacterial culture (OD) as the GY23-PMH group. 600 =0.5 or 1.0), after centrifugation to collect the bacterial cells, resuspend them in an equal volume of sterile water to obtain a bacterial suspension for application.

[0114] (1) Effects of hydrogel bacterial agents on the growth of hydroponic rice

[0115] After surface disinfection, rice seeds are soaked at 30℃ for 24 hours to promote germination, and then transferred to solutions containing appropriate amounts of sterile water, PMH, GY23, and GY23-PMH (where GY23 has an OD value of 24%). 600 In petri dishes with a concentration of 0.5 μm, the shoots were cultured at 25°C under conditions of a light-dark ratio of 12 h:12 h and a light intensity of 5000 Lux until the shoot length reached 0.5–1.0 cm. They were then transplanted into 1L black hydroponic boxes and cultured in a gradient using nutrient solution from the International Rice Research Institute (IRRI) (increased successively at 1 / 4, 1 / 2, and 1x concentrations for 5 days, 4 days, and 5 days respectively, for a total of 14 days). The water was changed every 2–3 days, and the appropriate solutions (sterile water, PMH, GY23, GY23-PMH, where the OD of GY23 is 0.5 μm) were added during each solution change. 600 =1.0), each treatment was applied directly to the hydroponic box, and the pH value was strictly controlled between 5.0 and 5.5 during the cultivation period, and proper ventilation was maintained.

[0116] The results are as follows Figure 10 , 11 As shown in the figure. The results indicated that the GY23-PMH treatment had a more significant promoting effect on rice seedling growth compared with the GY23 treatment alone. Compared with the CK group, the GY23-PMH treatment significantly increased root length, stem height, and overall plant height by 34.45%, 53.72%, and 47.68%, respectively, while root activity, total chlorophyll content, soluble protein, and soluble total sugar content also significantly increased by 59.88%, 4.30%, 5.24%, and 47.86%, respectively. Notably, there were no significant differences in the physiological indicators of the plants in the PMH treatment group compared with the CK group, indicating that PMH itself had no direct effect on plant growth. Compared with the GY23 strain alone, the GY23-PMH treatment further enhanced root development, increasing root length and root activity to 1.15 and 1.05 times that of the GY23 treatment, respectively. This result may be because PMH, as a carrier, enhances the survival ability of the strain in the rhizosphere, enabling the slow release of the bacteria and its sustained effect, thereby more effectively stimulating root development.

[0117] (2) Effects of hydrogel bacterial agents on rice root development

[0118] Roots are vital absorptive organs in plants, responsible for extracting water and various nutrients from the soil. A well-developed root system is fundamental for high crop yields. Root scanning and quantitative analysis of hydroponic rice roots were performed using a root scanner, and the results are shown in Table 2.

[0119] Table 2 Effects of hydrogel bacterial agents on rice roots

[0120]

[0121] The results showed that both GY23 and GY23-PMH treatments significantly promoted root development in rice. Compared with the control group (CK), the GY23-PMH treatment increased total root length, root surface area, root volume, number of root tips, number of branches, and number of intersections by 78.17%, 70.11%, 65.38%, 57.43%, 63.25%, and 87.05%, respectively. Compared with GY23 alone, the GY23-PMH treatment showed the most significant increase in root surface area and root volume, reaching 1.14 and 1.23 times that of the GY23 group, respectively. These results indicate that hydrogel inoculants may enhance the rice's ability to absorb water and nutrients by promoting root growth in surface area and root branching.

[0122] (3) Effects of hydrogel bacterial agents on the growth of potted rice seedlings

[0123] To evaluate the effects of GY23-PMH in the soil environment, a pot experiment was conducted. Bottomless plastic pots (21 cm in diameter, 17 cm in height) were used and filled with sterilized nutrient soil. Healthy seedlings grown hydroponically for 14 days (using the same hydroponic cultivation method as described above for rice) were transplanted at a rate of 3 seedlings per hole, one hole per pot per treatment, maintaining a water layer of 3–5 cm. On the day of transplanting, each pot was treated with 50 mL of the corresponding solution (sterile water, PMH, GY23, GY23-PMH, where GY23 has an OD value of [missing information]). 600 =1.0), and each treatment was applied directly to the soil. Subsequently, the corresponding solution was applied every 15 days. Pot experiments were conducted in an artificial climate chamber, with the temperature controlled between 25℃ and 30℃. After 3 months of cultivation under a light-dark ratio of 14 h:10 h and a light intensity of 5000 Lux, various indicators of the potted rice seedlings were measured.

[0124] like Figure 12 As shown, the results indicated that the physiological indicators of the PMH-treated group were not significantly different from those of the CK group, thus ruling out the direct effect of PMH itself on plant growth. Compared with the CK group, the GY23-PMH treatment increased the number of tillers, aboveground fresh and dry weight, total chlorophyll content, soluble protein, and soluble total sugar in rice by 35.90%, 46.69%, and 43.29%, 3.60%, 11.14%, and 38.65%, respectively. Compared with the treatment of strain GY23 alone, the rice treated with GY23-PMH showed significant increases in aboveground fresh weight, total chlorophyll content, and soluble protein and soluble total sugar content in leaves, reaching 1.14, 1.01, 1.03, and 1.16 times that of the GY23 group, respectively. These results indicate that PMH, as a carrier of strain GY23, can not only help strain GY23 adapt to the environment more effectively but also enhance its growth-promoting effect.

[0125] (4) Effects of hydrogel bacterial agents on the yield of potted rice

[0126] Another experiment was conducted, with the same cultivation conditions as the aforementioned potted seedlings for the first 3 months (from the growth period to the heading stage). Subsequently, the seedlings were cultivated at 25℃~30℃, a light-dark ratio of 12 h:12 h, and a light intensity of 5000 Lux until maturity, and the rice yield was measured.

[0127] The results are as follows Figure 13 , 14As shown, both inoculated strains GY23 and GY23-PMH significantly increased rice yield, mainly manifested in increased indicators related to panicle development. Compared with the control group (CK), GY23 treatment increased panicle length and grains per panicle by 20.69% and 20.63%, respectively, while GY23-PMH treatment increased them by 23.17% and 29.55%, respectively, showing a more significant promoting effect. GY23-PMH showed particularly outstanding performance in panicle weight and effective tillering. Compared with the single strain GY23 treatment, GY23-PMH treatment increased fresh weight per panicle, dry weight per panicle, and number of panicles per pot by 24.03%, 16.73%, and 14.29%, respectively. The increase in the number of panicles per pot directly reflects the promoting effect of the inoculant on effective tillering in rice, while the increase in fresh weight and dry weight per panicle indicates more robust panicle development and more complete grain filling. The results showed that the GY23-PMH inoculant was superior to single-strain treatment in promoting rice panicle development, increasing the number of grains per panicle, and promoting effective tillering, thus exhibiting better yield-increasing effects.

[0128] 2. The growth-promoting effect of hydrogel bacterial agents on Shanghai bok choy

[0129] To verify the broad-spectrum growth-promoting effect of GY23-PMH, it was applied to Shanghai bok choy. The experiment also included four treatment groups: sterile water control (CK), PMH group, GY23 group, and GY23-PMH group. Shanghai bok choy seeds were sterilized with 1% NaClO for 1 min, then soaked at 30℃ for 24 h to promote germination, and then germinated in solutions containing the corresponding solutions (sterile water, PMH, GY23, and GY23-PMH, where GY23 had an OD value of [missing value]). 600 Germination was carried out in petri dishes with a concentration of 0.5 g / cm³, under controlled temperature of 25°C and suitable light conditions. When the seedlings reached a height of 2–3 cm, they were transplanted into sterile soil. On the day of transplanting and every 15 days thereafter, the appropriate solution (containing OD₂₃ of GY23) was applied. 600 =1.0), each treatment was applied directly to the soil. Forty-five days after planting, the Shanghai bok choy plants were dug up, rinsed with water, and various physiological indicators of the plant tissues were measured.

[0130] The results are as follows Figure 15 , 16As shown, compared with the CK group, the various physiological and biochemical indicators of Shanghai bok choy were significantly improved after treatment with GY23-PMH. Specifically, plant height, fresh weight of aboveground and root parts, dry weight of aboveground and root parts, number of leaves, and total chlorophyll content increased significantly by 28.41%, 104.02%, 92.54%, 118.52%, 131.31%, 33.33%, and 10.92%, respectively. Simultaneously, the content of nutrients in Shanghai bok choy leaves, such as soluble protein, total soluble sugar, and free amino acids, increased significantly by 14.53%, 56.30%, and 57.28%, respectively, compared to the CK group. Compared with GY23 treatment alone, GY23-PMH inoculant had a more significant growth-promoting effect on Shanghai bok choy. The Shanghai bok choy treated with GY23-PMH showed significantly higher plant height, aboveground and root fresh weight, aboveground and root dry weight, and total chlorophyll content compared to the GY23 treatment group, increasing to 1.15, 1.12 and 1.47, 1.16 and 1.61 and 1.09 times that of the GY23 group, respectively. This indicates that GY23-PMH also promotes the growth of Shanghai bok choy. Simultaneously, the soluble protein and total soluble sugar content in the leaves of Shanghai bok choy treated with GY23-PMH were also significantly higher than those in the GY23 treatment group, increasing by 5.81% and 19.68% respectively, demonstrating the application potential of this microbial agent in improving vegetable quality.

[0131] The above results indicate that GY23-PMH can not only promote the growth of rice, but also effectively improve the nutritional quality of the common vegetable crop, Shanghai bok choy, indicating that the microbial agent has good broad-spectrum host activity, which lays the foundation for its promotion and application on different crops.

[0132] 3. The impact of hydrogel bacterial agents on soil properties

[0133] Soil pH is an important indicator of soil chemical properties, directly affecting soil nutrient availability and microbial activity. Soil enzyme activity is an important indicator reflecting soil biological activity and nutrient transformation capacity. Urease participates in soil nitrogen transformation, while phosphatase is closely related to organophosphorus mineralization. Different treatments also significantly affected soil enzyme activity. The content of available soil nutrients is directly related to the nutrient supply capacity of crops; therefore, the contents of alkaline-available nitrogen and available phosphorus were measured. The content of alkaline-available nitrogen reflects the soil's nitrogen supply capacity. Based on this, the following experiment was conducted:

[0134] The soil samples after planting Shanghai bok choy in step 2 were tested, and the following groups were set up: blank unplanted group (soil from the same source and batch, without Shanghai bok choy or any treatment); CK group (soil from the CK group in step 2 after planting Shanghai bok choy); PMH group (soil from the PMH group in step 2 after planting Shanghai bok choy); GY23 group (soil from the GY23 group in step 2 after planting Shanghai bok choy); and GY23-PMH group (soil from the GY23-PMH group in step 2 after planting Shanghai bok choy).

[0135] Air-dried soil samples that passed through a 1 mm sieve were taken from each group and the following parameters were determined: pH (KCl extraction method), urease activity (sodium phenolate-sodium hypochlorite colorimetric method), neutral phosphatase activity (sodium phenyl phosphate colorimetric method), alkaline nitrogen content (alkaline diffusion method), and available phosphorus content (NaHCO3 extraction-molybdenum antimony colorimetric method). Each sample was tested in triplicate. The results are shown in Table 3.

[0136] Table 3. Effects of hydrogel bacterial agents on soil properties

[0137]

[0138] The results showed significant differences in soil pH under different treatments. Compared with the unplanted control group, the soil pH in the planted control (CK) group decreased by 5.50%, which may be related to the secretion of organic acids or selective absorption of cations by the roots during crop growth. Compared with the CK group, all treatments (PMH, GY23, and GY23-PMH) significantly increased soil pH. The pH of the GY23-PMH treatment was not significantly different from the unplanted control group, indicating that the application of microbial agents can alleviate soil acidification.

[0139] Regarding urease activity, both GY23 and GY23-PMH treatments significantly enhanced its activity, increasing by 2.73 times and 2.72 times respectively compared to the control group, indicating that GY23 and GY23-PMH have a strong potential to promote soil nitrogen transformation. Phosphatase activity, measured using neutral phosphatase as the indicator, showed a similar trend to urease, with GY23-PMH treatment showing the highest activity, followed by GY23 treatment. Both were significantly higher than the control and PMH treatments, indicating that applying GY23 strain alone or preparing it as a microbial inoculant can enhance the mineralization capacity of soil organic phosphorus. Specifically, the changes in soil phosphatase activity after GY23-PMH and GY23 treatments were not significant, while the phosphatase activity after GY23-PMH treatment was significantly increased by 71.39% compared to the control group. The increased phosphatase activity is beneficial for the mineralization and decomposition of soil organic phosphorus, enhancing the supply of available phosphorus in the soil, and is crucial for improving soil phosphorus nutrition.

[0140] The content of available nitrogen was highest in the control (no planting) treatment, followed by the GY23-PMH and GY23 treatments, all significantly higher than the control (CK). This indicates that both the GY23 treatment alone and the GY23-PMH treatment are beneficial for improving soil nitrogen availability. Specifically, the available nitrogen content in the GY23-PMH treatment increased by 8.84% compared to the CK, reaching the highest level among all treatments. Regarding available phosphorus, the GY23-PMH treatment also showed the highest content, significantly higher than the CK, increasing by 19.34% compared to the CK. There were no significant differences among the other treatments. Overall, the trends in available nutrient content in the soil were consistent with the changes in enzyme activity, further confirming the promoting effect of GY23-PMH on soil nutrient transformation.

[0141] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Niallia taxi GY23, characterized by: The described Niger strain GY23 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 23, 2026, with accession number CGMCC NO.38415.

2. The bacterial preparation made using the Niger GY23 of claim 1.

3. A hydrogel, characterized in that: The hydrogel contains the Niger bacteria GY23 as described in claim 1.

4. The hydrogel according to claim 3, characterized in that: The hydrogel also includes sodium alginate, carboxymethyl chitosan, and calcium chloride.

5. The method for preparing the hydrogel according to claim 3 or 4, characterized in that: The preparation method includes the following steps: (1) Prepare sodium alginate solution and carboxymethyl chitosan solution respectively; (2) Prepare a culture medium of Niger GY23, and collect the cells of Niger GY23 by centrifuging the culture medium; (3) The cells of the bacterium Niger GY23 were mixed with the sodium alginate solution to prepare a cell-sodium alginate mixture; (4) The bacterial cell-sodium alginate mixture is mixed with the carboxymethyl chitosan solution to obtain a bacterial cell-polymer mixture; (5) The bacterial cell-polymer mixture is mixed with calcium chloride solution to obtain the hydrogel.

6. The use of the Niger bacteria GY23 of claim 1 or the bacterial preparation of claim 2 in the production of IAA and / or siderophores.

7. The application of the Niger GY23 of claim 1, the bacterial preparation of claim 2, the hydrogel of claim 3 or 4, or the hydrogel prepared by the preparation method of claim 5 in promoting plant growth.

8. The application according to claim 7, characterized in that: The plants mentioned include rice and Shanghai bok choy.

9. The application of the Niger GY23 of claim 1, the bacterial preparation of claim 2, the hydrogel of claim 3 or 4, or the hydrogel prepared by the preparation method of claim 5 in soil improvement.

10. The application according to claim 9, characterized in that: The applications include enhancing the activity of urease and phosphatase in the soil, and increasing the content of available nitrogen and phosphorus in the soil.

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