Endophyte Microbacterium sp. Of Guangxi curcuma zedoary and application of endophyte Microbacterium sp.

Abstract

The invention relates to a zedoary kwangxi endophyte Microbacterium sp. And application thereof, and belongs to the technical field of agricultural microorganisms. Aiming at the problem that the phosphate-solubilizing function of Microbacterium sp from Guangxi curcuma zedoary is difficult to stably and efficiently express and maintain in the process of preparing a microbial preparation, the invention provides a bacterial strain with the preservation number of GDMCC No: 67431 and a method for preparing the phosphate-solubilizing microbial preparation from the bacterial strain, the method comprises the following steps: activating the bacterial strain to obtain a seed solution; transferring into an inorganic phosphorus liquid fermentation culture medium for shaking culture, centrifugally collecting thalli, finally mixing the thalli with a carrier material containing turf, diatomite and bentonite, and ventilating and drying to obtain the solid phosphorus-dissolving microbial preparation. The strain and the preparation thereof are mainly used for preparing microbial products with the functions of fixing nitrogen, dissolving phosphorus, producing iron carriers and promoting the growth of the curcuma kwangxi.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of endophytic fungus *Microbacterium* sp. from *Curcuma zedoaria* in Guangxi and its applications. Background Technology

[0002] As an important medicinal plant, the availability of phosphorus in the soil is often a limiting factor in the growth of *Curcuma zedoaria*. Utilizing endophytic bacteria with phosphorus-solubilizing functions to prepare microbial preparations is a potential way to improve soil phosphorus utilization. However, in practical applications, the resources of specific endophytic strains with both good phosphorus-solubilizing function and strong adaptability from *Curcuma zedoaria* and other specific medicinal plants are still relatively limited, which to some extent restricts the development of highly efficient microbial preparations for this crop. On the other hand, even after obtaining functional strains, how to ensure that their phosphorus-solubilizing metabolic function is stably maintained during preparation and storage after being removed from the original plant endophytic environment, and ultimately effectively exerted after application, is also a common technical challenge. This is mainly because the strains are easily affected by multiple factors such as nutrient conditions and physicochemical environment during large-scale fermentation culture, collection, and subsequent binding and solidification with carriers, leading to a decrease in their physiological activity and unstable functional expression. Therefore, finding new specific functional strains and developing corresponding preparation methods to effectively maintain their activity is of great significance for developing highly efficient phosphorus-solubilizing microbial preparations suitable for *Curcuma zedoaria*. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide an endophytic strain of Curcuma guangxiensis, Microbacterium sp., which is capable of efficient phosphorus solubilization, exhibits a high content of readily available phosphorus in the fermentation broth, and also possesses the ability to fix nitrogen and produce iron carriers. It can be used to prepare microbial preparations that promote the growth of Curcuma guangxiensis.

[0005] To achieve these objectives and other advantages of the present invention, a strain of *Curcuma zedoaria* endophytic fungus *Microbacterium* sp. is provided. This strain was deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center; the address of the depository is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67431.

[0006] A method for preparing phosphate-solubilizing microbial preparations using the endophytic fungus *Microbacterium* sp. from *Curcuma zedoaria* includes the following steps: S1: The endophytic fungus Microbacterium sp. of Curcuma zedoaria in Guangxi was inoculated into NA solid medium for activation. Single colonies were picked from the activated NA solid medium and inoculated into a container containing liquid seed medium. The culture was carried out at 35-39℃ and shaken at 150-210 r / min for 24-48 hours to obtain the seed liquid. S2: Transfer the seed culture to the fermentation medium at an inoculation rate of 1% to 5% by volume. The fermentation medium is an inorganic phosphorus liquid medium. The fermentation is carried out at 35 to 39 °C with shaking at a speed of 150 to 210 r / min for 6 to 8 days. S3: After the fermentation culture is completed, centrifuge the fermentation broth at 4 ℃ at a speed of 8000~12000 r / min for 15~25 minutes and collect the bacterial cells; S4: Mix the collected microbial cells with the carrier material at a mass ratio of 1:1 to 5. The carrier material includes peat moss, diatomaceous earth, and bentonite. Dry the mixed material at 25 to 30 °C with an airflow speed of 0.5 to 1.5 m / s until the moisture content of the material is less than 10% by mass to obtain a solid phosphorus-solubilizing microbial preparation.

[0007] This invention employs a specific culture medium composition, culture temperature, and oscillation rate that match the original isolation and functional screening of the strain. During preparation, the environmental conditions required for its phosphate-solubilizing metabolic activity are reproduced and optimized, ensuring the efficient expression of the strain's phosphate-solubilizing function. Subsequently, functional cells are enriched through centrifugation and combined with a suitable carrier material, followed by drying and fixation under mild conditions. The synergistic effect of this series of steps allows the strain's phosphate-solubilizing activity to be effectively maintained in the formulation after being removed from its native plant environment, ultimately yielding a stable, easy-to-store, and easy-to-apply solid microbial preparation.

[0008] Preferably, the NA solid culture medium consists of 2.8-3.2 g of beef extract, 4.8-5.2 g of peptone, 2.3-2.7 g of glucose, 17-19 g of agar, and 1 L of water. The pH of the culture medium is 6.9-7.1, and it is used after being autoclaved for 18-22 minutes.

[0009] This invention provides a physiologically consistent and highly active initial strain for subsequent fermentation steps by precisely defining the proportions, pH range, and sterilization parameters of each component in the NA solid culture medium used for strain activation. The stability and balance of the culture medium components ensure that the strain can rapidly recover and grow uniformly during the activation phase, avoiding growth retardation or metabolic heterogeneity caused by fluctuations in basal nutrients. Standardized sterilization conditions and pH environment further eliminate interference from other microorganisms and acid / alkali stress, laying a reliable foundation for obtaining a high-quality seed culture, thereby ensuring the consistency and reproducibility of the starting point for subsequent fermentation processes.

[0010] Preferably, the liquid seed culture medium contains 3 g / L beef extract, 5 g / L peptone, and 2.5 g / L glucose, with a pH of 7.0, and is used after autoclaving for 18-22 minutes.

[0011] This invention provides a stable and balanced nutrient environment for the rapid proliferation of microorganisms by defining the standardized composition and sterilization conditions of the liquid seed culture medium. The clearly defined composition of this medium avoids batch-to-batch variations caused by complex organic matter, allowing the strains to rapidly adapt and enter the logarithmic growth phase during the pre-culture stage, resulting in high-density seed cultures with uniform physiological states. Precisely controlled pH and sterilization procedures further ensure the purity and reproducibility of the culture process, reducing the risk of microbial stress or mutation due to environmental fluctuations. This provides a high-activity, stable, and high-quality source of microbial strains for subsequent large-scale fermentation inoculation.

[0012] Preferably, in step S2, the inorganic phosphorus liquid fermentation medium contains the following raw material components: glucose 8~12 g / L, ammonium sulfate 0.8~1.2 g / L, sodium chloride 0.4~0.6 g / L, magnesium sulfate heptahydrate 0.2~0.3 g / L, potassium chloride 0.1~0.15 g / L, and calcium phosphate 3~5 g / L or iron phosphate 1~3 g / L; the initial pH of the medium is adjusted to 6.8~7.2.

[0013] The inorganic phosphorus liquid fermentation medium of this invention provides a specific nutrient environment conducive to the initiation and maintenance of phosphorus solubilization metabolism by offering specific types and concentrations of carbon sources, nitrogen sources, inorganic salts, and insoluble phosphates. Glucose serves as the primary carbon source, supporting cell growth and energy metabolism; ammonium sulfate provides the essential nitrogen source; sodium chloride, potassium chloride, and magnesium sulfate heptahydrate maintain suitable ion balance and enzymatic reaction conditions; and the selected calcium phosphate or iron phosphate, as the primary phosphorus source, effectively induces the expression of the strain's phosphate-solubilizing enzyme system due to its insoluble properties. With precise adjustment of the initial pH, this medium not only meets the basic growth requirements of the cells but also continuously stimulates and maintains their phosphate-solubilizing activity, thus providing a stable and efficient culture foundation for obtaining fermentation products with high phosphate-solubilizing activity.

[0014] Preferably, the shaking fermentation culture process includes a first culture stage and a second culture stage; The first culture stage was carried out at 35–39 °C for 48–72 hours. After the first culture stage, a sterile carbon-nitrogen mixture containing glucose and yeast extract was added to the fermentation system. The amount of glucose added was 0.5%–1.0% of the initial weight of the inorganic phosphorus liquid fermentation medium, and the amount of yeast extract added was 0.1%–0.3% of the initial weight of the inorganic phosphorus liquid fermentation medium. After adding the carbon-nitrogen mixture, the second culture stage begins, which continues at 35-39 ℃ until the total fermentation time reaches 6-8 days.

[0015] The two-stage fermentation process of this invention replenishes readily available carbon and organic nitrogen sources by adding a specific ratio of sterile carbon-nitrogen mixture during the late logarithmic growth phase of the cell. This operation mitigates the potential decline in cell metabolic activity caused by the gradual depletion of nutrients in the basal culture medium, particularly the rapid depletion of carbon sources. The supplemented nutrients support the cells in maintaining high metabolic activity, delaying the onset of death and enabling the strain to continuously perform phosphate solubilization throughout the extended fermentation cycle. This nutrient supplementation strategy based on key nodes in cell growth kinetics effectively coordinates the relationship between cell growth and functional expression, ensuring stable and efficient phosphate solubilization activity in the later stages of fermentation.

[0016] Preferably, in step S4, the preparation of the carrier material specifically includes: Crush the peat moss and pass it through a 40-60 mesh sieve, then dry it at 110-130 ℃ for 2-4 hours, and cool it for later use. Calcine the diatomaceous earth at 450-550 ℃ for 1.5-2.5 hours, cool it, grind it and pass it through a 100-200 mesh sieve for later use; Bentonite is dried at 80-100 °C for 3-5 hours, followed by sodium modification treatment. The sodium modification treatment involves adding a 4%-6% sodium carbonate solution to the dried bentonite, with a mass ratio of bentonite to sodium carbonate solution of 1:1.5-2.5. The mixture is stirred and reacted at 60-70 °C for 1-2 hours. After the reaction is completed, the bentonite is filtered, washed, dried at 80-100 °C, ground, and passed through a 200-300 mesh sieve to obtain sodium-modified bentonite. The dried peat, calcined and ground diatomaceous earth, and sodium bentonite were mixed at a dry weight ratio of (4~6):(2~4):(1~3) to obtain the composite carrier dry material. Sterile water is added to the dry composite carrier material and stirred to adjust the water content of the mixture to 25%~35% by mass, thereby obtaining the carrier material.

[0017] This invention utilizes targeted pretreatment of peat moss, diatomaceous earth, and bentonite—including drying and sieving of the peat moss, high-temperature calcination and grinding of the diatomaceous earth, and sodium modification of the bentonite—and blends them in specific proportions to construct a composite carrier matrix with a multi-level porous structure, suitable surface charge, and good water-holding capacity. When mixed with bacterial cells, this matrix achieves uniform dispersion and firm fixation of functional bacteria through multiple mechanisms such as physical adsorption, pore encapsulation, and ion exchange. The subsequent gentle, aerated drying process effectively reduces physical damage caused by rapid moisture removal. This entire carrier treatment and composite process synergistically optimizes the microenvironment of the bacteria during formulation processing and storage, thereby significantly improving the survival rate, long-term stability, and slow-release and colonization effects of the functional bacteria in the final solid product after application to soil.

[0018] Preferably, in step S4, before mixing the bacterial cells with the carrier material, a bacterial cell protectant is added to the bacterial cells, specifically including: Trehalose, skim milk powder, and glycerin were mixed in a mass ratio of (1.5~2.5):(1.0~2.0):(0.5~1.5), and sterile water was added to prepare a protective agent stock solution with a total solids mass concentration of 15%~25%. The pH of the protective agent stock solution was adjusted to 6.5~7.5, and after being filtered through a 0.22μm filter membrane for sterilization, the cell protectant was obtained. After collecting the bacterial cells in step S3, the bacterial cells are first resuspended in sterile 0.85% physiological saline to form a bacterial suspension, and the concentration of bacterial cells in the suspension is controlled within the range of 1×10⁻⁶. 9 ~5×10 9 CFU / mL; Under continuous stirring, the cell protectant was slowly added dropwise to the bacterial suspension at a volume ratio of 1:5~10, and the addition time was controlled at 10~20 minutes. After the addition is complete, the bacterial suspension containing the protectant is allowed to stand at 4 ℃ for 30-60 minutes. After standing, it is then mixed with the carrier material.

[0019] This invention pretreats the bacterial suspension with a composite protectant formulated from trehalose, skim milk powder, and glycerol in a specific ratio before mixing the bacterial cells with the carrier. Trehalose forms a stable protective layer on the bacterial surface, skim milk powder provides buffering and nutritional support, and glycerol regulates osmotic pressure and slows water loss. The synergistic effect of these three components pre-treats the bacterial cells' tolerance to changes in osmotic pressure, mechanical shearing, and dehydration stress. After treatment with the protectant and subsequent settling, the bacterial cells maintain cell membrane integrity and physiological activity more effectively during subsequent mixing with the carrier and aeration drying. This significantly reduces unnecessary loss of bacterial viability during processing, providing a more reliable guarantee for the long-term storage stability of the final formulation and its post-application efficacy.

[0020] Application of a strain of *Curcuma guangxiensis* endophytic bacterium *Microbacterium* sp. in the preparation of microbial preparations with nitrogen fixation, phosphorus solubilization, iron carrier production, and growth promotion functions of *Curcuma guangxiensis*.

[0021] The present invention has at least the following beneficial effects: The *Microbacterium sp.* endophytic bacteria of *Curcuma zedoaria* and its supporting preparation method provided by the present invention can obtain a unique functional strain with efficient phosphorus solubilization, self-generated nitrogen fixation, and iron-producing capabilities; through the application of a highly compatible fermentation medium, a two-stage fed-batch culture process, optimized construction of a composite carrier, and cell protection technology, the activity retention rate and metabolic stability of the functional bacteria during large-scale preparation are significantly improved; the final solid microbial preparation product not only has a high viable count and a long shelf life, but also adapts better to the soil environment after application, achieving the slow-release and long-lasting growth-promoting effects of the functional bacteria.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 This is a diagram illustrating the morphological characteristics of the endophytic bacterial strain of the present invention. Figure 2 This is a phylogenetic tree diagram of the endophytic bacterial strains of the present invention; Figure 3 The figure shows the qualitative detection results of the endophytic bacterial strains with nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functions of this invention; where A represents nitrogen fixation; B represents phosphorus solubilization; C represents potassium solubilization; and D represents siderophore. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0025] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0026] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0027] Test content 1. Test Methods 1.1 Isolation of endophytic bacteria Rinse the fresh and healthy rhizome tissues of Guangxi Curcuma zedoaria tissue culture seedlings with running water, air dry them naturally, weigh 1g each, spread them on PDA medium using the plate dilution method, incubate at 37 ℃ for 48 h, then pick different bacterial colonies to purify on NA plates and store them in a refrigerator at 4 ℃.

[0028] NA solid culture medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, 18 g agar, final pH 7.0±0.1, diluted to 1 L of water, autoclaved for 20 minutes.

[0029] 1.2 Screening and purification of culturable endophytic bacteria capable of nitrogen fixation, phosphorus solubilization, potassium solubilization, and siderophore production. Purified endophytic bacterial colonies were inoculated onto Ashby's nitrogen-free medium, inorganic phosphate medium, and potassium feldspar medium, respectively, and then incubated at 25 °C for qualitative analysis. Strains capable of growing on Ashby's nitrogen-free medium were identified as free-living nitrogen-fixing bacteria; while strains with phosphate or potassium solubilization functions would form phosphate-solubilizing or potassium-solubilizing zones on inorganic phosphate and potassium feldspar medium, respectively. Siderophore production capacity was determined using the MAS-CAS assay. Strains exhibiting siderophore production capacity were screened based on the production of an orange-yellow color on CAS medium. The ratio of the diameter of the yellow transparent zone to the colony diameter was used to determine the siderophore production capacity of the strain.

[0030] 1.3 Quantitative Detection of Nitrogen-Fixing, Phosphorus-Solubilizing, and Potassium-Solubilizing Functional Bacteria in Endophytic Culture The screened and purified microorganisms were inoculated into corresponding nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing liquid media, with three replicates for each treatment. They were then cultured on a shaker at 37 ℃ and 180 r / min for 7 days. After culture, the soluble ammonium nitrogen content in the fermentation broth was determined using indophenol blue spectrophotometry, and the soluble phosphorus content was determined using the molybdenum-antimony colorimetric method. To evaluate the potassium-solubilizing function of the potassium-solubilizing bacteria, the fermentation broth was sent to Beijing Yangou Technology Co., Ltd., which used inductively coupled plasma mass spectrometry to determine the potassium content in the fermentation broth. The results reflected the potassium-solubilizing function of the strain. The regression equation for the ammonium nitrogen content in the culture medium was y = 0.0157 + 0.0917x, R0. 2 =0.99. The regression equation for available phosphorus concentration is y = -0.0382 + 0.507x, R0 2 =0.99. Substituting the absorbance of the strain into the equation, we obtained the total amount of ammonium nitrogen and available phosphorus in the fermentation supernatant of the strain.

[0031] 1.4 Morphological characteristics Endophytic bacteria *Microbacterium* sp. were streaked onto NA medium and incubated at 37 °C for 3 days. Colony morphology was recorded. Gram staining was performed simultaneously, and colony morphology characteristics were observed and recorded under an optical microscope.

[0032] 1.5 Molecular systematics characteristics Using the Mighty Amp DNA Polymerase Ver.3 kit, colonies of antagonistic bacteria were directly subjected to colony PCR. The 16S rRNA gene was amplified by PCR using universal bacterial primers 27F (SEQ ID NO.1: 5′-agagtttgatcctggctcag-3′) and 1492R (SEQ ID NO.2: 5′-ggttaccttgttacgactt-3′). The PCR reaction system and conditions followed the kit instructions. The obtained PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced sequences were compared with high-similarity sequences downloaded from NCBI using BLAST. A phylogenetic tree of the 16S rRNA gene was constructed using MEGA 6.0 software to determine the taxonomic position of the endophytic bacterial strains.

[0033] 2. Experimental Results 2.1 Morphological characteristics and molecular identification of endophytic bacteria Microbacterium sp. strain like Figure 1 As shown, the strain Microbacterium sp. is transparent in color and has a smooth, unwrinkled surface; the bacteria are mostly round in shape. It is Gram-positive and the cells are rod-shaped.

[0034] The 16S rRNA gene sequence was amplified by PCR and sequenced using universal primers. The 16S rRNA gene sequence is shown in SEQ ID NO.3, with a nucleotide sequence size of 1219 bp. Alignment analysis of this sequence with sequences showing high similarity in NCBI revealed a high similarity of 99% between the strain and the compared *Microbacterium* genus. A phylogenetic tree was constructed using MEGA 6.0 software based on the 16S rRNA gene sequences of the strain with 99% similarity. The results are shown below. Figure 2 As shown, the strain clustered with other Microbacterium species in one branch. Based on traditional morphological characteristics and molecular phylogenetic analysis, it was identified as *Microbacterium* sp. This strain was deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Guangdong Institute of Scientific Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 67431.

[0035]

[0036] 2.2 Qualitative detection results of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functional strains Depend on Figure 3 As shown, the endophytic bacterium *Microbacterium* sp. can grow on Ashby's nitrogen-free medium and produce a clear zone, indicating that *Microbacterium* sp. has autotrophic nitrogen fixation capabilities; the endophytic bacterium *Microbacterium* sp. can grow on phosphate-solubilizing medium, indicating that this bacterium has a certain phosphate-solubilizing ability; the strain *Microbacterium* sp. can grow on potassium-solubilizing medium, but no potassium-solubilizing zone is formed; the strain *Microbacterium* sp. can grow on siderophore-producing medium and form a pale yellow clear zone, with a siderophore-producing capacity of 1.21 cm, indicating that the strain *Microbacterium* sp. has the ability to produce siderophores.

[0037] 2.3 Quantitative detection results of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functional strains The results showed that *Microbacterium sp.* is an endophytic bacterium with nitrogen fixation, phosphorus solubilization, and potassium solubilization functions. The nitrogen-fixing fermentation broth of *Microbacterium sp.* contained 46.67 mg / L of ammonium nitrogen, 62.32 mg / L of available phosphorus, and 22.82 mg / L of available potassium. Quantitative analysis of liquid fermentation indicated that although no obvious potassium-solubilizing zone was observed on solid plates, this strain significantly increased the soluble potassium content in the culture medium through metabolic activity, demonstrating potassium solubilization function.

[0038] Example 1 A method for preparing phosphate-solubilizing microbial preparations using the endophytic bacterium *Microbacterium* sp. (GDMCC No: 67431) from *Curcuma zedoaria*, comprising the following specific steps: 1. Strain activation and seed culture preparation Preparation and sterilization of NA solid culture medium: Weigh 3.0 g of beef extract, 5.0 g of peptone, 2.5 g of glucose, and 18.0 g of agar, and dissolve them in 1 L of deionized water. After thorough stirring and dissolution, adjust the pH of the culture medium to 7.0. Dispense the prepared culture medium into Erlenmeyer flasks and autoclave at 121 °C for 20 minutes. After sterilization, allow the culture medium to cool to approximately 50-60 °C, and pour it into sterile petri dishes under aseptic conditions to prepare NA solid plates.

[0039] Strain activation: A small amount of bacterial growth was picked up from the slant of the preserved endophytic fungus Microbacterium sp. (GDMCC No:67431) of Curcuma zedoaria from Guangxi and streaked onto a NA solid plate. The inoculated plate was then inverted and incubated at 37°C for 48 hours.

[0040] Preparation and sterilization of liquid seed culture medium: Weigh 3.0 g of beef extract, 5.0 g of peptone, and 2.5 g of glucose, dissolve them in 1 L of deionized water, and adjust the pH to 7.0. Dispense into 250 mL Erlenmeyer flasks, 100 mL per flask, autoclave at 121 ℃ for 20 minutes, and cool for later use.

[0041] Seed culture: Pick a single, plump colony from the activated NA plate and aseptically inoculate it into an Erlenmeyer flask containing 100 mL of liquid seed culture medium. Place the Erlenmeyer flask in a constant temperature shaker at 37 ℃ and 180 r / min and incubate for 36 hours to obtain a homogeneous, turbid seed culture.

[0042] 2. Fermentation culture and cell collection Fermentation culture: At an inoculum rate of 2% (v / v), 2 mL of the above seed culture was aseptically transferred to another 500 mL Erlenmeyer flask containing 100 mL of inorganic phosphorus liquid fermentation medium as the fermentation medium. The Erlenmeyer flask was placed in a constant temperature shaker at 37℃ and 180 r / min for shaking fermentation culture, with a total culture time of 7 days. The inorganic phosphorus liquid fermentation medium contained: glucose 10 g / L, ammonium sulfate 1.0 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium chloride 0.12 g / L, and calcium phosphate 4 g / L; the initial pH of the medium was 7.0, and it was used after autoclaving at 121℃ for 20 minutes.

[0043] Cell collection: After fermentation, transfer all fermentation broth to sterile centrifuge tubes and centrifuge at 10,000 r / min for 20 minutes at 4 ℃. Carefully discard the supernatant to obtain the cell precipitate.

[0044] 3. Carrier mixing and drying Carrier material preparation: The carrier material used in this embodiment is a simple physical mixture of commercial-grade peat moss, diatomaceous earth, and bentonite without special pretreatment. The above three carrier raw materials are weighed in a mass ratio of 1:1:1 and pre-mixed evenly under aseptic conditions.

[0045] Mixing of bacterial cells and carrier: The bacterial precipitate collected in step 2 is mixed with the above-mentioned mixed carrier material at a mass ratio of bacterial wet weight to carrier dry weight of 1:3. In a sterile container, the mixture is stirred thoroughly with a sterile glass rod for about 10 minutes to allow the bacterial cells and carrier material to initially combine and form a moist clump.

[0046] Drying: Spread the uniformly mixed, moist material evenly on a sterile tray and place it in a clean fume hood at 28 ℃, where it is dried by airflow at a speed of approximately 1.0 m / s. Turn the material regularly until the moisture content drops below 8% (as determined by gravimetric method), resulting in a dry, loose, solid phosphate-solubilizing microbial preparation.

[0047] Example 2 The method of Example 1 is used, except that the shaking fermentation culture process includes a first culture stage and a second culture stage; The first culture stage was carried out at 35 ℃ for 60 hours. After the first culture stage, a sterile carbon-nitrogen mixture containing glucose and yeast extract was added to the fermentation system. The amount of glucose added was 0.7% of the initial weight of the inorganic phosphorus liquid fermentation medium, and the amount of yeast extract added was 0.2% of the initial weight of the inorganic phosphorus liquid fermentation medium. After adding the carbon-nitrogen mixture, the second culture stage begins, which continues at 35 °C until the total fermentation time reaches 7 days.

[0048] Example 3 The method used in Example 2 differs in that the preparation of the carrier material specifically includes: The peat moss was crushed and passed through a 50-mesh sieve, then dried at 120 ℃ for 3 hours, and cooled for later use. Diatomaceous earth was calcined at 500 ℃ for 2.0 hours, cooled, ground, and passed through a 150-mesh sieve for later use. Bentonite was dried at 90 °C for 4 hours, followed by sodium modification treatment. The sodium modification treatment involved adding a 5% sodium carbonate solution to the dried bentonite, with a mass ratio of bentonite to sodium carbonate solution of 1:2.0. The mixture was stirred and reacted at 65 °C for 1.5 hours. After the reaction was completed, the bentonite was filtered, washed, dried at 90 °C, ground, and passed through a 250-mesh sieve to obtain sodium-modified bentonite. The dried peat moss, calcined and ground diatomaceous earth and sodium bentonite were mixed at a dry weight ratio of 5:3:2 to obtain the composite carrier dry material. Sterile water was added to the dry composite carrier material and stirred to adjust the moisture content of the mixture to 30% by mass, thus obtaining the carrier material. The obtained carrier material was mixed with the bacterial cells and placed in a clean fume hood at 28 °C for aeration and drying at an airflow rate of approximately 1.0 m / s. The material was turned periodically until the moisture content dropped below 8% (determined by gravimetric method), yielding a dry, loose, solid phosphate-solubilizing microbial preparation.

[0049] Example 4 The method of Example 3 is used, except that a cell protectant is added to the bacterial cells before mixing them with the carrier material, specifically including: Trehalose, skim milk powder, and glycerin were mixed in a mass ratio of 2:1.5:1.5, and sterile water was added to prepare a protective agent stock solution with a total solids mass concentration of 20%. The pH of the protective agent stock solution was adjusted to 7.0, and after being filtered through a 0.22μm filter membrane for sterilization, the cell protectant was obtained. After collecting the bacterial cells, they were first resuspended in sterile 0.85% physiological saline to form a bacterial suspension, with the bacterial cell concentration in the suspension controlled within the range of approximately 3 × 10⁻⁶. 9 CFU / mL; Under continuous stirring, the cell protectant was slowly added dropwise to the bacterial suspension at a volume ratio of 1:7, and the addition time was controlled at 15 minutes. After the addition is complete, the bacterial suspension containing the protectant is allowed to stand at 4 °C for 50 minutes. After standing, it is then mixed with the carrier material.

[0050] Effect test I. Stability evaluation of the formulation Method: The solid preparations prepared in Examples 1-4 were placed in sealed aluminum foil bags and stored at 25 °C and 60% relative humidity for 0, 1, 3, 6 and 12 months.

[0051] Testing indicators: Viable bacteria count (CFU / g): Plate count method.

[0052] Moisture content (%): Drying method.

[0053] Appearance: Whether it clumps, changes color, or has an odor.

[0054] The results are shown in Table 1.

[0055] Table 1 Stability test results As shown in Table 1, with the gradual optimization of the preparation process, the initial viable count of the formulation showed a significant increasing trend. In Example 4, the viable count immediately after drying reached as high as 6.3 × 10⁻⁶.9 The CFU / g was significantly higher than 2.2 × 10⁻⁶ in Example 1. 9 The CFU / g count was mainly attributed to the optimization of the fermentation medium, the two-stage fed-batch process that maintained high cell activity, and the introduction of a protectant that effectively reduced cell loss during centrifugation and initial drying. During the 12-month storage period, the viable cell counts of the formulations in all examples showed a natural decrease, but the rate of decrease differed significantly: Example 1, due to the use of a simple mixed carrier and the absence of a protectant, saw its viable cell count decrease to 2.5 × 10⁻⁶ after 12 months. 8 The CFU / g count was low, while in Example 4, under the dual protection of the composite carrier and the protectant, the viable count remained at 3.8 × 10⁻⁶ after 12 months. 9 The CFU / g concentration exhibited excellent storage stability. The moisture content of all formulations remained low (≤9.5%) during storage, and no clumping, significant discoloration, or off-odors were observed. This indicates that the ventilation drying process and carrier pretreatment method described in this invention can effectively control product moisture and maintain good physical properties, providing a suitable environment for the long-term survival of functional bacteria.

[0056] II. Evaluation of Usage Effect (Pot Experiment) Materials: Tissue culture seedlings of Curcuma zedoaria from Guangxi (30 days old), 1 seedling per pot, 10 pots per treatment.

[0057] CK group: No medication was administered; Preparations for Examples 1-4: Apply 2 g per pot (mixed into the substrate).

[0058] Cultivation conditions: Cultivate in a greenhouse for 60 days.

[0059] Measurement indicators: The increases in plant height, root length, and fresh weight of *Curcuma zedoaria* tissue culture seedlings before and after culture were measured. Root vitality (TTC method); Available phosphorus content in soil (mg / kg).

[0060] The results are shown in Table 2.

[0061] Table 2 Effect Evaluation Experiment As shown in Table 2, the plants treated in Example 1 exhibited significant increases in plant height, root length, and fresh weight, reaching 18.5 cm, 13.2 cm, and 6.8 g, respectively. Simultaneously, the available phosphorus content in the soil increased to 19.2 mg / kg, indicating that the plants possessed basic phosphorus-solubilizing and growth-promoting functions. Example 2, through optimization of the fermentation medium and the adoption of a two-stage feeding process, further improved the metabolic activity of the functional bacteria, with all growth indicators and available phosphorus content in the soil significantly superior to those of Example 1. Example 3, based on Example 2, introduced a pretreated composite carrier. This carrier, through optimized pore structure and surface properties, better protected and slowly released the functional bacteria, resulting in a root activity of 85.6 μg TTF / (g·h) and a significant increase in available phosphorus content in the soil to 46.8 mg / kg, demonstrating the positive effect of carrier improvement on bacterial colonization and functional expression. The most outstanding effect was observed in Example 4, which added a microbial cell protectant treatment to the composite carrier. After 60 days in pots, the plant height, root length, and fresh weight reached 31.2 cm, 23.1 cm, and 14.5 g, respectively, with root activity reaching 98.7 μg TTF / (g·h) and soil available phosphorus content increasing to 60.3 mg / kg. These results corroborate the conclusion in Table 1 that Example 4 showed the best storage stability, indicating that the formulation prepared by this invention through an optimized fermentation-composite carrier-microbial cell protection synergistic process not only maintains a high viable bacterial count for a long period but also continuously and efficiently exerts its phosphorus-solubilizing function after application to the soil, significantly promoting root development and plant growth of *Curcuma zedoaria* in Guangxi. This provides a reliable technical solution for developing high-efficiency, specialized microbial fertilizers.

[0062] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An endophytic fungus of Curcuma zedoaria from Guangxi Microbacterium sp., characterized in that, This strain was deposited on December 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection; the address of the depository is Guangdong Institute of Scientific Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67431.

2. A method utilizing the endophytic fungus of Curcuma zedoaria as described in claim 1. Microbacterium A method for preparing phosphate-solubilizing microbial agents, characterized in that... Includes the following steps: S1: Incorporate endophytic fungi from Curcuma zedoaria. Microbacterium sp . The strain was activated by inoculating it into NA solid medium. Single colonies were picked from the activated NA solid medium and inoculated into a container containing liquid seed medium. The culture was carried out at 35-39 °C with shaking at 150-210 r / min for 24-48 hours to obtain the seed liquid. S2: Transfer the seed culture to the fermentation medium at an inoculation rate of 1% to 5% by volume. The fermentation medium is an inorganic phosphorus liquid medium. The fermentation is carried out at 35 to 39 °C with shaking at a speed of 150 to 210 r / min for 6 to 8 days. S3: After the fermentation culture is completed, centrifuge the fermentation broth at 4 ℃ at a speed of 8000~12000 r / min for 15~25 minutes and collect the bacterial cells; S4: Mix the collected microbial cells with the carrier material at a mass ratio of 1:1 to 5. The carrier material includes peat moss, diatomaceous earth, and bentonite. Dry the mixed material at 25 to 30 °C with an airflow speed of 0.5 to 1.5 m / s until the moisture content of the material is less than 10% by mass to obtain a solid phosphorus-solubilizing microbial preparation.

3. The method of utilizing endophytic fungi from Curcuma zedoaria according to claim 2 Microbacterium A method for preparing phosphate-solubilizing microbial agents, characterized in that... NA solid medium consists of 2.8-3.2 g beef extract, 4.8-5.2 g peptone, 2.3-2.7 g glucose, 17-19 g agar, and 1 L water. The pH of the medium is 6.9-7.

1. It is used after being autoclaved for 18-22 minutes.

4. The method of utilizing endophytic fungi from Curcuma zedoaria according to claim 2. Microbacterium A method for preparing phosphate-solubilizing microbial agents, characterized in that... The liquid seed culture medium contains 3 g / L beef extract, 5 g / L peptone, and 2.5 g / L glucose. The pH of the culture medium is 7.

0. It is used after being autoclaved for 18-22 minutes.

5. The method of utilizing endophytic fungi from Curcuma zedoaria according to claim 2. Microbacterium sp . A method for preparing phosphate-solubilizing microbial agents, characterized in that, In step S2, the inorganic phosphorus liquid fermentation medium contains the following raw material components: glucose 8~12 g / L, ammonium sulfate 0.8~1.2 g / L, sodium chloride 0.4~0.6 g / L, magnesium sulfate heptahydrate 0.2~0.3 g / L, potassium chloride 0.1~0.15 g / L, and calcium phosphate 3~5 g / L or iron phosphate 1~3 g / L; the initial pH of the medium is adjusted to 6.8~7.

2.

6. The method of utilizing endophytic fungi from Curcuma zedoaria according to claim 5 Microbacterium sp . A method for preparing phosphate-solubilizing microbial agents, characterized in that, The shaking fermentation process includes a first culture stage and a second culture stage; The first culture stage was carried out at 35–39 °C for 48–72 hours. After the first culture stage, a sterile carbon-nitrogen mixture containing glucose and yeast extract was added to the fermentation system. The amount of glucose added was 0.5%–1.0% of the initial weight of the inorganic phosphorus liquid fermentation medium, and the amount of yeast extract added was 0.1%–0.3% of the initial weight of the inorganic phosphorus liquid fermentation medium. After adding the carbon-nitrogen mixture, the second culture stage begins, which continues at 35-39 ℃ until the total fermentation time reaches 6-8 days.

7. The method of utilizing endophytic fungi from Curcuma zedoaria according to claim 2 Microbacterium sp . A method for preparing phosphate-solubilizing microbial agents, characterized in that, In step S4, the preparation of the support material specifically includes: Crush the peat moss and pass it through a 40-60 mesh sieve, then dry it at 110-130 ℃ for 2-4 hours, and cool it for later use. Calcine the diatomaceous earth at 450-550 ℃ for 1.5-2.5 hours, cool it, grind it and pass it through a 100-200 mesh sieve for later use; Bentonite is dried at 80-100 °C for 3-5 hours, followed by sodium modification treatment. The sodium modification treatment involves adding a 4%-6% sodium carbonate solution to the dried bentonite, with a mass ratio of bentonite to sodium carbonate solution of 1:1.5-2.

5. The mixture is stirred and reacted at 60-70 °C for 1-2 hours. After the reaction is completed, the bentonite is filtered, washed, dried at 80-100 °C, ground, and passed through a 200-300 mesh sieve to obtain sodium-modified bentonite. The dried peat, calcined and ground diatomaceous earth, and sodium bentonite were mixed at a dry weight ratio of (4~6):(2~4):(1~3) to obtain the composite carrier dry material. Sterile water is added to the dry composite carrier material and stirred to adjust the water content of the mixture to 25%~35% by mass, thereby obtaining the carrier material.

8. The method of utilizing endophytic fungi from Curcuma zedoaria according to claim 7 Microbacterium sp . A method for preparing phosphate-solubilizing microbial agents, characterized in that, In step S4, before the bacterial cells are mixed with the carrier material, a bacterial cell protectant is added to the bacterial cells, specifically including: Trehalose, skim milk powder, and glycerin were mixed in a mass ratio of (1.5~2.5):(1.0~2.0):(0.5~1.5), and sterile water was added to prepare a protective agent stock solution with a total solids mass concentration of 15%~25%. The pH of the protective agent stock solution was adjusted to 6.5~7.5, and after being filtered through a 0.22μm filter membrane for sterilization, the cell protectant was obtained. After collecting the bacterial cells in step S3, the bacterial cells are first resuspended in sterile 0.85% physiological saline to form a bacterial suspension, and the concentration of bacterial cells in the suspension is controlled within the range of 1×10⁻⁶. 9 ~5×10 9 CFU / mL; Under continuous stirring, the cell protectant was slowly added dropwise to the bacterial suspension at a volume ratio of 1:5~10, and the addition time was controlled at 10~20 minutes. After the addition is complete, the bacterial suspension containing the protectant is allowed to stand at 4 ℃ for 30-60 minutes. After standing, it is then mixed with the carrier material.

9. The endophytic fungi of Curcuma zedoaria as described in claim 1 Microbacterium sp . Application in the preparation of microbial preparations with functions of nitrogen fixation, phosphorus solubilization, iron production, and promotion of the growth of Curcuma zedoaria in Guangxi.

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