A strain of serratia plymuthica, microcapsule preparation thereof, and preparation method and application thereof

Abstract

The application discloses a Serratia plymuthica, a microcapsule of the Serratia plymuthica, and a preparation method and application of the Serratia plymuthica, and the preservation number of the Serratia plymuthica CM9 is CGMCC No.39835.The Serratia plymuthica CM9 is a growth-promoting bacterium isolated from strawberry rhizosphere soil, and has growth-promoting properties such as nitrogen fixation, phosphorus dissolution, potassium solution, iron carrier production and IAA production.Meanwhile, the Serratia plymuthica-sodium alginate-chitosan composite microcapsule is prepared by an extrusion method, and the growth-promoting rates of the CM9 microcapsule inoculant on the plant height, root length, fresh weight and dry weight of strawberries are 55.98%, 45.41%, 86.88% and 90.83% respectively, and the growth-promoting effect is remarkable.The strain and the microcapsule inoculant provided by the application can be applied to strawberry cultivation, and can be used as a green and efficient microbial growth promoter, effectively improve the yield and quality of strawberries, and have good commercial development potential.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, specifically to a strain of Serratia plymouthii, its microcapsules, preparation methods, and applications. Background Technology

[0002] Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial bacteria that colonize the rhizosphere of plants and promote plant growth. They possess excellent properties such as nitrogen fixation in conjunction with plants, dissolving and releasing insoluble phosphorus in the soil, producing plant growth hormones (such as IAA), secreting siderophores, and inhibiting pathogens. However, currently commercially available PGPR inoculants are generally applied directly to the soil in liquid or solid powder form. Because the inoculants are directly exposed to the soil environment, they suffer from low survival rates, rapid decline in effective viable counts, short duration of effectiveness, and poor stability when faced with multiple stresses such as soil nutrient depletion, competition from soil-borne microorganisms, and predation by protozoa. This significantly restricts their widespread application in agricultural production.

[0003] Strawberry, a perennial herbaceous plant belonging to the genus *Fragaria* of the Rosaceae family, is widely cultivated in my country due to its nutritious fruit, high economic value, and short cultivation cycle. In recent years, with the continuous expansion of strawberry planting areas, the problem of continuous cropping has become increasingly prominent. Long-term continuous cropping leads to the deterioration of soil physical and chemical properties, imbalance of beneficial microbial communities, and exacerbation of soil-borne diseases. Simultaneously, the excessive use of chemical fertilizers not only causes soil acidification and secondary salinization but also reduces the content of soluble solids and vitamin C in strawberry fruits, seriously affecting fruit quality. These factors have become key bottlenecks restricting the high-quality development of the strawberry industry. Against this backdrop, using growth-promoting microbial agents to replace some chemical fertilizers, achieving reduced fertilizer application and improved quality and efficiency in strawberry cultivation, represents a green and sustainable development path with promising application prospects.

[0004] Therefore, screening for superior strawberry strains with stable growth-promoting effects and developing formulation technologies that can extend their field efficacy are urgent technical problems to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Serratia plymouthii, its microcapsule formulation, preparation method, and application. This strain has multiple growth-promoting functions, including nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and IAA production. Preparing it into a microcapsule formulation can effectively improve the survival rate and duration of action of the strain in soil. When applied to strawberry cultivation, the microcapsule formulation can significantly promote plant growth and improve fruit quality, and is expected to be developed into a green microbial inoculant product that can replace chemical fertilizers.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] This invention provides a strain of Serratia plymouthii CM9, classified and named Serratia plymouthii, which was deposited at the China General Microbiological Culture Collection Center on July 23, 2026, with accession number CGMCC No. 39835.

[0008] To achieve the objectives of this invention, the present invention also provides a microcapsule formulation comprising the aforementioned Serratia pylori CM9.

[0009] To achieve the objective of this invention, this invention also provides a method for preparing the above-mentioned microcapsule formulation, comprising the following steps:

[0010] (1) Sodium alginate solution, chitosan solution and rice starch gelatinization liquid are mixed to obtain sodium alginate-chitosan-rice starch gelatinization liquid, and then Serratia pylori CM9 bacterial solution is added to obtain Serratia pylori CM9 mixed treatment liquid.

[0011] (2) The Serratia pylori CM9 mixed treatment solution was dripped into a calcium chloride solution to cross-link and harden, forming gel particles;

[0012] (3) Wash the cross-linked gel particles to obtain the microcapsule formulation.

[0013] Preferably, in step (1), the mass fraction of the sodium alginate solution is 1% to 3%, the mass fraction of the chitosan solution is 2.5%, and the concentration of the rice starch gelatinized liquid is 3 mg / mL; the volume ratio between the sodium alginate solution, the chitosan solution, and the rice starch gelatinized liquid is 50:25:3.

[0014] Preferably, in step (1), the volume ratio of the Serratia pylori CM9 bacterial solution to the sodium alginate-chitosan-rice starch gelatinized solution is 1:3.12.

[0015] Preferably, in step (2), the mass fraction of the calcium chloride solution is 3%; the cross-linking hardening temperature is 4°C and the time is 24 h.

[0016] Preferably, in step (2), when the mixed treatment liquid is added to the calcium chloride solution, the drop height is 5-8 cm from the liquid surface; in step (3), the washing is rinsing with sterile water 2-3 times.

[0017] To achieve the objectives of this invention, the present invention also provides the application of the above-mentioned microcapsule formulation in promoting strawberry growth.

[0018] Furthermore, the promotion of strawberry growth is achieved through the growth-promoting properties of Serratia pylori CM9, including nitrogen fixation, phosphorus solubilization, potassium solubilization, iron carrier production, and IAA production.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention provides a novel strain of Serratia plymouthii with excellent growth-promoting properties. The Serratia plymouthii CM9 described in this invention was isolated and screened from strawberry rhizosphere soil. This strain has multiple growth-promoting functions, including nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and indoleacetic acid (IAA) production. It can provide multiple pathways for plant growth promotion and has good value for strain resource development.

[0021] (2) This invention uses sodium alginate-chitosan-rice starch gelatinized liquid as the wall material to prepare Serratia plymouthi composite microcapsules by extrusion. This composite wall material is safe, non-toxic, widely available, and inexpensive. The extrusion preparation process is simple, mild, and easy to industrialize, with minimal damage to the activity of the bacterial strain. The Serratia plymouthi microcapsules produced by this invention have stronger viability and stress resistance than traditional liquid live bacteria preparations, making them more suitable for production and application. This solves the problems of short shelf life, poor environmental adaptability, and insufficient long-term effectiveness of current rhizosphere growth promoters.

[0022] (3) The Serratia plymouthii microcapsules prepared in this invention have a significant growth-promoting effect on strawberries. The results of the pot growth-promoting test showed that after applying the Serratia plymouthii CM9 microcapsule agent, the plant height, root length, fresh weight and dry weight of strawberries increased by 55.98%, 45.41%, 86.88% and 90.83% respectively, with a significant growth-promoting effect. This indicates that the microcapsule agent provided by this invention can effectively promote the growth of strawberry plants and has good application prospects and industrial development potential in strawberry cultivation. It can provide technical support for achieving fertilizer reduction and green production in strawberry cultivation. Attached Figure Description

[0023] Figure 1 The results show the determination of nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and IAA production capabilities of strain CM9.

[0024] Figure 2 The images show the colony morphology and Gram staining results of strain CM9 on NB solid medium.

[0025] Figure 3 Phylogenetic tree diagram of 16S rDNA (A) and gyrB (B) of strain CM9.

[0026] Figure 4 The effect of microcapsule inoculum of strain CM9 on the growth-promoting effect of potted strawberries. Detailed Implementation

[0027] The *Serratia pylori* CM9 provided by this invention was deposited on July 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39835, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] To further illustrate the beneficial effects of the present invention, the following specific embodiments are provided. It should be noted that these embodiments are intended to help better understand the present invention and are by no means limited to its scope. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Reagents or instruments used in the following embodiments, unless otherwise specified, are all conventional products that can be purchased through legitimate channels.

[0029] Example 1: Isolation, screening and identification of rhizosphere growth-promoting bacteria

[0030] 1. Isolation and purification of rhizosphere growth-promoting bacteria

[0031] Strawberry rhizosphere soil samples were collected from the Shilixiang strawberry plantation in Mengjin District, Luoyang City, Henan Province, and brought back to the laboratory. These samples were then serially diluted with sterile water to obtain a concentration of 10. -2 -10 -6 For dilution, take 100 μL of the appropriate concentration of dilution and spread it on an NA plate. Incubate in the dark at 28°C with the plate inverted. Pick single colonies with different morphology, color, size, and other characteristics from the plate and purify them on NA plates until a pure culture of bacterial strain is obtained.

[0032] 2. Screening of rhizosphere growth-promoting bacteria

[0033] The isolated and purified strains were inoculated onto Ashby nitrogen-free medium, organic phosphorus (YM) medium, inorganic phosphorus (NBRIP) medium, and potassium feldspar medium, and cultured at 28°C for 3 days. The growth status of the strains was observed, and the results are as follows: Figure 1 As shown. Strain CM9 possesses nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing abilities. Strain CM9 was spot-inoculated onto a CAS blue plate, as shown... Figure 1 An orange halo around the colony indicates that the strain has the ability to produce siderophores; the larger and darker the yellow halo, the stronger the siderophore-producing ability. The Salkowski colorimetric method was used to determine the IAA production capacity of strain CM9, and the results are as follows: Figure 1 As shown, strain CM9 has the ability to produce IAA, and the amount of IAA secreted by CM9 is 26.12 ug / mL.

[0034] 3. Identification of rhizosphere growth-promoting bacteria CM9

[0035] This invention primarily employs a combination of bacterial morphology observation and molecular biology techniques to identify strain CM9. The experimental results are as follows: Figure 2 As shown, strain CM9 grows well on NA medium, producing round or oval, moist colonies with smooth surfaces and edges; they are white and opaque. Gram staining is Gram-negative; the bacteria are short rods, without spores or capsules.

[0036] A single colony of *Serratia pylori* CM9 was dissolved in 10 μL ddH2O as a template. Using primer pairs 27F / 1492R and UP-1 / UP-2r (primer concentration 10 mM) from Table 1, the 16S rDNA and gyrB gene fragments of CM9 were amplified by PCR. The 25 μL PCR amplification system was as follows: 12.5 μL 2×Taq PCR Master Mix, 1 μL each primer, 1 μL template DNA, and 9.5 μL ddH2O. The amplification program is shown in Table 1. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. CM9 was found to possess the 16S rDNA gene sequence shown in SEQ ID No. 1 and the gyrB gene sequence shown in SEQ ID No. 2. After obtaining the target sequences, BLAST alignment analysis was performed using the NCBI database, and homologous sequences were downloaded. A phylogenetic tree was constructed using MEGA 7.0 software and the Neighbor-Joining method. Figure 3 The taxonomic classification of strain CM9 was determined. The 16S rRNA sequence and gyrB gene of strain CM9 showed high homology with *Serratia plymuthica*, with a similarity of up to 99%. Based on morphological characteristics and molecular biological identification, strain CM9 was identified as *Serratia plymuthica*.

[0037]

[0038] Example 2: Preparation of rhizosphere growth-promoting bacteria CM9 microcapsules

[0039] 1. Microcapsule preparation method

[0040] Preparation of the encapsulating agent: Accurately weigh 10 g of rice and place it in 100 mL of distilled water. Heat and stir in a boiling water bath for 50 min to fully gelatinize the starch. Cool to room temperature and centrifuge at 4000 rpm for 10 min. Collect the supernatant, which is the rice starch gelatinized liquid (3 mg / mL). Prepare a 2.5% chitosan solution. Prepare sodium alginate solutions with concentrations of 1%, 2%, and 3%.

[0041] Preparation of Serratia pylori CM9 bacterial suspension: A single colony of Serratia pylori CM9 was inoculated onto NB medium and cultured at 30°C with shaking at 150 rpm for 24 h. The resulting culture was centrifuged at 5000 rpm for 5 min, washed repeatedly, and resuspended in phosphate-buffered saline (PBS) to allow OD to reach the target concentration. 600 The value reaches 1.

[0042] Preparation of crosslinking agent: Prepare a 3% CaCl2 solution for later use.

[0043] Microcapsules were prepared using an extrusion method: First, 50 mL of chitosan solution and 6 mL of rice starch gelatinized liquid were added sequentially to 100 mL of sodium alginate solution of different concentrations to obtain sodium alginate-chitosan-rice starch gelatinized liquids of different concentrations. Then, 50 mL of *Serratia pylori* CM9 bacterial solution was added, and the mixture was stirred evenly with a magnetic stirrer to obtain *Serratia pylori* CM9 mixed treatment solutions, namely, 1% sodium alginate-chitosan-rice starch gelatinized liquid-bacterial solution composite solution, 2% sodium alginate-chitosan-rice starch gelatinized liquid-bacterial solution composite solution, and 3% sodium alginate-chitosan-rice starch gelatinized liquid-bacterial solution composite solution. Using a 10 mL disposable syringe, each group of sodium alginate-chitosan-rice starch gelatinized liquid-bacterial solution composite solution was slowly dripped into 3% CaCl2 solution from a height of 5-8 cm above the liquid surface. After being dropped in, the composite droplet immediately forms small spherical gel particles. All particles are cross-linked and hardened at 4°C for 24 hours to ensure complete cross-linking. After cross-linking, the gel particles are rinsed 2-3 times with sterile water to remove uncross-linked CaCl2. The prepared microcapsule bacterial agent is then placed in a sterile petri dish and stored at 4°C for later use.

[0044] 2. Measurement of various indicators of microcapsules

[0045] Particle size: Observe the morphological characteristics of microcapsules prepared with different sodium alginate concentrations, record the overall morphology, tailing phenomenon and adhesion state of each group of microcapsules; randomly select 100 structurally intact microcapsules and arrange them in an orderly manner, measure the total length of the whole with a ruler, and calculate the average particle size of a single microcapsule.

[0046] Mechanical strength: Four microcapsule particles of similar size were selected and placed horizontally on a glass plate. A plastic petri dish of known weight was placed on top of the microcapsules. The weight was gradually increased in the petri dish until the microcapsules were observed to undergo obvious deformation. The total weight (G) was recorded and the particle mechanical strength (g) was calculated as G / 4.

[0047] Encapsulation efficiency, viable cell count, and fold increase: 0.5 g of microcapsules were taken before and after proliferation, respectively, and placed in 10 mL of 10% sodium citrate solution. The mixture was vigorously shaken using a vortex mixer until the particles were completely dissolved, releasing the encapsulated bacteria. The solution was serially diluted using the dilution plating method and spread onto NA medium. After incubation at 30°C for 48 h, colony counting was performed, and the viable cell count (cfu / g) per gram of microcapsule was calculated. The encapsulation efficiency and fold increase were calculated using Formulas 1 and 2.

[0048] Encapsulation rate (%) = (Number of viable bacteria in microcapsules before proliferation / Total number of viable bacteria in bacterial solution added during preparation) × 100%, Formula 1; Proliferation ratio = Number of viable bacteria in microcapsules after proliferation / Number of viable bacteria in microcapsules before proliferation, Formula 2.

[0049] The physicochemical properties of the Serratia pylori CM9 microcapsule inoculum were analyzed, and the results are shown in Table 2. In terms of viable bacterial encapsulation, the 2% sodium alginate treatment group showed the best performance, with an average particle size of 3.14 mm and an encapsulation rate of 87.53%, significantly higher than the 1% sodium alginate treatment group (80.26%) and the 3% sodium alginate treatment group (67.34%). Simultaneously, the 2% sodium alginate treatment group had the highest number of viable bacteria in the microcapsules, reaching 1.16 × 10⁻⁶. 9 cfu / g, the multiplication rate was 8.33 times.

[0050]

[0051] Example 3: Effects of CM9 microcapsule inoculum on strawberry plant growth

[0052] 1. Experimental materials: The strawberry variety tested was “Hongyan”, and the transplanted seedlings were provided by Shilixiang Strawberry Base in Mengjin District; the CM9 liquid inoculant and microcapsule inoculant tested were Serratia pylori CM9 bacterial solution prepared in Example 2 and the microcapsule inoculant prepared in this example.

[0053] 2. Experimental Design: Healthy and uniform strawberry seedlings were selected and planted in 22 cm × 22 cm pots, one seedling per pot. The potting soil was general horticultural potting soil that had never been used to grow strawberries. Three treatments were set up: a water control group (irrigated with sterile water), a CM9 liquid microbial agent treatment group (50 mL / pot / time), and a CM9 microcapsule microbial agent treatment group (containing microcapsule particles with an equal number of live bacteria). Each treatment group consisted of 3 pots, with 3 replicates. The first irrigation was performed one week after transplanting and the treatments were repeated weekly for a total of 4 times. After 30 days of growth, the growth of the strawberries was observed, and their morphological indicators, mainly plant height, root length, and fresh weight, were measured to evaluate the growth-promoting effect of the treatments.

[0054] 3. Test Results

[0055] After 30 days of growth under different treatments, strawberry plants showed significant differences in their growth status. For example... Figure 4 As shown, the water-treated control group exhibited weak growth, fewer leaves, lighter green leaves, and a stunted overall plant size. Strawberry plants treated with CM9 microcapsule inoculant and liquid inoculant showed vigorous growth, dark green leaves, and larger leaf area. In particular, the microcapsule-treated group showed faster new leaf emergence, increased number of runners, and more spread-out plants. Furthermore, the water-treated control group had poor root growth, with thin, short, and sparse fibrous roots, resulting in an underdeveloped root system. The CM9 liquid inoculant treatment group showed an increase in the number of lateral and fibrous roots, while the microcapsule-treated group showed a significantly increased number of lateral and fibrous roots, resulting in a well-developed root system and effectively improving the absorption area of ​​the strawberry plant roots. As shown in Table 3, compared with the control, the CM9 liquid inoculant treatment group had growth-promoting efficiencies of 53.59%, 30.03%, 58.04%, and 79.82% on strawberry seedling height, root length, fresh weight, and dry weight, respectively, while the CM9 microcapsule inoculant had growth-promoting efficiencies of 55.98%, 45.41%, 86.88%, and 90.83% on strawberry seedling height, root length, fresh weight, and dry weight, respectively. Its growth-promoting effect was better than that of the liquid inoculant treatment group.

[0056]

[0057] In summary, this invention provides a method for preparing and applying *Serratia pylori* CM9 and its microcapsules. *Serratia pylori* composite microcapsules were prepared by extrusion. The *Serratia pylori* microcapsules produced by this invention exhibit stronger viability and stress resistance than traditional liquid live bacteria preparations, making them more suitable for production applications. This invention solves the problems of short shelf life, poor environmental adaptability, and insufficient long-term effectiveness of current rhizosphere growth-promoting bacteria agents, effectively addressing the technical pain points in the use of rhizosphere growth-promoting bacteria. Furthermore, the *Serratia pylori* CM9 microcapsule agent provided by this invention can significantly increase the plant height, root length, and fresh weight of strawberry seedlings, demonstrating a significant growth-promoting effect and broad application prospects in strawberry production.

[0058] The above descriptions are merely some embodiments of the present invention. It should be noted that those skilled in the art can make other modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A strain of Serratia plymouthii CM9, characterized in that, The strain was classified and named *Serratiaplymuthica*, and was deposited on July 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39835.

2. A microcapsule formulation, characterized in that, It includes Serratia pylori CM9 as described in claim 1.

3. A method for preparing the microcapsule formulation according to claim 2, characterized in that, Includes the following steps: (1) Sodium alginate solution, chitosan solution and rice starch gelatinization liquid are mixed to obtain sodium alginate-chitosan-rice starch gelatinization liquid, and then Serratia pylori CM9 bacterial solution is added to obtain Serratia pylori CM9 mixed treatment liquid. (2) The Serratia pylori CM9 mixed treatment solution was dripped into a calcium chloride solution to cross-link and harden, forming gel particles; (3) Wash the cross-linked gel particles to obtain the microcapsule formulation.

4. The method for preparing the microcapsule formulation according to claim 3, characterized in that, In step (1), the mass fraction of the sodium alginate solution is 1% to 3%, the mass fraction of the chitosan solution is 2.5%, and the concentration of the rice starch gelatinized liquid is 3 mg / mL; the volume ratio between the sodium alginate solution, the chitosan solution, and the rice starch gelatinized liquid is 50:25:

3.

5. The method for preparing the microcapsule formulation according to claim 3, characterized in that, In step (1), the volume ratio of the Serratia pylori CM9 bacterial solution to the sodium alginate-chitosan-rice starch gelatinized solution is 1:3.

12.

6. The method for preparing the microcapsule formulation according to claim 3, characterized in that, In step (2), the mass fraction of the calcium chloride solution is 3%; the cross-linking hardening temperature is 4°C and the time is 24 h.

7. The method for preparing the microcapsule formulation according to claim 3, characterized in that, In step (2), when the mixed treatment solution is added to the calcium chloride solution, the drop height is 5-8 cm from the liquid surface; in step (3), the washing is rinsing with sterile water 2-3 times.

8. The use of the microcapsule formulation according to claim 2 or the microcapsule formulation prepared by any one of claims 3-7 in promoting strawberry growth.

9. The application according to claim 8, characterized in that, The promotion of strawberry growth is achieved through the growth-promoting properties of Serratia pylori CM9, including nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and IAA production.

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