Pantoea H1 as well as demonstration method and application thereof in agriculture assistance

Abstract

The invention relates to the technical field of microorganisms, and discloses pantoea H1 and a demonstration method and application thereof in agriculture assistance, the pantoea H1 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.36875. The invention further provides a microbial agent containing the bacterial strain, a preparation method and application of the bacterial strain in crop planting, the bacterial strain has efficient dual phosphorus solubilizing capacity, indissolvable inorganic phosphorus can be dissolved, organophosphorus can be mineralized, and the effectiveness of soil phosphorus is improved; meanwhile, the microbial agent has excellent saline-alkaline tolerance, can still keep high activity under adversity, and by applying the microbial agent in the seedling stage of crops, the rhizosphere microenvironment can be remarkably improved, root development and plant growth of the crops such as corn and mung beans can be promoted, the emergence rate and yield of the crops in soil such as saline-alkaline land can be increased, and the crop yield can be increased. And an excellent microbial germplasm resource is provided for activating soil nutrients, reducing weight and improving efficiency.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of pantothenic acid H1 and its demonstration methods and applications in assisting farmers. Background Technology

[0002] Phosphorus is the second most essential nutrient for plant growth, playing a crucial role in crop energy metabolism, signal transduction, and growth and development. In agricultural production, most phosphorus in the soil exists in an insoluble form that is difficult for crops to absorb and utilize. To maintain crop yields, large amounts of chemical phosphate fertilizers are typically applied during agricultural planting. However, phosphate fertilizers applied to the soil are easily fixed by metal ions such as calcium, iron, and aluminum in the soil, with approximately 75%-90% of the phosphorus rapidly converting into unusable forms and accumulating in the soil. This phenomenon not only leads to extremely low utilization rates of phosphate fertilizers in the current season, resulting in a serious waste of non-renewable phosphate rock resources, but also causes excessive phosphorus accumulation to be lost through surface runoff, easily leading to environmental pollution risks such as eutrophication of water bodies.

[0003] Utilizing phosphate-solubilizing microorganisms to convert insoluble inorganic and organic phosphorus in soil into soluble phosphorus that crops can absorb is considered a safe, economical, and effective biological approach to solving the aforementioned problems. Activated sludge, as a rich resource of microorganisms, contains abundant functional microbial strains; however, the development of phosphate-solubilizing strains with agricultural application potential is currently insufficient. Although various phosphate-solubilizing bacteria have been isolated using existing technologies, many challenges remain in practical agricultural applications. On the one hand, the existing phosphate-solubilizing microbial strains are relatively limited in variety and generally suffer from poor environmental adaptability, especially under adverse soil conditions such as saline-alkali soils, where exogenous strains often fail to effectively colonize or maintain high biological activity, leading to unstable field application effects. On the other hand, some strains have a single phosphorus activation mechanism, making it difficult to simultaneously achieve efficient dissolution of inorganic phosphorus and full mineralization of organic phosphorus in the soil, thus failing to maximize the extraction and utilization of accumulated phosphorus reserves in the soil. Therefore, screening and obtaining a superior strain with both highly efficient dual phosphate-solubilizing capabilities and good environmental tolerance is of significant application value for developing new high-efficiency biofertilizers, activating soil nutrients, and ensuring food security. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of Pantothenia glutinosa H1 and its demonstration method and application in assisting farmers. Existing phosphorus-solubilizing microorganisms have poor environmental adaptability (especially salt and alkali tolerance) and a single phosphorus-solubilizing mechanism, which leads to the problem that the insoluble phosphorus accumulated in the soil cannot be effectively activated and utilized, and the crop yield increase effect is unstable.

[0005] To achieve the above objectives, the present invention provides a Pantoea conspicua H1 strain and its demonstration method and application in assisting farmers. In the first aspect, the present invention provides a Pantoea conspicua H1 strain, employing the following technical solution: A strain of Pantoea conspicua H1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36875, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on December 2, 2025.

[0006] By adopting the above technical solution, the pantothecin H1 strain provided by the present invention has the following significant technical effects: Dual phosphorus-solubilizing properties: This strain possesses the dual ability to efficiently dissolve both insoluble inorganic phosphorus (such as tricalcium phosphate) and mineralized organic phosphorus (such as calcium phytate). During metabolism, this strain can alter the pH of the environment and secrete related enzymes to convert phosphorus fixed in the soil into water-soluble phosphorus that can be absorbed and utilized by crops, thereby improving the availability of phosphorus in the soil.

[0007] Salt and alkali tolerance: This strain, selected from a specific environment, exhibits good tolerance to high pH (8.5) and high salt concentration (60 mmol / L mixed saline-alkali) environments. Under salt and alkali stress, this strain can still maintain high biological activity and reproductive capacity, making it suitable for colonization in saline-alkali soil environments.

[0008] Preferably, the 16S of the pantothenic acid strain H1 is... rRNA The gene sequence is shown in SEQ ID No. 1. By adopting the above technical solution, the molecular biological classification characteristics of this strain have been clarified, ensuring the singleness of the strain source and the stability of the genetic background, which facilitates the rapid identification and tracking of the strain through genetic methods.

[0009] Secondly, the present invention provides a microbial inoculant containing the above-mentioned strain of Pantotheca H1, employing the following technical solution: A microbial agent comprising a strain of pantothecin H1 and an agriculturally acceptable carrier; the carrier being a liquid culture medium carrier or a solid matrix carrier.

[0010] By adopting the above technical solution, the strain is provided with the necessary nutritional support and physical survival space by using a carrier, which extends the shelf life of the strain and facilitates its transportation and application in field production.

[0011] Preferably, the microbial agent is prepared by a method comprising the following steps: inoculating a strain of Pantotheca H1 into a liquid culture medium and culturing it for 8-24 hours at a temperature of 25℃-30℃ and a rotation speed of 160rpm-200rpm to obtain a liquid microbial agent; or, mixing the liquid microbial agent with a solid substrate carrier at a mass ratio of 1:2-1:4, fermenting and drying it at 30℃-35℃ to obtain a solid microbial agent. By adopting the above technical solution, the specific temperature control and fermentation parameters (25℃-30℃, 8-24 hours) keep the bacteria in the middle and late stages of the logarithmic growth phase, at which time the bacteria metabolism is vigorous and the biomass accumulation reaches its peak, resulting in a high number of effective viable bacteria and avoiding bacterial death due to over-cultivation. The composting process in the solid fermentation process promotes the adsorption and binding of bacteria and carrier, improving the survival rate of the agent in the soil.

[0012] Preferably, the liquid culture medium carrier comprises tryptone, yeast extract, and sodium chloride; the solid matrix carrier comprises peat moss and wheat bran, wherein the mass ratio of peat moss to wheat bran is (2-4):1. By adopting the above technical solution, the liquid carrier components provide abundant carbon and nitrogen sources, maintaining high bacterial activity; the peat moss in the solid carrier has good adsorption and water retention properties, while the wheat bran provides slow-release organic nutrients. The microenvironment formed by the combination of the two is conducive to the rapid colonization of the bacterial strain in the early stage of soil application.

[0013] Thirdly, this invention provides a demonstration method for using the aforementioned pan-mycelium H1 strain in agricultural assistance, employing the following technical solution: A demonstration method for using Pan-Mycobacterium H1 in assisting farmers includes the following steps: S1. Preparation of microbial agent: providing the above-mentioned Pan-Mycobacterium H1 or microbial agent; S2. Application treatment: applying the Pan-Mycobacterium H1 or microbial agent to crops or planting soil by means of seed soaking, root irrigation or soil mixing.

[0014] By adopting the above technical solution, this invention utilizes the rhizosphere growth-promoting mechanism of a strain of Pantotheca H1 to achieve increased crop yield and soil improvement. The specific mechanism of action is as follows: Rhizosphere colonization and acidification-induced phosphorus reduction: A strain of Pantothecin H1 colonized the crop rhizosphere soil. During metabolism, the strain secreted small-molecule organic acids such as gluconic acid and citric acid, leading to a decrease in the pH of the rhizosphere microenvironment. High concentrations of hydrogen ions (H+)... + It exchanges with calcium ions in insoluble phosphates (such as Ca3(PO4)2), disrupting the phosphate crystal lattice structure and thus releasing hydrogen phosphate ions (HPO4). 2- or H2PO4 - ).

[0015] Enzymatic hydrolysis and mineralization of organic phosphorus: The strain secretes extracellular enzymes such as acid phosphatase and phytase, which act on organic phosphorus compounds (such as phytates) in the soil, catalyzing the hydrolysis and breakage of phosphoester bonds, mineralizing organic phosphorus into inorganic phosphorus, and significantly increasing the available phosphorus content in the soil.

[0016] Improving the microecology of saline-alkali soil: In saline-alkali soil, the organic acids secreted by the strains can neutralize the excessive alkalinity around the roots, reduce the pH value of the rhizosphere, and alleviate the corrosive and toxic effects of carbonates on crop roots. At the same time, the improved rhizosphere physicochemical environment promotes the absorption of nutrients such as nitrogen and phosphorus by crops and improves the crops' tolerance to saline-alkali stress.

[0017] Preferably, in step S2, the specific method of application treatment is selected from any of the following: (1) Root irrigation treatment: Dilute the liquid microbial agent with water 100-200 times and irrigate along the soil around the roots of the crop. The application amount is equivalent to 1-2L of the original solution per acre. (2) Seed soaking treatment: Immerse the crop seeds in liquid microbial agent for 2-4 hours under dark conditions, take them out and dry them in the shade before sowing; (3) Soil treatment: Mix solid microbial agents with organic fertilizer and apply them in strips in the planting furrow.

[0018] By adopting the above technical solutions, different application methods are adapted to different agricultural production scenarios. Seed soaking treatment enables the bacterial strain to colonize the seed coat and radicle in the early stage of seed germination, providing early protection; root irrigation treatment ensures that the bacterial solution directly acts on the dense root area, improving the utilization efficiency of the bacterial strain; soil treatment combined with the application of organic fertilizer provides a continuous source of nutrition for the bacterial strain and prolongs the effect time.

[0019] Preferably, the application treatment is carried out during the seedling stage of the crop; the application treatment includes at least two consecutive applications with an interval of 5-7 days. By adopting the above technical solution, applying the treatment during the seedling stage (such as the three-leaf and one-heart stage of corn, or the first pair of true leaves stage of mung beans) captures the critical window period for crop root development and nutrient requirements. The intermittent continuous application can replenish the number of microbial communities lost due to environmental competition, maintain the population dominance of one strain of Pantotheca H1 in the rhizosphere soil, and ensure a continuous phosphorus-soluble growth-promoting effect.

[0020] Preferably, the crop is corn or mung bean; the planting soil is saline-alkali land, specifically saline-alkali soil with a pH of 8.0-9.0. By adopting the above technical solution, the applicability of this method under specific crops and adverse soil conditions has been verified, particularly in moderately saline-alkali land, where biological improvement methods overcome the problem of low utilization of chemical fertilizers.

[0021] Fourthly, the present invention provides an application of the above-mentioned Pantotheca H1 strain in the preparation of bio-fertilizers or soil conditioners, employing the following technical solution: Application of a single strain of Pantotheca H1 in the preparation of biofertilizers or soil conditioners.

[0022] By employing the above-mentioned technical solution, a wild strain of Pantothenia glutinosa H1, possessing specific phosphorus-solubilizing and stress-resistance functions, was transformed into a standardized agricultural input. This application not only developed new microbial germplasm resources but also provided an environmentally friendly and efficient biological solution to problems in agricultural production such as soil phosphorus fixation, difficulties in utilizing saline-alkali land, and excessive application of chemical fertilizers.

[0023] Preferably, the number of viable Pantotheca H1 strains in the bio-fertilizer or soil conditioner is ≥1.0 × 10⁻⁶. 8 CFU / mL, or ≥2.0×10 8 CFU / g.

[0024] By adopting the above technical solution, the quality indicators of the product are defined. The high concentration of effective live bacteria ensures that the microbial community after application to the soil occupies an ecological niche advantage in competition with native microorganisms, thus ensuring the stability and effectiveness of bio-fertilizers or soil conditioners in actual field applications.

[0025] This invention provides a strain of pan-mycelium H1 and its demonstration method and application in assisting farmers. It has the following beneficial effects: The present invention provides a strain of Pantotheca H1 with excellent phosphorus activation ability, exhibiting significant dissolution and mineralization effects on common insoluble inorganic phosphorus (tricalcium phosphate) and organic phosphorus (calcium phytate) in soil. This strain lowers the environmental pH by secreting organic acids during metabolism, using acidolysis to disrupt the inorganic phosphate lattice, while simultaneously secreting phosphatases to catalyze the hydrolysis of organic phosphorus. This dual mechanism effectively releases potential phosphorus resources fixed in the soil, converting ineffective phosphorus into water-soluble phosphorus that crops can directly absorb and utilize, thereby reducing reliance on chemical phosphate fertilizers in agricultural production and alleviating soil compaction and degradation caused by excessive fertilization.

[0026] The strains in this invention were screened from specific environments and possess natural tolerance to high pH and salinity stress. They maintain high reproductive activity and colonization capacity even in moderately saline-alkali environments (pH 8.5). When applied to saline-alkali soils, the metabolic acid-producing activity of these strains in the rhizosphere effectively neutralizes alkaline substances, regulates the rhizosphere pH balance, and improves the physicochemical environment for root growth. This improvement in the rhizosphere microecology alleviates the toxic effects of saline-alkali stress on crop roots, increases the germination rate and biomass accumulation of crops such as mung beans under adverse conditions, and provides an effective technical means for the bioremediation and utilization of saline-alkali land.

[0027] This invention relates to a microbial inoculant prepared based on a strain of Pantotheca H1. When applied in conjunction with seedling irrigation or seed soaking, it can improve the agronomic traits and yield composition of crops. Field trials have shown that this technique effectively promotes root elongation and plant height in maize and mung beans, optimizes the root-to-shoot ratio, reduces tip barrenness in maize, and increases single ear weight and 100-grain weight. Through the synergistic effect of improving rhizosphere nutrient supply and enhancing plant resistance, this strain can significantly increase crop yield per acre, achieving improved quality and efficiency in agricultural production. Attached Figure Description

[0028] Figure 1 The pan-bacterium H1 of this invention is based on 16S rRNA A schematic diagram of a phylogenetic tree of gene sequences; Among them, the appendix Figure 1 Evolutionary relationship analysis of strain H1 with other bacterial groups—based on 16S rRNA Gene sequences were used to construct phylogenetic trees using the neighbor-joining method. All phylogenetic analyses were performed using MEGA11 software. BLAST comparison and phylogenetic analysis were performed on strain H1's 16S... rRNA The sequence is 100% homologous to Pantoea conspicua, and the two cluster together in the phylogenetic tree. Based on the classical classification threshold, H1 is preliminarily identified as this species. Figure 2 This is a schematic diagram illustrating the determination of the phosphorus solubility effect of pantothenic acid H1 in this invention; Figure 3 This is a schematic diagram of the Gram staining results of Pantotheca H1 of the present invention; Figure 4 This is a schematic diagram illustrating the growth-promoting effect of Pantothecin H1 on potted mung beans according to the present invention. Figure 5 This is a schematic diagram showing the growth of corn after implementing Pantotheca H1 according to the present invention; Figure 6 This is a schematic diagram illustrating the effect of the pantothecin H1 strain of the present invention on corn fruits. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Preparation Examples 1-5: Preparation Example 1: Screening, isolation and identification of a pantothenic acid strain H1 This preparation example describes the process of obtaining a strain of *Pantoea conspicua* H1. Activated sludge was collected from the aeration tank of a wastewater treatment plant. 1.0 g of activated sludge was weighed and added to a conical flask containing 20 mL of sterile distilled water. The flask was shaken and incubated at room temperature for 30 minutes to prepare a suspension. The supernatant was collected and serially diluted with sterile water to concentrations of 10-fold, 100-fold, and 1000-fold. 100 μL of each dilution was spread onto LB agar plates, which were then inverted and incubated at 28°C for 24 hours. After colonies grew, single colonies were picked based on their morphology and purified by streak plating three times on LB agar plates to obtain the purified strain. The purified strain was inoculated onto organophosphorus selection medium (containing calcium phytate) plates and incubated at 28°C for 72 hours. The diameter of the phosphate-solubilizing zone (D) and the colony diameter (d) were measured. A superior strain with a D / d value of 4.25 was selected and named H1.

[0031] Identification of strain H1: After culturing on LB plates for 3 days, Gram staining and microscopic examination were performed. The results showed that the bacterial cells were red, indicating that it was a Gram-negative bacterium. Genomic DNA was extracted from the strain and analyzed using 16S... rRNA PCR amplification and Sanger sequencing were performed using universal primers 27F and 1942R to obtain the sequence shown in SEQ ID No. 1. The sequence was then BLAST-aligned in the NCBI database, and a phylogenetic tree was constructed, confirming that strain H1 is *Pantoea conspicua*. This strain was mixed with 80% glycerol at a 1:4 volume ratio and stored at -80°C.

[0032] Preparation Example 2: Preparation of a Pantotheca H1 liquid microbial inoculant (standard process) This preparation example corresponds to the bacterial culture preparation method mainly used in subsequent examples and test examples. A single strain of *Pantheraea perlatum* H1 glycerol tube, stored at -80°C, was streaked onto an LB agar plate using an inoculation loop and activated by incubation at 37°C for 24 hours. A single colony was picked and inoculated into a 250mL Erlenmeyer flask containing 50mL of LB liquid medium, and cultured with shaking at 37°C and 180rpm for 12 hours to obtain the seed culture. The seed culture was then transferred at a 1% (v / v) inoculation rate to a 500mL Erlenmeyer flask containing 200mL of fresh LB liquid medium and cultured with shaking at 37°C and 180rpm until the optical density (OD) of the bacterial culture reached the desired value. 600 Once the concentration reaches 1.0, cultivation is stopped. The resulting fermentation broth is a liquid microbial inoculum of Pantotheca H1, with an effective viable count of 1.5 × 10⁻⁶. 8 CFU / mL.

[0033] Preparation Example 3: Preparation of a liquid microbial inoculant of Pantotheca H1 strain (low-temperature long-term process) This preparation example aims to verify the feasibility of culturing this strain at lower temperatures. One activated *Pantheraea perfringens* H1 strain from Preparation Example 2 was inoculated into LB broth to prepare a seed culture. The seed culture was inoculated into fresh LB broth at an inoculation rate of 2% (v / v), the incubation temperature was adjusted to 28°C, the shaker speed was set to 160 rpm, and the incubation time was extended to 24 hours. After incubation, the OD of the bacterial culture was measured. 600 The value was 1.1, and the number of viable bacteria in the obtained liquid was 1.2 × 10⁻⁶. 8 CFU / mL. The microbial agent prepared by this process is used to support the generalization regarding the lower fermentation temperature range in the claims.

[0034] Preparation Example 4: Preparation of a Pantotheca H1 liquid microbial inoculant (high temperature short time process) This preparation example aims to verify the rapid fermentation ability of this strain at a high inoculum size. One activated *Pantotheca acuminata* H1 strain from Preparation Example 2 was inoculated into LB broth to prepare a seed culture. The seed culture was inoculated into fresh LB broth at an inoculum size of 5% (v / v), and the culture temperature was set at 30°C, the shaker speed at 200 rpm, and the culture time was shortened to 10 hours. After culture, the OD of the bacterial culture was measured. 600 The value was 0.95, and the effective viable bacteria count of the resulting liquid was 1.4 × 10⁻⁶. 8 CFU / mL. The bacterial agent prepared by this process is used to support the generalizations regarding different inoculum amounts and incubation times in the claims.

[0035] Preparation Example 5: Preparation of a solid microbial fertilizer made from a strain of Pantotheca H1 This preparation example demonstrates the process for preparing a solid dosage form of this strain. Commercially available peat moss and wheat bran were mixed at a mass ratio of 3:1 as a carrier. The mixture was pulverized and passed through a 60-mesh sieve, then autoclaved at 121°C for 30 minutes and dried for later use. The OD obtained in Preparation Example 2 was taken... 600 One strain of Pantotheca H1 liquid inoculant (1.0 μL) was mixed with a sterilized and cooled carrier at a liquid-to-solid mass ratio of 1:3. The mixture was thoroughly stirred under aseptic conditions and then fermented in a sterile room at 30°C for 48 hours, turning the pile twice during fermentation. After fermentation, the mixture was dried at 35°C until the moisture content was 10%, then pulverized and packaged to obtain one strain of Pantotheca H1 solid microbial fertilizer. The effective viable count was determined to be 2.0 × 10⁻⁶. 8 CFU / g. This preparation example serves to support the generalization regarding the microbial agent formulation and carrier in the claims.

[0036] Examples 1-5: As attached Figure 1Example 1: This example provides a method for dissolving inorganic phosphorus using a strain of *Pantotheca acuminata* H1, comprising the following steps: 1 mL of bacterial suspension was taken from a liquid microbial agent of *Pantotheca acuminata* H1 obtained in Preparation Example 2, placed in a sterile centrifuge tube, centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were resuspended and washed with sterile distilled water. This washing was repeated three times, and finally, the suspension was resuspended with sterile distilled water to the original volume to obtain an inoculum suspension. A 250 mL Erlenmeyer flask containing 50 mL of inorganic phosphorus screening medium (with 3.0 g / L-5.0 g / L tricalcium phosphate as the sole phosphorus source) was inoculated with the above inoculum suspension at an inoculation rate of 1% (v / v). The inoculated Erlenmeyer flask was placed in a constant temperature shaker at 28℃±1℃ and cultured at 180 rpm in the dark for 5-7 days. A treatment with an equal volume of sterile distilled water was set up as a blank control. Samples were taken periodically during the culture period for subsequent determination of the water-soluble phosphorus content in the fermentation broth.

[0037] As attached Figure 2 Example 2: This example provides a method for dissolving organophosphates using a strain of *Pantheraea perlatum* H1, comprising the following steps: The washing of the bacterial cells and the preparation of the inoculum suspension are the same as in Example 1. A 250 mL Erlenmeyer flask containing 50 mL of organophosphate screening medium (with 3.0 g / L-5.0 g / L calcium phytate as the sole phosphorus source) is inoculated with the inoculum suspension at a rate of 1% (v / v). The Erlenmeyer flask is placed in a constant temperature shaker at 28℃±1℃ and cultured at 180 rpm in the dark for 5-7 days. A treatment with an equal volume of sterile distilled water is provided as a blank control. After the culture is completed, the supernatant is collected by centrifugation and used for subsequent determination of the water-soluble phosphorus content in the fermentation broth.

[0038] As attached Figure 3Example 3: This example provides a potted application method for promoting mung bean growth under salt-alkali stress using a strain of Pantothenic Acid H1, including the following steps: Select commercially available mung bean seeds, choosing plump, uniformly sized seeds free from pests and diseases, and rinse them with sterile water after surface disinfection. Prepare planting soil by filling planting pots with sieved soil, ensuring each pot contains the same amount of soil. Prepare a salt-alkali stress simulation solution with a solute molar ratio of NaCl:Na2SO4:NaHCO3:Na2CO3=1:9:9:1, a total concentration of 60mmol / L, and adjust the pH to 8.5±0.1. Before sowing, soak the potting soil with this salt-alkali solution, and sow 5 seeds per pot when the soil moisture is suitable. After the seedlings have grown their first pair of true leaves, thin them out, retaining 3 seedlings of uniform growth per pot. Use the Pantothenic Acid H1 liquid inoculant prepared in Example 2 as a treatment agent, applying 10mL of the solution to the soil around the roots of the seedlings. A blank control group was set up, receiving an equal volume of sterile water. After inoculation, the potted plants were placed in a light incubator and cultured at 28°C. A second root drenching was performed 7 days after the first inoculation, with the same amount of water applied. The culture period lasted a total of 30 days, after which plant height, root length, and biomass were measured.

[0039] As attached Figure 4 As shown in Example 4: This example provides a field application method for improving maize yield and soil quality using a single strain of Pantotheca H1, including the following steps: The maize variety used was Zhengdan 958. A randomized block design was used, with treatment and control groups. During the maize seedling stage, a liquid inoculant of Pantotheca H1 (effective viable count ≥1.0 × 10⁻⁶) provided in Example 2 was used. 8 The bacterial solution (CFU / mL) was diluted 100-200 times with water to prepare a diluted bacterial solution. The diluted solution was applied to the root zone of maize in the treatment group via root irrigation, at a rate equivalent to 1-2 L of the original solution per acre. The control group received the same amount of water. Field management followed conventional agricultural practices, with no additional chemical phosphate fertilizer applied throughout the growing season. At maize maturity and harvest, yield measurements were taken for each plot, including plant height, ear height, ear length, ear diameter, tip barrenness length, ear weight, and yield per acre. Rhizosphere soil samples were also collected to determine the content of available phosphorus and alkaline nitrogen.

[0040] As attached Figure 5 Appendix Figure 6As shown in Example 5: This example provides a method for seed soaking treatment using a strain of Pantotheca H1, comprising the following steps: using a liquid inoculant of Pantotheca H1 prepared in Preparation Example 3 or Preparation Example 4. Corn or mung bean seeds are selected, and after surface disinfection and cleaning, the seeds are immersed in a container containing the H1 liquid inoculant, with the liquid level 1-2 cm above the seeds. Soaking is carried out at room temperature in the dark for 2-4 hours, with gentle stirring to ensure even contact. After soaking, the seeds are removed and placed in a cool place to dry until the surface is free of water stains, and then sown. Seeds soaked in sterile water are used as a blank control. This example colonizes the bacterial strain on the seed surface through soaking, aiming to provide growth promotion and stress resistance protection in the early stages of seed germination, supporting the method of use regarding seed soaking in the claims.

[0041] Comparative Examples 1-5: Comparative Example 1: Compared with Example 1, the difference is that an equal volume of sterile distilled water was used instead of one strain of pantothenic acid H1 liquid microbial agent, and all other aspects are the same.

[0042] Comparative Example 2: Compared with Example 1, the difference is that an equal volume of sterile LB liquid culture medium (without bacterial cells) was used instead of one strain of pantothenic acid H1 liquid microbial agent, and all other aspects are the same.

[0043] Comparative Example 3: Compared with Example 3, the difference is that an equal volume of sterile distilled water was used instead of one strain of pantothenic acid H1 liquid microbial agent, and all other aspects are the same.

[0044] Comparative Example 4: Compared with Example 3, the difference is that the Pantotheca H1 liquid microbial agent used was first sterilized by high-pressure steam at 121°C for 20 minutes before use (i.e., using inactivated bacterial solution), and the rest were the same.

[0045] Comparative Example 5: Compared with Example 4, the difference is that an equal volume of water was used instead of one strain of Pantotheca H1 to dilute the bacterial solution, while the rest were the same.

[0046] Test Example 1-3: Test Example 1: Determination of the phosphorus-solubilizing ability of a strain of Pantothenia glutinosa H1 Experimental results: The phosphorus-solubilizing characteristics of a pantothenic bacterium H1 strain were verified using the plate solubilization zone method and liquid fermentation method.

[0047] Plate phosphate solubility test: Following the method in Preparation Example 1, the activated H1 strain was inoculated onto solid screening media containing tricalcium phosphate (inorganic phosphorus) and calcium phytate (organic phosphorus), respectively, and incubated at 28°C for 72 h. The diameter of the phosphate solubility zone (D) and the colony diameter (d) were measured, and the D / d value was calculated. A treatment inoculated with an equal volume of sterile water served as a blank control.

[0048] Liquid fermentation experiment: Conducted according to the procedures of Example 1 (inorganic phosphorus) and Example 2 (organic phosphorus). The experimental setup was as follows: H1 treatment group: Inoculated with a live strain of pantothenic acid H1.

[0049] CK control group: Inoculated with an equal volume of sterile distilled water (competition ratio 1).

[0050] Substrate control group: Inoculated with an equal volume of sterile LB liquid medium (corresponding to ratio 2). Each treatment was set up in 3 replicates. After 5 days of culture, the fermentation broth was centrifuged, the pH value of the supernatant was measured, and the soluble phosphorus content was determined by the molybdenum antimony colorimetric method.

[0051] Experimental results: The measurement results are shown in Table 1.

[0052]

[0053] Results and conclusions: As shown in Table 1, under the condition that the only phosphorus source is insoluble inorganic phosphorus (tricalcium phosphate) or organic phosphorus (calcium phytate), the pH value of the fermentation broth of the CK control group and the substrate control group did not change significantly, and the soluble phosphorus content remained at an extremely low level (<10mg / L), indicating that the background phosphorus content introduced by the natural environment or culture medium was extremely low, and there was no spontaneous phosphorus dissolution phenomenon.

[0054] After 5 days of cultivation, the pH of the fermentation broth in the treatment group inoculated with a single strain of pantothenic acid H1 decreased significantly. Specifically, the pH of the inorganic phosphorus system decreased from 7.20 to 4.42, and the organic phosphorus system decreased to 5.12. Along with the decrease in pH, the soluble phosphorus content in the fermentation broth increased substantially, reaching 421.3 mg / L in the inorganic phosphorus system and 315.7 mg / L in the organic phosphorus system. The transparent phosphorus-soluble zones observed in the plate experiments (D / d values ​​of 4.25 and 3.68, respectively) were consistent with the results of liquid fermentation.

[0055] Data shows that a strain of Pantotheca H1 can secrete organic acids during its metabolism, leading to acidification of the culture environment. This acidification process converts insoluble inorganic phosphates into soluble phosphorus. Simultaneously, this strain possesses the ability to mineralize organic phosphorus sources such as calcium phytate. A strain of Pantotheca H1 exhibits highly efficient dual phosphorus-solubilizing activity, enabling the activation of potential phosphorus resources in the soil.

[0056] Test Example 2: Determination of the growth-promoting effect of mung beans under salt-alkali stress Experimental Description: This test aims to verify the actual promoting effect of a strain of Pantotheca H1 on crop growth under saline-alkali stress, and to exclude the nutritional interference of the organic matter components of the bacteria itself.

[0057] Experimental grouping and treatment: Pot experiments were conducted strictly in accordance with the methods of Example 3, Comparative Example 3 and Comparative Example 4.

[0058] Example 3 (live bacteria treatment): One strain of Pantotheca H1 live bacteria solution was applied as a root irrigation.

[0059] Comparative Example 3 (blank control): Sterile water was applied to the roots.

[0060] Comparative Example 4 (Inactivated Control): H1 bacterial solution inactivated by high temperature was applied by root irrigation.

[0061] Stress conditions: Soil in all treatment groups was infiltrated with a 60 mmol / L saline-alkali simulated solution (pH 8.5) to simulate a moderately saline-alkali environment.

[0062] Measurement indicators: Germination rate: The number of seedlings that emerge is counted on the 7th day after sowing, and the germination rate is calculated.

[0063] Biomass indicators: After 30 days of cultivation, carefully remove the plants, wash off the soil from the roots, and dry the surface moisture. Use a ruler to measure the plant height (from the base of the stem to the growing point) and root length (length of the taproot), and use an electronic balance to weigh the fresh weight of the plants. Take 3 plants from each pot and calculate the average value, repeating the process 3 times.

[0064] Experimental results: The measurement data are shown in Table 2.

[0065]

[0066] Results and Conclusions: According to the data in Table 2, under mixed saline-alkali stress conditions of pH 8.5 and 60 mmol / L, the growth of mung bean plants in Comparative Example 3 (blank control) was significantly inhibited, with low germination rate, poor root development, and less biomass accumulation. The data for Comparative Example 4 (inactivated bacteria) were similar to those of the blank control group, with no significant differences between the two. This indicates that simply adding bacterial cell lysate or culture medium components is insufficient to alleviate the damage caused by saline-alkali stress to crops.

[0067] Example 3 (H1 live bacteria) showed significantly better performance in all agronomic indicators compared to the two control groups. Compared to the blank control, after applying one strain of pantothenic acid H1 live bacteria, the germination rate of mung beans increased by about 40%, plant height increased by about 71.8%, root length increased by about 90.4%, and fresh weight increased by about 144.7%.

[0068] The above results confirm that a strain of Pantotheca H1 can maintain high biological activity even under saline-alkali conditions. Its growth-promoting mechanism lies in the colonization and metabolic activities of living microorganisms in the rhizosphere: on the one hand, it regulates the pH of the rhizosphere microenvironment by secreting organic acids, alleviating alkaline stress; on the other hand, the living bacteria continuously convert phosphorus fixed in the soil into forms absorbable by plants, promoting root elongation and nutrient absorption, thereby enhancing the plant's survival ability and biomass accumulation under adverse conditions. The fact that the inactivated bacterial solution failed further confirms that the effect of this technology stems from the life activities of the bacterial strain rather than the material components themselves.

[0069] Test Example 3: Maize Field Yield and Rhizosphere Soil Nutrient Determination Experimental Description: This test aims to verify the effects of a strain of Pantotheca H1 on maize agronomic traits, yield composition, and soil fertility under actual field planting conditions.

[0070] Experimental setup: Field trials were conducted according to the methods of Example 4 (H1 treatment group) and Comparative Example 5 (blank control group). The soil fertility was uniform in the experimental sites, and routine management was consistent.

[0071] Sampling and testing: Sampling and testing were conducted during the corn maturity and harvest period.

[0072] Agronomic traits were investigated: 10 maize plants were randomly selected from each plot, and plant height (from ground to the tip of the tassel), ear length, and tip length were measured.

[0073] Yield determination: Actual yield measurement was conducted for each plot. The number of effective ears in each plot was counted, the weight of fresh ears was measured, and after threshing and drying, the weight of 100 grains and the yield per mu (calculated based on a standard moisture content of 14%) were calculated.

[0074] Soil nutrient analysis: Soil samples from the rhizosphere layer (0-20 cm) of maize in each plot were collected using a multi-point mixing method. After air-drying and sieving, the available phosphorus content was determined using the sodium bicarbonate extraction-molybdenum antimony colorimetric method, and the available nitrogen content was determined using the alkaline hydrolysis diffusion method.

[0075] Experimental results: The measurement data are shown in Table 3.

[0076]

[0077] Results and conclusions: As shown in Table 3, the field measurement data showed that the treatment group treated with one strain of Panax notoginseng H1 liquid inoculant was superior to the water control group in all indicators.

[0078] At the soil nutrient level, the rhizosphere soil available phosphorus content in the treatment group of Example 4 reached 26.95 mg / kg, an increase of 46.31% compared to the control group. This data directly demonstrates that after colonization in the complex field soil environment, a strain of Pantotheca H1 can effectively secrete organic acids and phosphatases, converting insoluble phosphorus fixed by calcium, iron, and aluminum in the soil into available phosphorus that crops can directly absorb and utilize. Simultaneously, the soil alkaline nitrogen content also increased to a certain extent, indicating that the strain's activity improved the rhizosphere microecological environment and promoted nitrogen cycling.

[0079] The improved availability of soil nutrients was directly reflected in the growth and development of maize. Compared with the control group, maize treated with H1 inoculant showed increased plant height and vigorous vegetative growth; during the reproductive growth stage, ear length increased significantly, and tip barrenness length was greatly shortened (by approximately 64.84%), indicating sufficient grain filling and improved pollination, fertilization, and nutrient supply. Among the final yield components, the combined increase in single ear weight and 100-kernel weight resulted in a yield of 734.8 kg / mu, a 19.44% increase compared to conventional planting.

[0080] In summary, a strain of Pantothecin H1 possesses excellent phosphorus-solubilizing and growth-promoting properties. By activating existing soil nutrients and improving root nutrition, it can increase crop yield.

[0081] Appendix: Strain 16S rRNA Sequence (SEQ ID No.1):

Claims

1. A strain of pantothecin H1, characterized in that, The aforementioned Pantotheca H1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36875.

2. The pantothecin H1 strain according to claim 1, characterized in that, The 16S of the pantothecin H1 strain rRNA The gene sequence is shown in SEQ ID No.

1.

3. A microbial inoculant containing a strain of Pantotheca H1 as described in claim 1 or 2, characterized in that, The microbial agent comprises a strain of Pantotheca H1 and an agriculturally acceptable carrier. The carrier is a liquid culture medium carrier or a solid matrix carrier.

4. The microbial agent according to claim 3, characterized in that, The microbial inoculant is prepared by a method comprising the following steps: A strain of Pantotheca H1 was inoculated into a liquid culture medium and cultured for 8-24 hours at a temperature of 25℃-30℃ and a rotation speed of 160rpm-200rpm to obtain a liquid microbial agent. The liquid microbial agent is mixed with a solid matrix carrier at a mass ratio of 1:2 to 1:4, and fermented and dried at 30℃-35℃ to obtain a solid microbial agent.

5. The microbial agent according to claim 3, characterized in that, The liquid culture medium carrier contains tryptone, yeast extract, and sodium chloride; The solid matrix carrier comprises peat moss and wheat bran, wherein the mass ratio of peat moss to wheat bran is (2-4):

1.

6. A demonstration method for using a strain of pantothenic acid H1 as described in claim 1 or 2 in agricultural assistance, characterized in that, Includes the following steps: S1. Preparation of inoculum: Provide one strain of Pantotheca H1 or a microbial inoculum; S2. Application treatment: Apply the aforementioned Pantotheca H1 strain or microbial agent to crops or planting soil by means of seed soaking, root irrigation, or soil mixing.

7. The method according to claim 6, characterized in that, In step S2, the specific method of application treatment is selected from any of the following: (1) Root irrigation treatment: Dilute the liquid microbial agent with water 100-200 times and irrigate along the soil around the roots of the crop. The application amount is equivalent to 1-2L of the original solution per acre. (2) Seed soaking treatment: Immerse the crop seeds in liquid microbial agent for 2-4 hours under dark conditions, take them out and dry them in the shade before sowing; (3) Soil treatment: Mix solid microbial agents with organic fertilizer and apply them in strips in the planting furrow.

8. The demonstration method of using a strain of pantothenic acid H1 in assisting farmers according to claim 6, characterized in that, The application treatment is carried out during the seedling stage of the crop; the application treatment includes at least two consecutive applications with an interval of 5-7 days.

9. The demonstration method of a strain of Pantotheca H1 in assisting farmers according to claim 6, characterized in that, The crop is corn or mung bean; the planting soil is saline-alkali land, which is saline-alkali soil with a pH of 8.0-9.

0.

10. The use of a strain of Pantotheca H1 according to any one of claims 1-2 in the preparation of biofertilizers or soil conditioners.

Images