Kangxi curcuma zedoary endophyte Bacillus sp. And application thereof

Abstract

The invention discloses a zedoary kwangxi endophyte Bacillus sp. And application thereof, and belongs to the technical field of microorganisms. The invention aims to solve the technical problem that the tissue culture seedlings of the curcuma kwangxi grow slowly. According to the key point of the technical scheme, the invention provides a zedoary kwangxi endophyte Bacillus sp. With the preservation number of GDMCC No: 67430, and a method for promoting the growth of tissue culture seedlings by applying the strain. The method comprises the following steps: carrying out activation culture on a strain, carrying out nitrogen fixation fermentation to obtain a fermentation solution, soaking rhizome tissues of tissue culture seedlings by using the diluted fermentation solution, inoculating the soaked tissue culture seedlings into a tissue culture medium, culturing and the like. The endophyte and the application method thereof are mainly used for preparing a microbial preparation with the functions of nitrogen fixation, phosphate solubilization and siderophore production so as to effectively promote the growth of Guangxi curcuma zedoary.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an endophytic fungus of Curcuma zedoaria from Guangxi. Bacillus sp. and its applications. Background Technology

[0002] Slow growth of tissue culture seedlings is a common technical problem in the cultivation and propagation of Curcuma zedoaria in Guangxi, which to some extent restricts the large-scale production and application of seedlings. The reasons for slow growth are multifaceted, among which the lack of efficient and stable beneficial microbial communities in the plant's rhizosphere and internal environment, particularly the relative scarcity of functional endophytic bacteria that can establish a good symbiotic relationship with Curcuma zedoaria and directly provide it with nutritional support, is a significant limiting factor. While some microorganisms are known to have the potential to promote plant growth, specific growth-promoting endophytic bacteria resources with clear taxonomic status, defined functions, and stable availability for Curcuma zedoaria, a specific medicinal plant, remain relatively scarce. Isolating and screening specific endophytic strains that can efficiently fix nitrogen and solubilize phosphorus and adapt to the internal environment of Curcuma zedoaria is difficult because the composition of plant endophytic communities is complex, the isolation, purification, and functional identification of functional strains are labor-intensive, and the genetic stability, functional activity, and host affinity of the screened strains often require long-term verification. Therefore, obtaining a specific endophytic bacterium for promoting growth of Curcuma zedoaria from Guangxi that has been properly preserved, clearly classified, and has a clear functional orientation is of great significance for solving the nutrient supply bottleneck for the growth of its tissue culture seedlings. 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 zedoaria from Guangxi. Bacillus sp. can fix nitrogen, solubilize phosphorus and produce iron carriers, thus effectively promoting the growth of Guangxi Curcuma zedoaria tissue culture seedlings.

[0005] To achieve these objectives and other advantages of the present invention, an endophytic fungus of Curcuma zedoaria from Guangxi is provided. Bacillus 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: 67430.

[0006] A method for promoting the growth of tissue culture seedlings of Curcuma zedoaria from Guangxi includes the following steps: S1: The endophytic fungi of the aforementioned Curcuma zedoaria BacillusThe sp. strain was inoculated into NA liquid medium and cultured with shaking at 35-40 ℃ and 160-200 r / min for 48-72 hours to obtain activated bacterial solution; S2: Inoculate the activated bacterial solution into the nitrogen-fixing fermentation medium and incubate it on a shaker at 35~39 ℃ and 170~190 r / min for 6~8 days to obtain the fermentation broth; S3: After rinsing the root and stem tissue of the Guangxi Curcuma zedoaria tissue culture seedlings with sterile water, soak them in fermentation liquid diluted 10 to 20 times for 1 to 3 hours. S4: Soaked Guangxi Curcuma zedoaria tissue culture seedlings were inoculated into tissue culture medium and cultured at 23~27 ℃, light intensity of 1500~2500 lux, and light duration of 12~16 h / d.

[0007] This invention is based on the endophytic fungi of Curcuma zedoaria. Bacillus The nitrogen-fixing and phosphorus-solubilizing functions of *Curcuma zedoaria* sp. are utilized through liquid fermentation to allow for its proliferation and metabolism, producing soluble ammonium nitrogen and available phosphorus that can be directly absorbed and utilized by plants. Immersing the rhizomes of tissue-cultured seedlings in this diluted fermentation solution allows these nutrients to directly adhere to and penetrate the tissue surface, providing initial nutritional support for subsequent in vitro culture. After inoculation, the strain and its metabolites continue to function in the tissue culture environment, providing continuous nutritional support for the early growth of *Curcuma zedoaria* tissue-cultured seedlings, thus effectively overcoming their slow growth. This method has clear operational steps, mild fermentation conditions, and is easy to implement in conventional tissue culture laboratories, providing a direct and feasible microbial auxiliary means to address the slow growth of *Curcuma zedoaria* tissue-cultured seedlings.

[0008] Preferably, the NA liquid 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, and 1 L of water, with a pH of 6.9-7.1, and is used after autoclaving for 18-22 minutes.

[0009] The components of this invention, such as beef extract, peptone, glucose, and agar, are all within specific ranges. Combined with near-neutral pH conditions and appropriate sterilization time, they constitute a nutritionally balanced and environmentally stable culture medium system. This formulation represents the endophytic bacteria... Bacillus The activation and proliferation of *Sp.* provided the necessary carbon, nitrogen, minerals, and coagulation support, effectively promoting rapid and robust cell growth, thus laying the foundation for the subsequent preparation of highly active fermentation broth. By optimizing the culture medium composition and physicochemical parameters, the stability and reproducibility of the strain activation phase were ensured, avoiding problems such as poor strain growth or decreased activity caused by improper culture medium composition or pH fluctuations.

[0010] Preferably, the nitrogen-fixing fermentation medium is Ashby nitrogen-free liquid medium, which consists of 19-21 g mannitol, 0.4-0.6 g KH2PO4, 0.1-0.3 g MgSO4·7H2O, 0.1-0.3 g NaCl, 0.05-0.15 g CaSO4·2H2O, 1.8-2.2 g CaCO3, and 1 L water, with a pH of 6.8-7.2, and is used after autoclaving for 18-22 minutes.

[0011] The Ashby nitrogen-free liquid culture medium defined in this invention uses mannitol as the main carbon source and contains specific proportions of phosphates, magnesium salts, sodium salts, calcium salts, and calcium carbonate, but does not contain bound nitrogen sources. This composition ratio can support the growth of the endophytic bacteria. Bacillus The fermentation process of *Sp.* provides the necessary carbon skeleton and mineral nutrients while creating a nitrogen-deficient selective stress environment, thereby directionally inducing and enhancing the strain's biological nitrogen fixation metabolic activity. The addition of calcium carbonate to the culture medium helps buffer the pH, maintaining it within a suitable near-neutral range, providing a stable physicochemical environment for the strain's sustained fermentation. Optimized sterilization conditions effectively kill contaminating microorganisms without damaging key components of the culture medium, ensuring a pure and efficient fermentation process. This specifically composed culture medium provides a reliable and reproducible basis for preparing fermentation broths with high nitrogen fixation activity.

[0012] Preferably, the method further includes solidifying the fermentation broth from step S2 to prepare endophytic bacteria. Bacillus Solid carriers of sp. and its metabolites; The preparation method of the solid support specifically includes: The fermentation broth was mixed with sodium alginate solution at a volume ratio of 1:1~3 to obtain a mixed bacterial solution. Add 1% to 3% of the total mass of bentonite and 0.5% to 1.5% of glycerol to the mixed bacterial solution, stir evenly, and then introduce it into a calcium chloride solution with a mass concentration of 2% to 4% by dripping or spraying to solidify and form gel microspheres with a particle size of 1 to 3 mm. The gel microspheres were separated and dried at a low temperature of 30-40 °C until the water content was 10%-20% to obtain the solid carrier. In step S4, the solid carrier is premixed with the tissue culture medium at a ratio of 5-15 g per liter of tissue culture medium, and then the tissue culture seedlings are inoculated and cultured.

[0013] When liquid fermentation broth is directly applied to tissue culture systems, its active ingredients are easily lost due to irrigation or osmosis, making it difficult to maintain an effective concentration in the rhizosphere. By mixing the fermentation broth with sodium alginate solution, adding bentonite and glycerol, and then introducing calcium chloride solution for solidification, gel microspheres with a certain mechanical strength can be formed, thereby encapsulating the bacteria and their metabolites within them. The addition of bentonite enhances the structural stability and ion exchange capacity of the microspheres, while glycerol helps maintain bacterial activity during drying. The solid carrier obtained after low-temperature drying has a suitable moisture content, facilitating storage and use. During tissue culture, this solid carrier is pre-mixed with the culture medium, allowing it to slowly dissolve and release the encapsulated nutrients and active bacteria during cultivation. This provides a more sustained and stable supply of nutrients and microbial support for the tissue culture seedlings, prolonging the growth-promoting effect and improving nutrient utilization efficiency.

[0014] Preferably, during the preparation of the solid carrier, the gel microspheres are subjected to multi-layer coating to form a sustained-release unit with a core-shell structure; The specific components of the multi-layer coating process include: The gel microspheres are immersed in a chitosan acetate solution with a mass concentration of 0.5%~1.5% for 10~30 seconds and then removed. They are then treated with glutaraldehyde (crosslinking agent) vapor for 5~15 minutes to form a first semi-permeable membrane on the surface of the microspheres, thus obtaining membrane-containing gel microspheres. Membrane-containing gel microspheres were dispersed in a solution containing GR24, a plant rhizosphere signaling molecule and an analogue of 5-deoxystrylolactone, at a concentration of 10. -8 ~10 -6 mol / L, gentle shaking adsorption for 30-60 minutes to obtain gel microspheres containing rhizosphere signaling molecules; Gel microspheres containing rhizosphere signaling molecules were transferred into a film-forming solution composed of sodium alginate and nano-sized diatomaceous earth. A second porous protective film was formed on the surface of the microspheres by electrostatic spraying. The mass ratio of sodium alginate to nano-diatomaceous earth was 3:1 to 5:1, and the thickness of the second porous protective film was 20 to 50 micrometers. In tissue culture microenvironments, simple gel carriers still face challenges such as rapid release of internal active substances, susceptibility to bacterial contamination, and difficulty in guiding roots to actively approach the carrier. By multi-layer coating gel microspheres, a slow-release unit with a core-shell structure can be constructed. First, the surface of the microspheres is impregnated and cross-linked with chitosan acetate solution to form a selectively permeable semi-permeable membrane. This membrane effectively regulates the diffusion rate of internal nutrients and bacteria outward, preventing premature release. Subsequently, the microspheres are placed in a solution containing the plant rhizosphere signaling molecule GR24, allowing the signaling molecule to adsorb onto the surface of the microspheres or into the membrane. These signaling molecules actively induce the roots of *Curcuma zedoaria* in Guangxi to grow and branch towards the carrier. Finally, using electrostatic spraying technology, a porous protective membrane composed of sodium alginate and nano-diatomaceous earth is coated onto the outer layer of the microspheres. This membrane provides a physical barrier for the internal structure, enhancing its resistance to bacterial degradation. The resulting core-shell structure enables programmed slow release of nutrients and microorganisms, and through the guidance of signaling molecules, it promotes the formation of closely interacting micro-regions between plant roots and the carrier, thereby significantly extending the duration of the growth-promoting effect and improving the successful colonization and spatial competitiveness of functional strains in the rhizosphere.

[0015] Preferably, the second porous protective membrane is an environmentally responsive membrane, so that the second porous protective membrane has the ability to dynamically adjust its permeability according to changes in the rhizosphere microenvironment; The specific methods for preparing the film-forming solution for the second porous protective membrane include: Gelatin and citric acid were modified by amidation to synthesize a pH-responsive gelatin derivative, wherein the molar ratio of gelatin to citric acid was 1:0.05~0.15; the pH-responsive gelatin derivative was then blended with sodium alginate and nano-diatomaceous earth at a mass ratio of (0.5~1.5):3:1 to obtain a film-forming solution.

[0016] Conventional slow-release carriers typically have a fixed release rate, which is difficult to match in real time with the dynamic physiological activities and nutrient requirements of plant roots. This can lead to the premature or delayed release of nutrients or signaling molecules, reducing utilization efficiency. To address this issue, the film-forming solution was modified by reacting gelatin with citric acid to synthesize a pH-responsive gelatin derivative, which was then blended with sodium alginate and nano-diatomaceous earth to prepare the film-forming solution. The second porous protective membrane, formed by electrostatic spraying, maintains low permeability in the initially near-neutral tissue culture environment. When the roots of *Curcuma zedoaria* tissue culture seedlings grow and secrete organic acids, causing a decrease in the pH value of the rhizosphere microzone, the pH-responsive gelatin derivative in the membrane swells, increasing the membrane's porosity and pore size, thereby accelerating the directional release of embedded signaling molecules and nutrients into the rhizosphere. Conversely, when root activity weakens and the microenvironment pH rises, the membrane structure tends to recover, and the release rate slows down. This design enables the carrier release behavior to be dynamically correlated with the intensity of root physiological activity, achieving adaptive and precise supply with faster release when demand is high and slower release when demand is low, thereby optimizing the delivery efficiency of growth-promoting factors to the target root zone in both time and space.

[0017] Preferably, in step S4, the tissue culture medium is a symbiotic functionalized medium, and the preparation method of the symbiotic functionalized medium includes: Based on 1 / 2 MS medium without organic nitrogen sources and phosphates, the following components were added: Calcium lignosulfonate, concentration 0.05~0.15 g / L; Calcium magnesium phytate, concentration 0.1~0.3 g / L; Volcanic rock microparticles, with a particle size of 50-150 micrometers, were pretreated with 0.1 mol / L citric acid solution to a concentration of 5-15 g / L. Aqueous extracts from the roots of healthy Curcuma zedoaria plants in Guangxi were added at 1% to 3% (v / v) of the total volume of the culture medium. The pH of the symbiotic functionalized culture medium is 6.0–6.5, and the coagulant used has a gel strength of 800–1000 g / cm³. 2 The low-acyl gellan gum is used at a concentration of 2.2~2.6 g / L and is sterilized by autoclaving before use. The preparation method of water extract from the roots of healthy Curcuma zedoaria plants in Guangxi is as follows: roots of healthy Curcuma zedoaria plants from non-tissue culture are taken and extracted with sterile water at a mass-to-volume ratio of 1:20~50 g / mL at 40-50 ℃ for 1-2 hours, and then filtered to remove bacteria to obtain water extract from the roots of healthy Curcuma zedoaria plants in Guangxi.

[0018] When *Curcuma zedoaria* tissue culture seedlings are inoculated into traditional media rich in readily available nitrogen and phosphorus, excessive external nutrients may inhibit the expression of nitrogen fixation and phosphorus solubilization functions of endophytic bacteria, weakening the plant's dependence on the strain and potentially leading to functional degradation of the strain in the long term. Therefore, a specially designed symbiotic functionalized medium was used: based on 1 / 2 MS medium free of organic nitrogen and phosphate, calcium lignosulfonate was added as a slow-release carbon source, calcium magnesium phytate as a slow-release phosphorus source and root exudate inducer, and citric acid-pretreated volcanic rock microparticles were introduced as physical support and a trace element library. Simultaneously, a small amount of root water extract from healthy *Curcuma zedoaria* plants was added to provide host-specific signaling substances. The medium used low-acyl gellan gum as a solidifying agent, and the pH was adjusted to a slightly acidic range. This medium, by reducing the content of readily available nutrients, forces the plant to rely more on the nutrient supply function of endophytic bacteria; the added slow-release substrate continuously stimulates the endophytic bacteria to exert nitrogen fixation and phosphorus solubilization metabolic activities; and the root water extract and optimized physical microenvironment jointly enhance the recognition and interaction between the plant and the strain. Thus, this culture medium reconstructs the rhizosphere environment from three dimensions: nutrition, signaling, and physical structure, promoting and maintaining a close and efficient functional symbiotic relationship between endophytic bacteria and *Curcuma zedoaria* tissue culture seedlings from Guangxi.

[0019] Endophytic fungi of Curcuma zedoaria in Guangxi Bacillus Application of sp. in the preparation of microbial preparations with nitrogen fixation, phosphorus solubilization, iron-producing properties, and promotion of the growth of Curcuma zedoaria in Guangxi.

[0020] The present invention has at least the following beneficial effects: First, the endophytic fungus of Curcuma zedoaria provided in this invention... Bacillus The sp. has been preserved and identified, and has a clear taxonomic position and genetic stability. It also has multiple growth-promoting functions such as nitrogen fixation, phosphorus solubilization and iron production, providing a specific and reliable microbial resource for directly and effectively solving the problem of slow growth of Turmeric tissue culture seedlings in Guangxi.

[0021] Secondly, by solidifying the fermentation broth of the strain and further constructing a slow-release carrier with a core-shell structure and environmental responsiveness, this invention significantly prolongs the action time of active nutrients and functional strains in the tissue culture system, realizes the programmed and intelligent release of nutrients and signals, improves nutrient utilization efficiency, and enhances the colonization competitiveness of the strains in the rhizosphere.

[0022] Third, the symbiotic functionalized culture medium designed in this invention, through the synergistic design of nutrient ratio, specific signaling substances and physical structure, can directionally stimulate the functional expression of endophytic bacteria while reducing the amount of readily available nutrients, optimize the rhizosphere microenvironment, strengthen and maintain the functional symbiotic relationship between plants and endophytic bacteria, thereby ensuring and improving the long-term effect and stability of this growth-promoting method.

[0023] 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

[0024] 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

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Test content 1. Test Methods 1.1 Isolation of endophytic bacteria Rinse the rhizome tissue of fresh and healthy Guangxi Curcuma zedoaria tissue culture seedlings with running water, air dry them naturally, weigh 1 g, 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 ℃.

[0029] NA liquid culture medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, final pH 7.0±0.1, diluted to 1L water, autoclaved for 20 minutes.

[0030] 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.

[0031] 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.

[0032] 1.4 Morphological characteristics Endophytic bacteria ①-1 were streaked onto NA medium and incubated at 37 ℃ for 3 days. Colony morphology was recorded. Gram staining was performed simultaneously, and colony morphology characteristics were observed and recorded under an optical microscope.

[0033] 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.

[0034] 2. Experimental Results 2.1 Morphological characteristics and molecular identification of endophytic bacteria strain ①-1 like Figure 1 As shown, strain ①-1 is milky white, cloudy and opaque, with an unwrinkled surface; the bacteria are mostly round. It is Gram-positive and the cells are rod-shaped.

[0035] 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 1261 bp. Alignment analysis of this sequence with sequences showing high similarity in NCBI revealed a high similarity of 99% between the strain and the corresponding Bacillus 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 Bacillus bacteria in one branch. Based on traditional morphological characteristics and molecular phylogenetic tree analysis, it was identified as... Bacillus 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: 67430.

[0036]

[0037] 2.2 Qualitative detection results of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functional strains Depend on Figure 3 As shown, endophytic bacteria Bacillus The ability of sp. to grow on Ashby nitrogen-free medium and produce a clear zone indicates that it is an endophytic bacterium. Bacillus sp. possesses autotrophic nitrogen fixation capabilities; endophytic bacteria can grow on phosphate-solubilizing media, and the strains... Bacillus The presence of a phosphate-solubilizing zone indicates that this bacterium possesses a certain phosphate-solubilizing ability; strain Bacillus sp. can grow on potassium-solubilizing medium, but does not exhibit a potassium-solubilizing zone; strain Bacillus sp. can grow on siderogenic carriers and form a pale yellow transparent zone. The siderogenic capacity is 1.15 cm, indicating that the strain... Bacillus sp. has the ability to produce iron carriers.

[0038] 2.3 Quantitative detection results of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing strains The results showed that the strain Bacillus sp. Endophytic bacteria possessing nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functions; strains Bacillus The nitrogen-fixing fermentation broth of the strain contained 54.19 mg / L of ammonium nitrogen, 37.31 mg / L of available phosphorus, and 20.48 mg / L of available potassium. Quantitative analysis of the liquid fermentation showed that although no obvious potassium-solubilizing zone was observed on the solid plate, this strain could significantly increase the content of soluble potassium in the culture medium through metabolic activity, thus possessing potassium-solubilizing function.

[0039] Example 1 I. Activation and Culture of Strains 1. Culture medium preparation: Prepare NA liquid culture medium. Accurately 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 stir well. Adjust the pH of the culture medium to 7.0 ± 0.1. Dispense the liquid culture medium into 250 mL Erlenmeyer flasks, 100 mL per flask, and autoclave at 121 ℃ for 20 minutes. Cool before use.

[0040] 2. Inoculation and Culture: In a sterile operating room, inoculate from the preserved... Bacillus In a glycerol tube containing sp. GDMCC No. 67430, a suitable amount of bacterial growth was picked up with an inoculation loop and inoculated into the sterilized NA liquid medium. The inoculated Erlenmeyer flask was placed in a constant temperature shaking incubator at 37 ℃ and a shaking speed of 180 r / min for 60 hours to obtain an activated bacterial solution. At this point, the bacterial solution was uniformly turbid, indicating that the strain had proliferated well.

[0041] II. Nitrogen-fixing fermentation culture 1. Culture Medium Preparation: Prepare Ashby's nitrogen-free liquid medium. Accurately weigh 20.0 g mannitol, 0.5 g KH₂PO₄, 0.2 g MgSO₄·7H₂O, 0.2 g NaCl, 0.1 g CaSO₄·2H₂O, and 2.0 g CaCO₃, and dissolve them in 1 L of deionized water. Stir until fully dissolved or suspended. Adjust the pH of the medium to 7.0 ± 0.2. Dispense 200 mL into 500 mL Erlenmeyer flasks, autoclave at 121 °C for 20 minutes, and cool before use.

[0042] 2. Inoculation and Fermentation: The activated bacterial culture was inoculated at a rate of 5% (v / v) into the above-mentioned Ashby nitrogen-free liquid medium under aseptic conditions. The Erlenmeyer flask was placed in a constant-temperature shaking incubator at 37 ℃ and a shaking speed of 180 r / min for 7 consecutive days to obtain the fermentation broth. After the culture was completed, the medium remained turbid, indicating that the strain grew well under nitrogen-free conditions and underwent nitrogen fixation metabolism.

[0043] III. Treatment of Guangxi Curcuma zedoaria tissue culture seedlings 1. Preparation of tissue culture seedlings: Select sterile tissue culture seedlings of Curcuma zedoaria from Guangxi with basically uniform growth and no pollution (subculture for about 30 days). Carefully remove them with sterile forceps in a clean bench and place their rhizomes in a sterile culture dish.

[0044] 2. Fermentation broth dilution and soaking: The obtained fermentation broth was diluted 15 times with sterile water under aseptic conditions to obtain a soaking solution. The prepared Rhizomes of Curcuma zedoaria tissue culture seedlings were completely immersed in the soaking solution and left to soak at room temperature (about 25°C) for 2 hours.

[0045] IV. Inoculation and Culture 1. Preparation of tissue culture medium: In this example, basic 1 / 2 MS solid medium is used as the tissue culture medium. After preparation and sterilization according to standard methods, it is poured into tissue culture flasks or petri dishes under aseptic conditions and allowed to solidify for later use.

[0046] 2. Inoculation: Remove the soaked *Curcuma zedoaria* tissue culture seedlings from the soaking solution and gently blot away excess liquid with sterile filter paper. Then, inoculate them onto prepared 1 / 2 MS solid medium, one seedling per bottle.

[0047] 3. Culture: Place the inoculated tissue culture bottles in the tissue culture room for culture. The culture conditions are set as follows: temperature 25±1℃, light intensity 2000 lux, light duration 14 hours and dark duration 10 hours per day.

[0048] Example 2 The method of Example 1 is used, except that it further includes solidification of the fermentation broth obtained from nitrogen-fixing fermentation to prepare endophytic bacteria. Bacillus Solid carriers of sp. and its metabolites; The preparation method of the solid support specifically includes: The fermentation broth was mixed with sodium alginate solution at a volume ratio of 1:2 to obtain a mixed bacterial solution. Add 2% bentonite and 1.0% glycerol by total mass to the mixed bacterial solution, stir evenly, and then introduce it into a 3% calcium chloride solution by dripping or spraying to solidify and form gel microspheres with a particle size of 2 mm. The gel microspheres were separated and dried at 35 °C to a water content of 15% to obtain the solid carrier. The solid carrier was pre-mixed with the tissue culture medium at a ratio of 10 g per liter of tissue culture medium, sterilized, and then inoculated and cultured as tissue culture seedlings.

[0049] Example 3 The method of Example 2 is used, except that the gel microspheres are subjected to multi-layer coating treatment during the preparation of the solid carrier to form a sustained-release unit with a core-shell structure. The specific components of the multi-layer coating process include: The gel microspheres were immersed in a 1.0% chitosan acetate solution for 20 seconds and then removed. They were then treated with glutaraldehyde vapor (0.5%-2% v / v) for 10 minutes to form a first semi-permeable membrane on the surface of the microspheres, thus obtaining membrane-containing gel microspheres. Membrane-containing gel microspheres were dispersed in a solution containing GR24, a plant rhizosphere signaling molecule and an analogue of 5-deoxystrylolactone, at a concentration of 10. -7 mol / L, gentle shaking adsorption for 40 minutes to obtain gel microspheres containing rhizosphere signaling molecules; Gel microspheres containing rhizosphere signaling molecules were transferred into a film-forming solution composed of sodium alginate and nano-sized diatomaceous earth. A second porous protective film was formed on the surface of the microspheres by electrostatic spraying. The concentration of sodium alginate in the film-forming solution was 1.5% (w / v), the mass ratio of sodium alginate to nano-diatomaceous earth was 4:1, and the thickness of the second porous protective film was 30 micrometers. After drying at 35 °C to a moisture content of 15%, the microspheres were pre-mixed with tissue culture medium, sterilized, and then inoculated into tissue culture seedlings.

[0050] Example 4 The method of Example 3 is adopted, except that the second porous protective membrane is an environmentally responsive membrane, so that the second porous protective membrane has the ability to dynamically adjust its permeability according to changes in the rhizosphere microenvironment; The specific methods for preparing the film-forming solution for the second porous protective membrane include: Gelatin and citric acid were modified via an amidation reaction to synthesize a pH-responsive gelatin derivative, wherein the molar ratio of gelatin to citric acid was 1:0.1. The pH-responsive gelatin derivative was then added to a film-forming solution prepared from sodium alginate and nano-diatomaceous earth, with a sodium alginate concentration of 1.5% (w / v) and a mass ratio of pH-responsive gelatin derivative, sodium alginate, and nano-diatomaceous earth of 1.5:3:1. This film-forming solution was then electrostatically sprayed onto the surface of gel microspheres to form a second porous protective membrane, thus creating an environmentally responsive film.

[0051] The specific conditions for the amidation reaction are as follows: Reaction system: Dissolve gelatin in an appropriate amount of deionized water to prepare an 8% (w / w) solution, and preheat to 55°C to ensure complete dissolution. While continuously stirring, add citric acid in a molar ratio of 1:0.1 to the gelatin.

[0052] pH adjustment: Use 1 mol / L hydrochloric acid or acetic acid to adjust the pH of the reaction system to a slightly acidic environment of 4.2 to promote the reaction between carboxyl and amino groups.

[0053] Catalyst: 1% of the total mass of the reactants is added as a carbodiimide catalyst (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC) to efficiently catalyze the formation of amide bonds.

[0054] Reaction temperature and time: The reaction system was heated to 80 °C and reacted for 5 hours under continuous gentle stirring.

[0055] Post-processing: After the reaction was completed, the product solution was cooled and dialyzed in deionized water (molecular weight cutoff 13000 Da) for 36 hours to remove unreacted citric acid, catalyst, and small molecule byproducts. Finally, the modified pH-responsive gelatin derivative solid was obtained by freeze-drying.

[0056] Example 5 The method of Example 3 is used, except that in step S4, the tissue culture medium is a symbiotic functionalized medium, and the preparation method of the symbiotic functionalized medium includes: Based on 1 / 2 MS medium without organic nitrogen sources and phosphates, the following components were added: Calcium lignosulfonate, concentration 0.10 g / L; Calcium magnesium phytate, concentration 0.2 g / L; Volcanic rock microparticles, with a particle size of 100 micrometers, were pretreated with 0.1 mol / L citric acid solution to a concentration of 10 g / L. Aqueous extracts from the roots of healthy Curcuma zedoaria plants in Guangxi were added at 2% (v / v) of the total culture medium volume. The pH of the symbiotic functionalized culture medium was 6.5, and the coagulant used had a gel strength of 1000 g / cm³. 2 The low-acyl gellan gum is used at a concentration of 2.5 g / L and is sterilized by autoclaving. The method for preparing the water extract of healthy roots of Curcuma zedoaria plants in Guangxi is as follows: roots of healthy, non-tissue culture Curcuma zedoaria plants are taken and extracted with sterile water at a mass-to-volume ratio of 1:30 g / mL at 45 ℃ for 1.5 hours. After filtration through a 0.22 μm filter membrane for sterilization, the water extract of healthy roots of Curcuma zedoaria plants in Guangxi is obtained.

[0057] control group The tissue culture seedlings of Curcuma zedoaria from Guangxi were directly inoculated onto the prepared 1 / 2 MS solid medium, with one seedling inoculated per bottle. Other culture conditions were the same as in Example 1.

[0058] After 30 days of continuous culture, the increases in plant height, leaf spread, and root length of *Curcuma zedoaria* tissue culture seedlings in Guangxi were measured and calculated, as shown in Table 1 below.

[0059] Increase in plant height = Plant height after cultivation - Plant height before cultivation; Increase in leaf spread = Leaf spread after cultivation - Leaf spread before cultivation; Increase in root length = Root length after cultivation - Root length before cultivation

[0060] Table 1 Growth index data Table 1 shows that after 30 days of continuous culture, the *Curcuma zedoaria* tissue culture seedlings treated with different growth-promoting methods exhibited superior growth compared to the control group in terms of plant height, leaf spread, and root length. The control group showed the lowest increases in all parameters, with increases of only 1.45±0.16 cm, 0.65±0.12 cm, and 0.80±0.18 cm in plant height, leaf spread, and root length, respectively. In contrast, all groups treated with the method described in this invention showed significant promoting effects. Example 1 increased the three parameters to 2.15±0.18 cm, 1.30±0.15 cm, and 1.60±0.20 cm, respectively. With the gradual optimization and integration of the technical solutions, the growth-promoting effect is further enhanced. The corresponding data for Example 2 were 2.77±0.22 cm, 1.83±0.18 cm, and 2.18±0.25 cm, respectively; Example 3 improved to 3.23±0.20 cm, 2.27±0.22 cm, and 2.72±0.30 cm; Example 4 achieved 3.40±0.25 cm, 2.40±0.24 cm, and 2.90±0.35 cm; Example 5 showed the best results, with the three indicators increasing to 3.71±0.30 cm, 2.71±0.26 cm, and 3.25±0.40 cm, respectively. The data results indicate that the series of methods provided by this invention can effectively and progressively improve the growth performance of *Curcuma zedoaria* tissue culture seedlings from Guangxi, and the data from each example show good reproducibility.

[0061] 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 Bacillus 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: 67430.

2. A method for promoting the growth of tissue culture seedlings of Curcuma zedoaria from Guangxi, characterized in that, Includes the following steps: S1: The endophytic fungus of Curcuma zedoaria as described in claim 1 Bacillus The sp. strain was inoculated into NA liquid medium and cultured with shaking at 35-40 ℃ and 160-200 r / min for 48-72 hours to obtain activated bacterial solution; S2: Inoculate the activated bacterial solution into the nitrogen-fixing fermentation medium and incubate it on a shaker at 35~39 ℃ and 170~190 r / min for 6~8 days to obtain the fermentation broth; S3: After rinsing the root and stem tissue of the Guangxi Curcuma zedoaria tissue culture seedlings with sterile water, soak them in fermentation liquid diluted 10 to 20 times for 1 to 3 hours. S4: Soaked Guangxi Curcuma zedoaria tissue culture seedlings were inoculated into tissue culture medium and cultured at 23~27 ℃, light intensity of 1500~2500 lux, and light duration of 12~16 h / d.

3. The method for promoting the growth of *Curcuma zedoaria* tissue culture seedlings according to claim 2, characterized in that, NA liquid culture medium consists of 2.8-3.2 g beef extract, 4.8-5.2 g peptone, 2.3-2.7 g glucose, and 1 L water. The pH of the culture medium is 6.9-7.

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

4. The method for promoting the growth of *Curcuma zedoaria* tissue culture seedlings according to claim 2, characterized in that, The nitrogen-fixing fermentation medium is Ashby nitrogen-free liquid medium, which consists of 19-21 g mannitol, 0.4-0.6 g KH2PO4, 0.1-0.3 g MgSO4·7H2O, 0.1-0.3 g NaCl, 0.05-0.15 g CaSO4·2H2O, 1.8-2.2 g CaCO3, and 1 L water, with a pH of 6.8-7.

2. It is used after autoclaving for 18-22 minutes.

5. The method for promoting the growth of *Curcuma zedoaria* tissue culture seedlings according to claim 2, characterized in that, The process also includes solidifying the fermentation broth from step S2 to prepare endophytic bacteria. Bacillus Solid carriers of sp. and its metabolites; The preparation method of the solid support specifically includes: The fermentation broth was mixed with sodium alginate solution at a volume ratio of 1:1~3 to obtain a mixed bacterial solution. Add 1% to 3% of the total mass of bentonite and 0.5% to 1.5% of glycerol to the mixed bacterial solution, stir evenly, and then introduce it into a calcium chloride solution with a mass concentration of 2% to 4% by dripping or spraying to solidify and form gel microspheres with a particle size of 1 to 3 mm. The gel microspheres were separated and dried at a low temperature of 30-40 °C until the water content was 10%-20% to obtain the solid carrier. In step S4, the solid carrier is premixed with the tissue culture medium at a ratio of 5-15 g per liter of tissue culture medium, and then the tissue culture seedlings are inoculated and cultured.

6. The method for promoting the growth of *Curcuma zedoaria* tissue culture seedlings according to claim 5, characterized in that, In preparing the solid carrier, the gel microspheres are subjected to multi-layer coating to form a sustained-release unit with a core-shell structure; The specific components of the multi-layer coating process include: The gel microspheres are immersed in a chitosan acetate solution with a mass concentration of 0.5%~1.5% for 10~30 seconds and then removed. They are then treated with glutaraldehyde vapor for 5~15 minutes to form a first semi-permeable membrane on the surface of the microspheres, thus obtaining membrane-containing gel microspheres. Membrane-containing gel microspheres were dispersed in a solution containing GR24, a plant rhizosphere signaling molecule and an analogue of 5-deoxystrylolactone, at a concentration of 10. -8 ~10 -6 mol / L, gentle shaking adsorption for 30-60 minutes to obtain gel microspheres containing rhizosphere signaling molecules; Gel microspheres containing rhizosphere signaling molecules were transferred into a film-forming solution composed of sodium alginate and nano-sized diatomaceous earth. A second porous protective film was formed on the surface of the microspheres by electrostatic spraying. The mass ratio of sodium alginate to nano-diatomaceous earth was 3:1 to 5:1, and the thickness of the second porous protective film was 20 to 50 micrometers. The method for promoting the growth of tissue culture seedlings of Curcuma zedoaria in Guangxi according to claim 6 is characterized in that the second porous protective membrane is an environmentally responsive membrane, so that the second porous protective membrane has the ability to dynamically adjust its permeability according to changes in the rhizosphere microenvironment; The specific methods for preparing the film-forming solution for the second porous protective membrane include: Gelatin and citric acid were modified by amidation to synthesize a pH-responsive gelatin derivative, wherein the molar ratio of gelatin to citric acid was 1:0.05~0.15; the pH-responsive gelatin derivative was then blended with sodium alginate and nano-diatomaceous earth at a mass ratio of (0.5~1.5):3:1 to obtain a film-forming solution.

7. The method for promoting the growth of *Curcuma zedoaria* tissue culture seedlings according to claim 2, characterized in that, In step S4, the tissue culture medium is a symbiotic functionalized medium, and the preparation method of the symbiotic functionalized medium includes: Based on 1 / 2 MS medium that does not contain organic nitrogen sources and phosphates, the following components were added: Calcium lignosulfonate, concentration 0.05~0.15 g / L; Calcium magnesium phytate, concentration 0.1~0.3 g / L; Volcanic rock microparticles, with a particle size of 50-150 micrometers, were pretreated with 0.1 mol / L citric acid solution to a concentration of 5-15 g / L. Aqueous extracts from the roots of healthy Curcuma zedoaria plants in Guangxi were added at 1% to 3% (v / v) of the total volume of the culture medium. The pH of the symbiotic functionalized culture medium is 6.0–6.5, and the coagulant used has a gel strength of 800–1000 g / cm³. 2 The low-acyl gellan gum is used at a concentration of 2.2~2.6 g / L and is sterilized by autoclaving before use. The preparation method of water extract from the roots of healthy Curcuma zedoaria plants in Guangxi is as follows: roots of healthy Curcuma zedoaria plants from non-tissue culture are taken and extracted with sterile water at a mass-to-volume ratio of 1:20~50 g / mL at 40-50 ℃ for 1-2 hours, and then filtered to remove bacteria to obtain water extract from the roots of healthy Curcuma zedoaria plants in Guangxi.

8. The endophytic fungi of Curcuma zedoaria as described in claim 1 Bacillus Application of sp. in the preparation of microbial preparations with nitrogen fixation, phosphorus solubilization, iron-producing properties, and promotion of the growth of Curcuma zedoaria in Guangxi.

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