Guangxi curcuma zedoary endophyte Peribacillus sp. And application of Guangxi curcuma zedoary endophyte Peribacillus sp.

Abstract

The invention discloses endophyte Peribacillus sp. Of curcuma kwangxi and application of the endophyte Peribacillus sp., and belongs to the technical field of agricultural microorganisms. Aiming at the technical problems that the existing single-function growth-promoting microbial agent is unstable in growth-promoting effect on the curcuma kwangsiensis and weak in rhizosphere colonization ability, the invention provides a curcuma kwangsiensis endophyte Peribacillus sp. With the preservation number of GDMCC No: 67432, and discloses a method for preparing a microbial preparation from the curcuma kwangsiensis endophyte Peribacillus sp., and the method is characterized by comprising the following steps: activating the strain, performing expanded fermentation to obtain a high-activity bacterial solution, adding the high-activity bacterial solution into the high-activity bacterial solution, and preparing the curcuma kwangsiensis endophyte with the preservation number of GDMCC No: 67432. The bacterial liquid is mixed with a composite carrier material composed of sodium alginate and humic acid, a solid microbial preparation is obtained through low-temperature drying, and finally the preparation is mixed with a turf perlite transplanting matrix in a specific proportion for use. The preparation prepared by the method can effectively enhance the survival, colonization and functional expression of functional bacteria in rhizosphere, and is mainly used for promoting the growth of tissue culture seedlings of Guangxi curcuma zedoary and preventing and treating diseases.

Description

Technical Field

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

[0002] Curcuma zedoaria is an important medicinal plant, but its tissue-cultured seedlings generally suffer from slow growth during cultivation, hindering rapid propagation and standardized production. Utilizing plant endophytic bacteria to promote host growth is a potential solution. Endophytic bacteria can colonize healthy plant tissues and typically possess various functions that promote plant growth, such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and the secretion of siderophores.

[0003] However, in practical applications, directly utilizing endophytic bacteria isolated from plants often faces the challenge of unstable effects. One major reason is that many growth-promoting agents consist of single-function strains or unoptimized strain combinations, resulting in limited adaptability and growth-promoting efficiency for specific host plants (such as Curcuma zedoaria), making it difficult to establish a stable dominant population in the complex rhizosphere environment. This leads to low colonization success rates of beneficial bacteria in the target crop rhizosphere after agent application, making it difficult to maintain population density, thus resulting in large fluctuations and poor reproducibility of growth-promoting effects.

[0004] The reasons for this weak colonization ability and unstable efficacy are complex. On the one hand, the strains themselves may lack tolerance to the stress of the new environment after transplantation; on the other hand, conventional formulation preparation methods may fail to adequately protect bacterial activity or provide a microenvironment conducive to bacterial survival and reproduction in the rhizosphere. Therefore, how to screen for specific functional strains highly adaptable to *Curcuma zedoaria* and develop formulation preparation methods that can effectively maintain bacterial viability and support their successful colonization in the rhizosphere of *Curcuma zedoaria* is key to improving the application efficacy of endophytic bacteria and is also a challenge encountered in current technical practice. Summary of the Invention

[0005] 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.

[0006] Another objective of this invention is to provide an endophytic strain of Curcuma zedoaria from Guangxi. Peribacillus sp., which can be used in the preparation of microbial preparations to promote the growth of Guangxi turmeric tissue culture seedlings, enhance their stress resistance, or improve their rhizosphere microenvironment. The strain has one or more functional potentials to promote plant growth, including nitrogen fixation, phosphorus solubilization, potassium solubilization, and iron production.

[0007] To achieve these objectives and other advantages of the present invention, an endophytic fungus of Curcuma zedoaria from Guangxi is provided. PeribacillusThis 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 GDMCCNo: 67432.

[0008] This strain was isolated from the rhizome tissue of healthy *Curcuma zedoaria* tissue culture seedlings from Guangxi. Based on observation of its culture characteristics, physiological and biochemical assays, and 16S rDNA sequence analysis, it was identified as... Peribacillus This strain is a bacterium. Studies have found that it can grow stably on artificial culture media and exhibits various physiological activities related to plant nutrient acquisition. Further functional verification showed that this strain possesses the ability to fix nitrogen autonomously, converting atmospheric nitrogen into plant-available ammonium nitrogen; simultaneously, it can dissolve insoluble inorganic phosphorus in the culture medium, releasing available phosphorus that can be absorbed by plants; in addition, this strain also has the ability to secrete siderophores, helping plants acquire iron under low-iron conditions. When this strain was prepared into a microbial preparation and applied to tissue-cultured *Curcuma zedoaria* seedlings in Guangxi, it effectively colonized the seedling roots. Through the synergistic effect of the above-mentioned multiple functions, it promoted the seedling's utilization of nutrients such as nitrogen, phosphorus, and iron, thereby significantly accelerating the growth rate of the transplanted seedlings and improving growth indicators such as plant height and leaf number, providing a new microbial resource for the cultivation of robust *Curcuma zedoaria* seedlings.

[0009] A method utilizing the endophytic fungi of Curcuma zedoaria Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, comprising the following steps: S1, the endophytic bacteria of the Guangxi Curcuma zedoaria Peribacillus sp. strains were inoculated into NA liquid medium for activation; S2, the activated strain was inoculated into the fermentation medium at a volume percentage of 1% to 5% for large-scale fermentation. The fermentation conditions were 30 to 37 ℃, 150 to 200 r / min, and 48 to 72 h. S3, the fermentation broth obtained in step S2 is mixed with the carrier material at a mass ratio of 1:1~3 and stirred evenly. The carrier material is composed of sodium alginate and humic acid at a mass ratio of 2:1~5:1. S4. The mixture obtained in step S3 is dried at a low temperature of 35~40 ℃ until the moisture content of the material is 15%~25% to obtain a solid microbial preparation. S5, the solid microbial preparation obtained in step S4 is mixed with the transplanting substrate of Guangxi Curcuma zedoaria tissue culture seedlings at a mass ratio of 1:50~100. The transplanting substrate is a mixture of peat moss and perlite at a volume ratio of 3~5:1.

[0010] This invention involves activating the bacterial strain in a liquid culture medium, then transferring it to a fermentation medium of a specific composition for large-scale cultivation to obtain a high concentration of active bacterial cells. Subsequently, the fermented bacterial broth is mixed with a composite carrier material composed of sodium alginate and humic acid in a specific ratio. Sodium alginate forms a gel network under mild conditions, encapsulating and physically protecting the bacterial cells, while humic acid, as a natural organic substance, provides both a porous structure for bacterial cell attachment and a continuous supply of nutrients. The mixed material undergoes a controlled low-temperature drying process to produce a solid preparation with stable moisture content. Finally, this solid preparation is mixed in a specific ratio with a transplanting substrate specifically designed for Curcuma zedoaria tissue culture seedlings in Guangxi. This specific substrate is composed of peat moss and perlite, possessing good air permeability and water retention. When used for the cultivation of Curcuma zedoaria tissue culture seedlings, the preparation prepared by this method enables the functional bacterial strains to more effectively resist environmental stress under the dual protection of the carrier and substrate, and to slowly release, revive, and proliferate in the rhizosphere as the substrate becomes moist. This significantly improved the colonization success rate and persistence of exogenous strains in the root system of target plants, thereby promoting the growth of Curcuma zedoaria seedlings more stably and improving their overall robustness through the multiple functions of the strains themselves, such as nitrogen fixation, phosphorus solubilization, and iron carrier production.

[0011] 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.

[0012] Preferably, the fermentation medium contains 5-20 g / L sucrose, 2-8 g / L yeast extract, 0.5-2 g / L dipotassium hydrogen phosphate, and 0.2-1 g / L magnesium sulfate.

[0013] Preferably, in step S2, the extended fermentation adopts a two-stage fermentation process; The first stage of fermentation was carried out in the fermentation medium under the following conditions: temperature 35-37 ℃, rotation speed 180-200 r / min, and fermentation time 24-36 h. In the second stage of fermentation, the fermentation broth obtained in the first stage is transferred to a protective medium at a volume percentage of 10% to 20% for further fermentation. The protective medium contains 2 to 5 g / L sucrose, 1 to 2 g / L yeast extract, 0.1 to 0.5 g / L dipotassium hydrogen phosphate, 0.1 to 0.3 g / L magnesium sulfate, and 5 to 15 g / L trehalose. The fermentation conditions are a temperature of 30 to 32 ℃, a rotation speed of 100 to 150 r / min, and a fermentation time of 24 to 36 h. The resulting bacterial solution is used in step S3.

[0014] To improve the preservation and transport adaptability of the formulation, this method employs a step-by-step cultivation strategy. First, high-density fermentation is carried out under nutrient-rich conditions to promote rapid and massive cell proliferation. Subsequently, the actively growing microbial community is transferred to a maintenance medium with a simple carbon source structure and added trehalose, and cultured further at lower temperatures and rotation speeds. During this stage, the microbial metabolic activity shifts towards adapting to adversity, and trehalose, as a compatible solute, gradually accumulates within the microorganisms, helping to stabilize cell membrane structure and protect the activity of biomolecules such as proteins. The microorganisms treated in this process exhibit enhanced intrinsic ability to withstand drying and temperature changes. When these adaptively cultured microorganisms are used in subsequent carrier adsorption and drying processes, they demonstrate higher survival rates. Therefore, the solid microbial agent obtained by this method shows a smaller decline in the number of viable bacteria after storage at room temperature for a period of time, maintaining its biological efficacy for a longer period and thus extending the product's effective shelf life.

[0015] Preferably, the bacterial broth obtained from fermentation is pretreated before being mixed with the carrier material. The specific pretreatment steps are as follows: Add 1% to 3% (w / w) of citrate-sodium citrate buffer solution to the bacterial culture to adjust the pH of the bacterial culture to 5.5 to 6.0; Add calcium chloride to the pH-adjusted bacterial solution to achieve a final concentration of 0.05–0.15 mol / L. Stir the solution at 25–30 °C for 10–20 minutes at a stirring speed of 30–60 r / min to complete the pretreatment of the bacterial solution.

[0016] This invention adds a pretreatment step before mixing the bacterial culture with the carrier. To address the changes in bacterial surface characteristics after specific cultivation, a buffer substance is added to the bacterial culture to adjust the pH to a slightly acidic range. Under this environment, the charge state of the bacterial cell surface changes, reducing mutual repulsion. Subsequently, an appropriate amount of calcium ions is introduced into the system and maintained for a period of time under low-speed stirring. Calcium ions can pre-bind with specific components on the bacterial surface and in the carrier, acting as a bridge. When this pretreated bacterial culture comes into contact with the carrier material containing sodium alginate, calcium ions can rapidly mediate the formation of a denser and more uniform ionic cross-linking network. This makes the bacteria more firmly and uniformly fixed within the carrier framework, reducing detachment during subsequent processing. The solid dosage form prepared through this treatment exhibits improved uniformity of internal bacterial distribution and better structural integrity during drying and storage, resulting in a more stable and sustained release of active bacteria during use, thus enhancing application reliability.

[0017] Preferably, step S3 specifically includes: Sodium alginate was prepared into an aqueous solution with a mass percentage concentration of 1.5% to 3.0%, and stirred at a temperature of 40 to 50 °C until completely dissolved. The fermentation broth was slowly added to the sodium alginate solution while stirring. The fermentation broth and sodium alginate solution were mixed at a volume ratio of 1:0.8~1.5. The stirring speed was controlled at 100~200 r / min and the mixing time was 5~15 minutes to obtain the bacteria-sodium alginate mixture. Humic acid is pre-ground and passed through an 80-100 mesh sieve to obtain fine humic acid powder; Under continuous stirring, fine humic acid powder is slowly and evenly sprinkled into the bacterial-sodium alginate mixture. The mass ratio of sodium alginate to humic acid is 2:1 to 5:1, and the mass ratio of fermentation broth to carrier material is 1:1 to 3. After all the fine humic acid powder is added, maintain the temperature of the mixture at 25~35 ℃ and continue stirring at 60~100 r / min for 20~40 minutes until the mixture presents a uniform paste. The paste-like mixture is left to stand and mature for 10-20 minutes, and then transferred to step S4 for low-temperature drying.

[0018] In the preparation of microbial agents, sodium alginate readily undergoes rapid gelation upon contact with cations in the fermentation broth. This characteristic easily leads to uneven mixing, localized lumps, and incomplete cell encapsulation when directly mixed with the fermentation broth and solid humic acid powder. To address this issue, the mixing process was systematically adjusted. First, sodium alginate was pre-prepared into a homogeneous solution and appropriately heated to reduce its viscosity and ensure complete dissolution. Subsequently, under continuous and gentle stirring, the fermentation broth was gradually added to the sodium alginate solution, allowing the cells to initially and uniformly disperse in the liquid matrix, avoiding premature gelation caused by excessively high instantaneous local concentrations. Next, finely sieved humic acid powder was slowly and evenly sprinkled into the mixture, allowing the humic acid particles to be gradually wetted and dispersed in the multiphase mixture. Under continuous stirring while maintaining a suitable temperature and controlling low shear force, the sodium alginate molecular chains and humic acid particles were fully bonded through physical adsorption and hydrogen bonding, and the entire system gradually transformed into a homogeneous paste. Finally, after a brief period of settling and maturation, the internal structure of the mixture becomes more stable. Through this optimized process, the bacterial cell distribution in the final carrier complex is more uniform, with no visible clumps or unencapsulated areas. This provides a material basis with a uniform structure and high encapsulation rate for subsequent drying and molding processes, ensuring the consistency and reliability of the active ingredients in the final formulation.

[0019] Preferably, step S5 specifically includes: After dry mixing of peat moss and perlite at a volume ratio of 3:1 to 5:1, spray water mist to adjust the moisture content to 25% to 35%, and let it stand for 4 to 8 hours to obtain the pretreated transplanting substrate. The solid microbial preparation was divided into two parts with a mass ratio of 1:1 to 2, and labeled as preparation A and preparation B, respectively. The preparation A is mixed with 30% to 40% of the total pretreated transplanting substrate by the first stage. The mixing mass ratio of preparation A to pretreated transplanting substrate is 1:10 to 20. The mixture is mixed in a drum mixer with a speed of 20 to 40 r / min for 5 to 10 minutes to obtain a high-concentration bacterial agent premixed substrate. The formulation B is mixed with the remaining 60% to 70% of the pretreated transplanting substrate in a second stage. The mixing mass ratio of formulation B to the pretreated transplanting substrate is 1:80 to 160. The mixture is mixed for 5 to 10 minutes in a drum mixer with the same rotation speed to obtain a low-concentration bacterial agent premixed substrate. In the planting container, first lay a low-concentration microbial agent premixed substrate that occupies 40% to 50% of the container volume, then lay all the high-concentration microbial agent premixed substrate on top, and finally cover with the remaining low-concentration microbial agent premixed substrate to complete the potting. After laying the high-concentration microbial agent premixed substrate and before covering it with the remaining low-concentration microbial agent premixed substrate, apply 10-20 mL of root-promoting nutrient solution to the roots of the Guangxi Curcuma zedoaria tissue culture seedlings. The root-promoting nutrient solution contains 0.01%-0.05% citric acid.

[0020] When mixing microbial agents with transplanting substrates, simple uniform mixing may result in random distribution of the agents within the substrate, limiting actual contact with seedling roots and hindering the rapid formation of a dominant rhizosphere microbial community during the critical colonization window after transplanting. Therefore, a layered mixing and application strategy was adopted. First, the solid microbial agent was divided into two parts, mixed separately with small and large amounts of moist substrate, creating two types of premixes: one with locally high concentrations and the other with overall low concentrations. In the planting container, a layer of low-concentration premix was first laid as a base, then the entire high-concentration premix was concentrated in the area where the roots were expected to extend, and finally, the remaining low-concentration premix was covered. This created a "high-concentration active layer" around the roots. Furthermore, after laying the high-concentration layer, a small amount of nutrient solution containing a low concentration of citric acid was applied to the roots of the transplanted tissue culture seedlings. Citric acid can gently regulate the pH of the rhizosphere microenvironment and chelate some metal ions, which helps stimulate root secretion and growth, while simultaneously activating the activity of microbial germination and migration from the carrier. Through this spatial concentration gradient design and the assistance of chemical signals, functional strains can come into contact with new roots earlier and more concentratedly, significantly improving their successful colonization efficiency on the root surface and in the rhizosphere, thereby establishing a mutually beneficial symbiotic relationship more quickly and providing continuous and stable growth support for the early growth of seedlings.

[0021] An endophytic fungus of Curcuma zedoaria from Guangxi Peribacillus Application of sp. in the preparation of microbial preparations with nitrogen fixation, phosphorus solubilization, iron carrier production and disease prevention functions.

[0022] This invention offers at least the following beneficial effects: First, by employing exclusive endophytic strains with multiple growth-promoting functions, it simultaneously improves the efficiency of *Curcuma zedoaria* in acquiring and utilizing key nutrients such as nitrogen, phosphorus, and iron, thereby promoting the growth vitality and biomass accumulation of its tissue-cultured seedlings at the root source. Second, the microbial agent preparation process, through optimized culture conditions and a composite carrier system, significantly enhances the tolerance of functional strains to environmental stresses such as dryness and temperature fluctuations, and ensures the uniform distribution and firm fixation of the bacteria in the carrier, directly improving the preservation stability and shelf life of the preparation. Finally, the accompanying application method, by constructing a rhizosphere microbial agent concentration gradient and combining it with root microenvironment regulation, effectively guides and promotes the early directional colonization of functional strains in the seedling roots, enabling them to establish a symbiotic relationship more quickly and continuously exert their growth-promoting effects, shortening the recovery time after transplanting, and providing a reliable guarantee for the stable cultivation of robust *Curcuma zedoaria* seedlings.

[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 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 ℃.

[0029] NA liquid culture medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, final pH 7.0±0.1, diluted to 1 L of 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 solubilizing 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 halo on CAS medium. The ratio of the orange-yellow halo diameter 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 Endogenous bacteria Peribacillussp. 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.

[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 Endophytic bacteria Peribacillus Morphological characteristics and molecular identification of sp. strains like Figure 1 As shown, strain Peribacillus sp. is milky yellow, cloudy and not transparent; the bacteria are mostly round. They are Gram-positive and 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 1219 bp. Alignment analysis of this sequence with sequences showing high similarity in NCBI revealed a high similarity of 99% between the strain and its corresponding Bacillus peritrichous. 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 peritrichous in one branch. Based on traditional morphological characteristics and molecular phylogenetic tree analysis, it was identified as... Peribacillus 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: 67432.

[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 Peribacillus The ability of sp. to grow on Ashby nitrogen-free medium and produce a clear zone indicates that it is an endophytic bacterium. Peribacillus sp. possesses autotrophic nitrogen fixation; endophytic bacteria Peribacillus The ability of this strain to grow on phosphate-solubilizing medium and exhibit phosphate-solubilizing zones indicates that it possesses a certain phosphate-solubilizing capacity. Peribacillus sp. can grow on potassium-solubilizing medium, but does not exhibit a potassium-solubilizing zone; strain Peribacillus sp. can grow on siderogenic carriers and form a pale yellow transparent zone. The siderogenic capacity is 1.13 cm, indicating that the strain... Peribacillus sp. has the ability to produce iron carriers.

[0038] 2.3 Quantitative detection results of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functional strains The results showed that the strain Peribacillus sp. Endophytic bacteria possessing nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing functions; strains Peribacillus The nitrogen-fixing fermentation broth of this strain contained 41.28 mg / L of ammonium nitrogen, 11.42 mg / L of available phosphorus, and 26.11 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, indicating that it possesses potassium-solubilizing function.

[0039] Example 1 A method utilizing the endophytic fungi of Curcuma zedoaria Peribacillus The specific steps for preparing disease-preventing and growth-promoting microbial agents are as follows: S1, strain activation: Endophytic fungi of Curcuma zedoaria from Guangxi, with preservation number GDMCC No: 67432. Peribacillus The sp. strain was inoculated into liquid NA medium and cultured with shaking at 37 ℃ and 180 r / min for 24 hours to complete activation.

[0040] The composition of NA medium is as follows: 3.0 g beef extract, 5.0 g peptone, 2.5 g glucose, 18 g agar, add water to make up to 1 L, adjust the pH to 7.0, and autoclave at 121 ℃ for 20 minutes before use.

[0041] S2, Expanded Fermentation: The activated bacterial solution from step S1 was transferred to the fermentation medium at an inoculum volume of 3% for expanded fermentation. The fermentation conditions were: temperature 35 ℃, rotation speed 180 r / min, and fermentation time 60 hours.

[0042] The fermentation medium consisted of 10 g / L sucrose, 5 g / L yeast extract, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, and water as the solvent, with a natural pH.

[0043] S3, Mixing with carrier material: Mix the fermentation broth obtained in step S2 with the carrier material at a mass ratio of 1:2, and stir with a stirrer at a speed of 150 r / min for 15 minutes to make it evenly mixed.

[0044] The carrier material is a uniform mixture of sodium alginate and humic acid in a mass ratio of 3:1.

[0045] S4, Low-temperature drying: The mixture obtained in step S3 is placed in a drying tray, spread into a thin layer, and placed in a forced-air drying oven for low-temperature drying at 38 ℃. The material is dried until the moisture content is approximately 20%, yielding a solid powdered microbial preparation.

[0046] S5, Mix with transplanting substrate: Mix the solid microbial preparation obtained in step S4 with the transplanting substrate of Guangxi Curcuma zedoaria tissue culture seedlings at a mass ratio of 1:75, and manually stir evenly.

[0047] The transplanting substrate is a premix of peat moss and perlite in a volume ratio of 4:1.

[0048] Example 2 The method of Example 1 is used, but the difference from Example 1 is that in step S2, the scale-up fermentation adopts a two-stage fermentation process; The first stage of fermentation was carried out in the fermentation medium under the following conditions: temperature 35 ℃, rotation speed 200 r / min, and fermentation time 30 h. In the second stage of fermentation, the fermentation broth obtained in the first stage was transferred to a protective medium at a volume percentage of 15% for further fermentation. The protective medium contained 3 g / L sucrose, 1.5 g / L yeast extract, 0.3 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, and 10 g / L trehalose. The fermentation conditions were 30 ℃, 120 r / min, and 30 h. The resulting bacterial solution was used in step S3.

[0049] Example 3 The method used in Example 2 differs from that in Example 2 in that the bacterial broth obtained from fermentation is pretreated before being mixed with the carrier material. The specific pretreatment steps are as follows: Add 2% (w / w) citrate-sodium citrate buffer to the bacterial culture to adjust the pH of the culture to 6.0; Calcium chloride was added to the pH-adjusted bacterial solution to achieve a final concentration of 0.1 mol / L. The solution was then stirred at 25 °C for 15 minutes at a stirring speed of 40 r / min to complete the pretreatment of the bacterial solution.

[0050] Example 4 The method used in Example 3 differs from that in Example 3 in that step S3 specifically includes: Sodium alginate was prepared into an aqueous solution with a mass percentage concentration of 2.0%, and stirred at 45 °C until completely dissolved. The fermentation broth was slowly added to the sodium alginate solution while stirring. The fermentation broth and sodium alginate solution were mixed at a volume ratio of 1:1. The stirring speed was controlled at 150 r / min and the mixing time was 10 minutes to obtain the bacterial-sodium alginate mixture. Humic acid is pre-ground and passed through an 80-mesh sieve to obtain fine humic acid powder; Under continuous stirring, fine humic acid powder is slowly and evenly sprinkled into the bacterial-sodium alginate mixture. The mass ratio of sodium alginate to humic acid is 3:1, and the mass ratio of fermentation broth to carrier material is 1:2. After all the fine humic acid powder has been added, maintain the temperature of the mixture at 30 ℃ and continue stirring at 80 r / min for 30 minutes until the mixture becomes a uniform paste. The paste-like mixture is left to stand and mature for 15 minutes, and then transferred to step S4 for low-temperature drying.

[0051] Example 5 After dry mixing of peat moss and perlite at a volume ratio of 4:1, water mist was sprayed to adjust the moisture content to 30%, and the mixture was allowed to stand for 6 hours to obtain the pretreated transplanting substrate. The solid microbial preparation prepared in Example 4 was divided into two parts with a mass ratio of 1:1, and labeled as preparation A and preparation B, respectively. The preparation A was mixed with 35% of the total pretreated transplanting substrate by mass in the first stage. The mixing mass ratio of preparation A to pretreated transplanting substrate was 1:15. The mixture was mixed in a drum mixer at 30 r / min for 5 to 10 minutes to obtain a high-concentration bacterial agent premixed substrate. The formulation B was mixed with the remaining 65% of the pretreated transplanting substrate in a second stage. The mixing mass ratio of formulation B to the pretreated transplanting substrate was 1:120. The mixture was mixed for 7 minutes in a drum mixer at the same speed to obtain a low-concentration bacterial agent premixed substrate. In the planting container, first lay a low-concentration microbial agent premixed substrate that occupies 45% of the container volume, transplant the Guangxi Curcuma zedoaria tissue culture seedlings, then lay all the high-concentration microbial agent premixed substrate on top, and finally cover with the remaining low-concentration microbial agent premixed substrate to complete the potting. After laying the high-concentration microbial agent premixed substrate and before covering it with the remaining low-concentration microbial agent premixed substrate, apply 15 mL of root-promoting nutrient solution containing 0.03% citric acid to the roots of the Guangxi Curcuma zedoaria tissue culture seedlings. Routine management was the same as the treatment group.

[0052] Application effect Treatment group: The transplanting substrate containing the microbial preparations prepared in Examples 1-4 was filled into seedling trays, and the tissue culture seedlings of Curcuma zedoaria were transplanted and then managed using conventional methods.

[0053] Comparison Group 1: No microbial agents were added to the transplanting substrate, and the daily management and treatment were the same as the treatment group.

[0054] Thirty days after transplanting, the seedlings in the treatment group showed increases in plant height, number of leaves, and root system compared to control group 1. The incidence rates of root rot, stem base rot, and wilt were also measured. The results are shown in Tables 1 and 2 below.

[0055] Table 1 Growth performance The data in the table represent the increase rate compared to comparison group 1.

[0056] As shown in Table 1, Example 1, using the basic process, exhibited a significant growth-promoting effect. Compared with the control group 1 without added microbial agents, its plant height, number of leaves, and root biomass increased by 20.5%, 18.2%, and 25.1%, respectively. With the gradual optimization of the preparation process, from Example 2 introducing two-stage fermentation, to Example 3 adding microbial pretreatment, and then to Example 4 optimizing the carrier mixing process, all three growth indicators showed a stable and significant upward trend, indicating that each process improvement effectively enhanced the activity, stability, and interaction efficiency of the functional bacteria in the formulation with the plants. When the optimized formulation of Example 4 was used in conjunction with the stratified application method (Example 5), the growth-promoting effect reached its peak, with the increase rates of various growth indicators further increasing to 34.7%, 31.2%, and 42.3%.

[0057] Table 2 Results of Air Defense Damage As shown in Table 2, the untreated seedlings in control group 1 exhibited disease incidence rates as high as 32.5%, 28.0%, and 22.3% for root rot, stem base rot, and wilt, respectively. After application of the formulation of this invention, the disease incidence was effectively controlled, and the control effect increased with optimization of the preparation process. The disease incidence rates in Examples 1 to 4 decreased sequentially. Finally, Example 5, which combined the optimal formulation and precise application method, achieved the best control effect against the three main diseases, reducing the disease incidence rates to 5.1%, 4.0%, and 3.5%, respectively.

[0058] 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 Peribacillus 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: 67432.

2. A method utilizing the endophytic fungus of Curcuma zedoaria as described in claim 1. Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, characterized in that... Includes the following steps: S1, the endophytic fungus of Curcuma zedoaria as described in claim 1 Peribacillus sp. strains were inoculated into NA liquid medium for activation; S2, the activated strain was inoculated into the fermentation medium at a volume percentage of 1% to 5% for large-scale fermentation. The fermentation conditions were 30 to 37 ℃, 150 to 200 r / min, and 48 to 72 h. S3, the fermentation broth obtained in step S2 is mixed with the carrier material at a mass ratio of 1:1~3 and stirred evenly. The carrier material is composed of sodium alginate and humic acid at a mass ratio of 2:1~5:

1. S4. The mixture obtained in step S3 is dried at a low temperature of 35~40 ℃ until the moisture content of the material is 15%~25% to obtain a solid microbial preparation. S5, the solid microbial preparation obtained in step S4 is mixed with the transplanting substrate of Guangxi Curcuma zedoaria tissue culture seedlings at a mass ratio of 1:50~100. The transplanting substrate is a mixture of peat moss and perlite at a volume ratio of 3~5:

1.

3. The endophytic fungi of Curcuma zedoaria according to claim 2 Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, 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 endophytic fungi of Curcuma zedoaria according to claim 2 Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, characterized in that... The fermentation medium contains 5-20 g / L sucrose, 2-8 g / L yeast extract, 0.5-2 g / L dipotassium hydrogen phosphate, and 0.2-1 g / L magnesium sulfate.

5. The endophytic fungi of Curcuma zedoaria according to claim 2 Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, characterized in that... In step S2, the extended fermentation adopts a two-stage fermentation process; The first stage of fermentation was carried out in the fermentation medium under the following conditions: temperature 35-37 ℃, rotation speed 180-200 r / min, and fermentation time 24-36 h. In the second stage of fermentation, the fermentation broth obtained in the first stage is transferred to a protective medium at a volume percentage of 10% to 20% for further fermentation. The protective medium contains 2 to 5 g / L sucrose, 1 to 2 g / L yeast extract, 0.1 to 0.5 g / L dipotassium hydrogen phosphate, 0.1 to 0.3 g / L magnesium sulfate, and 5 to 15 g / L trehalose. The fermentation conditions are a temperature of 30 to 32 °C, a rotation speed of 100 to 150 r / min, and a fermentation time of 24 to 36 h. The resulting bacterial solution is used in step S3.

6. The endophytic fungi of Curcuma zedoaria according to claim 5 Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, characterized in that... The bacterial broth obtained from fermentation is pretreated before being mixed with the carrier material. The specific steps of the pretreatment are as follows: Add 1% to 3% (w / w) of citrate-sodium citrate buffer solution to the bacterial culture to adjust the pH of the bacterial culture to 5.5 to 6.0; Add calcium chloride to the pH-adjusted bacterial solution to achieve a final concentration of 0.05–0.15 mol / L. Stir the solution at 25–30 °C for 10–20 minutes at a stirring speed of 30–60 r / min to complete the pretreatment of the bacterial solution.

7. The endophytic fungi of Curcuma zedoaria according to claim 2 Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, characterized in that... Step S3 specifically includes: Sodium alginate was prepared into an aqueous solution with a mass percentage concentration of 1.5% to 3.0%, and kept at a temperature of 40 to 50°C with stirring until completely dissolved. The fermentation broth was slowly added to the sodium alginate solution while stirring. The fermentation broth and sodium alginate solution were mixed at a volume ratio of 1:0.8~1.

5. The stirring speed was controlled at 100~200 r / min and the mixing time was 5~15 minutes to obtain the bacteria-sodium alginate mixture. Humic acid is pre-ground and passed through an 80-100 mesh sieve to obtain fine humic acid powder; Under continuous stirring, fine humic acid powder is slowly and evenly sprinkled into the bacterial-sodium alginate mixture. The mass ratio of sodium alginate to humic acid is 2:1 to 5:1, and the mass ratio of fermentation broth to carrier material is 1:1 to 3. After all the fine humic acid powder has been added, maintain the temperature of the mixture at 25~35 ℃ and continue stirring at 60~100 r / min for 20~40 minutes until the mixture becomes a uniform paste. The paste-like mixture is left to stand and mature for 10-20 minutes, and then transferred to step S4 for low-temperature drying.

8. The endophytic fungi of Curcuma zedoaria according to claim 2 Peribacillus A method for preparing disease-preventing and growth-promoting microbial agents, characterized in that... Step S5 specifically includes: After dry mixing of peat moss and perlite at a volume ratio of 3:1 to 5:1, spray water mist to adjust the moisture content to 25% to 35%, and let it stand for 4 to 8 hours to obtain the pretreated transplanting substrate. The solid microbial preparation was divided into two parts with a mass ratio of 1:1 to 2, and labeled as preparation A and preparation B, respectively. The preparation A is mixed with 30% to 40% of the total pretreated transplanting substrate by the first stage. The mixing mass ratio of preparation A to pretreated transplanting substrate is 1:10 to 20. The mixture is mixed in a drum mixer with a speed of 20 to 40 r / min for 5 to 10 minutes to obtain a high-concentration bacterial agent premixed substrate. The formulation B is mixed with the remaining 60% to 70% of the pretreated transplanting substrate in a second stage. The mixing mass ratio of formulation B to the pretreated transplanting substrate is 1:80 to 160. The mixture is mixed for 5 to 10 minutes in a drum mixer with the same rotation speed to obtain a low-concentration bacterial agent premixed substrate. In the planting container, first lay a low-concentration microbial agent premixed substrate that occupies 40% to 50% of the container volume, then lay all the high-concentration microbial agent premixed substrate on top, and finally cover with the remaining low-concentration microbial agent premixed substrate to complete the potting. After laying the high-concentration microbial agent premixed substrate and before covering it with the remaining low-concentration microbial agent premixed substrate, apply 10-20 mL of root-promoting nutrient solution to the roots of the Guangxi Curcuma zedoaria tissue culture seedlings. The root-promoting nutrient solution contains 0.01%-0.05% citric acid.

9. The endophytic fungi of Curcuma zedoaria as described in claim 1 Peribacillus Application of sp. in the preparation of microbial preparations with nitrogen fixation, phosphorus solubilization, iron carrier production and disease prevention functions.

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