A compound microbial agent and its application in licorice quality improvement in arid and saline-alkali land.

Abstract

This invention discloses a compound microbial inoculant and its application in licorice quality improvement in arid and saline-alkali land, belonging to the field of microbial technology and agricultural planting. The compound microbial inoculant includes *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis*; the viable count concentration of the compound microbial inoculant is 1-2 × 10⁻⁶. 10 CFU / mL. This compound microbial agent can significantly improve the yield and quality of licorice. Simultaneously, it can improve the fertility of saline-alkali soil by activating soil phosphorus and potassium, and enhancing the activity of urease and alkaline phosphatase. This invention solves the problems of weak rhizosphere colonization and low component conversion rate of existing microbial agents, providing an innovative technical solution for the efficient cultivation of licorice in arid saline-alkali lands.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microbial agent and its application in the quality improvement of licorice in saline-alkali land in arid areas. Background Technology

[0002] In the unique habitat of saline-alkali lands in arid regions, the synthesis of secondary metabolites in licorice (Glycyrrhiza uralensis) faces multiple environmental stresses. When soil salinization reaches moderate to severe levels, high concentrations of Na+ become a significant factor. + / Cl - Na + SO4 2- Ion toxicity and osmotic stress create a dual inhibition, leading to hindered root development, decreased photosynthetic efficiency, and a 30%-40% reduction in biomass accumulation compared to suitable environments. This becomes a key bottleneck limiting its application in saline-alkali land ecological restoration. Furthermore, the nutrient predicament in infertile soils, with available phosphorus content often below 5 mg / kg and organic matter content less than 0.6%, results in a 40%-60% decrease in the expression of CYP72A154, a key enzyme in glycyrrhizic acid synthesis. Even more severe is the high pH environment unique to saline-alkali land, which sharply reduces the rhizosphere microbial diversity index to below 2.1, lowering the abundance of nitrogen-fixing and phosphate-solubilizing bacteria by 2-3 orders of magnitude compared to normal soils, resulting in nitrogen and phosphorus nutrient conversion rates of less than 30%.

[0003] Existing microbial remediation systems face multiple technical bottlenecks in licorice cultivation in saline-alkali land: First, the metabolic networks of single-function strains (such as *Rhizobium sinense* or *Bacillus amyloliquefaciens*) have significant defects, making it difficult to construct systemic tolerance through the secretion of stress-resistance substances such as ACC deaminase (<15 μmol α-KA / mg protein / h) and siderophores (<60% CAS activity). Second, conventional microbial agents have insufficient colonization efficiency under saline-alkali stress, and their chemotactic receptors (such as CheA protein) are sluggish in response to unique signaling molecules secreted by licorice roots, resulting in a low chemotactic index. More importantly, single strains cannot reconstruct the material cycling system of the rhizosphere microenvironment, and their nitrogenase activity and phosphatase secretion are insufficient to meet the growth requirements of licorice.

[0004] To address the above issues, it is of great significance to explore how to overcome the stress threshold of moderate to severe saline soil on licorice growth based on the theory of rhizosphere microorganism-plant interaction and through multiple mechanisms such as screening and constructing novel compound microbial agents, improving the rhizosphere micro-ecological environment, enhancing the ability to regulate ion homeostasis, and inducing the expression of stress-resistant genes. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent and its application in the quality improvement of licorice in arid and saline-alkali land, thereby solving the problems existing in the prior art. This compound microbial agent can improve the germination rate and germination speed of licorice seeds, the overall length of licorice seedlings, the fresh weight of licorice seedlings, and the levels of endogenous hormones IAA and GA in licorice seedlings. S In addition to the total flavonoid content of licorice seedlings, this compound microbial agent can improve the fertility of saline-alkali soil by activating soil phosphorus and potassium and increasing the activity of urease and alkaline phosphatase.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] Technical Solution 1: A compound microbial agent comprising *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis*; the viable count concentration of the compound microbial agent is 1-2 × 10⁻⁶. 10 CFU / mL.

[0008] The compound microbial agent includes Arthrobacter globiformis, Pseudomonas filiformis, Sinorhizobium meliloti, and Bacillus subtilis.

[0009] *Arthrobacter spheroidae* was deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 26629 (patent number 202310246967.7); *Pseudomonas filamentosa* GSICC 31645 and *Rhizobium sinense* GSICC 31930 were purchased from the Gansu Branch of the China Industrial Microbiological Culture Collection Center; *Bacillus subtilis* CGMCC No. 24484 was purchased from the China General Microbiological Culture Collection Center.

[0010] Furthermore, in the compound microbial agent, the volume ratio of *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis* is 1:1:1:1.

[0011] Technical Solution 2: Application of the aforementioned compound microbial inoculant in licorice cultivation.

[0012] Technical Solution 3: Application of the aforementioned compound microbial agent in the improvement of saline-alkali soil.

[0013] The content of available phosphorus and potassium in the compound microbial agent of this invention was significantly higher than that in the control group at all three time periods after treatment, indicating the best activation effect on phosphorus and potassium nutrients. After two and five root drenchings following seed treatment, the activity of urease and alkaline phosphatase was significantly increased, promoting nitrogen and phosphorus cycling and improving soil fertility. The nitrate nitrogen content did not change significantly compared to the control group after seed treatment and two root drenchings, but after five root drenchings, the nitrate nitrogen content decreased compared to the previous period and was significantly lower than the negative control, improving the utilization rate of nitrogen in the rhizosphere of licorice. The content of available phosphorus, available potassium, and organic matter in the roots of licorice was significantly increased after seed treatment and two root drenchings compared to the control group. However, after five root drenchings, the content of these components decreased compared to two root drenchings, indicating that while increasing the content of these nutrients, they were also utilized and transformed.

[0014] Technical Solution 4: The application of the aforementioned microbial inoculant in promoting plant growth.

[0015] Technical Solution 5: Application of the aforementioned microbial inoculant in improving plant yield and quality.

[0016] The compound microbial agent provided by this invention can effectively improve the germination rate and germination speed of licorice seeds, the whole plant length of licorice seedlings, the fresh weight of licorice seedlings, and the levels of endogenous hormones IAA and GA in licorice seedlings. S And the total flavonoid content of licorice seedlings.

[0017] Furthermore, the plant includes licorice.

[0018] The present invention discloses the following technical effects:

[0019] This invention utilizes *Arthrobacter globosum*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis* as a compound microbial agent for seed embedding and root irrigation treatment of licorice, especially moderately saline-alkali land. The results show that this compound microbial agent significantly improves licorice yield and quality compared to the control group. Simultaneously, this compound microbial agent improves saline-alkali soil fertility by activating soil phosphorus and potassium, and increasing urease and alkaline phosphatase activity. This invention solves the problems of weak rhizosphere colonization and low component conversion rate of existing microbial agents, providing an innovative technical solution for the efficient cultivation of licorice in arid saline-alkali lands and providing technical support for the efficient utilization of saline-alkali land resources in arid regions. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The study investigated the effects of different treatment groups on the germination rate and germination percentage of licorice seeds. Groups A, B, C, and D were single-strain treatments of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense* (a type of rhizobium in alfalfa), and *Bacillus subtilis*, respectively. Groups A+C represented a mixed treatment of *Arthrobacter spheroides* and *Rhizobium sinense*. Groups A+D represented a mixed treatment of *Arthrobacter spheroides* and *Bacillus subtilis*. Groups A+C+D represented a mixed treatment of *Arthrobacter spheroides*, *Rhizobium sinense*, and *Bacillus subtilis*. Groups A+B+C+D represented a mixed treatment of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense*, and *Bacillus subtilis*. Group CK was the control group.

[0022] Figure 2 The study investigated the effects of different treatments on the whole-plant length and fresh weight of licorice seedlings. Groups A, B, C, and D were single-strain treatments of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense* (a type of rhizobium in alfalfa), and *Bacillus subtilis*, respectively. Groups A+C consisted of a mixed treatment of *Arthrobacter spheroides* and *Rhizobium sinense*. Groups A+D consisted of a mixed treatment of *Arthrobacter spheroides* and *Bacillus subtilis*. Groups A+C+D consisted of a mixed treatment of *Arthrobacter spheroides*, *Rhizobium sinense*, and *Bacillus subtilis*. Groups A+B+C+D consisted of a mixed treatment of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense*, and *Bacillus subtilis*. The control group (CK) was also included.

[0023] Figure 3 Different treatments for the effects of endogenous hormones IAA and GA on licorice seedlings S The effects of the study were investigated. Groups A, B, C, and D were single-strain treatments of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense* (a type of alfalfa rhizobia), and *Bacillus subtilis*, respectively. Groups A+C were mixed treatments of *Arthrobacter spheroides* and *Rhizobium sinense*. Groups A+D were mixed treatments of *Arthrobacter spheroides* and *Bacillus subtilis*. Groups A+C+D were mixed treatments of *Arthrobacter spheroides*, *Rhizobium sinense* (a type of alfalfa rhizobia), and *Bacillus subtilis*. Groups A+B+C+D were mixed treatments of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense* (a type of alfalfa rhizobia), and *Bacillus subtilis*. Group CK was the control group.

[0024] Figure 4 The study investigated the effects of different treatments on the total flavonoid content of licorice seedlings. Groups A, B, C, and D were single-strain treatments of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense* (a type of alfalfa rhizobia), and *Bacillus subtilis*, respectively. Groups A+C consisted of a mixed treatment of *Arthrobacter spheroides* and *Rhizobium sinense*. Groups A+D consisted of a mixed treatment of *Arthrobacter spheroides* and *Bacillus subtilis*. Groups A+C+D consisted of a mixed treatment of *Arthrobacter spheroides*, *Rhizobium sinense*, and *Bacillus subtilis*. Groups A+B+C+D consisted of a mixed treatment of *Arthrobacter spheroides*, *Pseudomonas filamentosa*, *Rhizobium sinense*, and *Bacillus subtilis*. The control group (CK) was also included.

[0025] Figure 5 This invention provides a comparison of licorice seedlings treated with the compound microbial agent of this invention by seed dressing and root irrigation one year after planting with a negative control. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, the experimental methods used in the examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0032] The compound microbial agent provided by this invention includes *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis*. *Arthrobacter spheroidae* is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 26629 (patent number 202310246967.7); *Pseudomonas filamentosa* GSICC 31645 and *Rhizobium sinense* GSICC 31930 were purchased from the Gansu Branch of the China Industrial Microbiological Culture Collection Center; and *Bacillus subtilis* CGMCC No. 24484 was purchased from the China General Microbiological Culture Collection Center.

[0033] Bacillus pilaris GSICC31638, Bacillus subtilis GSICC32855, Arthrobacter spheroides GSICC30147, and Pseudomonas filamentosa GSICC31645 were all purchased from the Gansu Branch of the China Industrial Microbial Culture Collection Center.

[0034] The licorice used was Ural licorice, and the seeds and seedlings were purchased from Minqin County Gendegen Traditional Chinese Medicine Planting and Development Co., Ltd. The experimental site was located in Xicha Town, Gaolan County, Lanzhou City, Gansu Province (saline-alkali land, pH 7.80, total salt content 4.08 g·kg⁻¹). -1 ).

[0035] Example 1

[0036] Preparation of bacterial suspensions of *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis*.

[0037] (1) Cultivation and fermentation of Arthrobacter spheroidae: 10.01 g·L⁻¹ yeast extract -1 Tryptone 14.78 g / L -1 MgSO4 5g·L -1 pH 8.09, culture temperature 37.07℃, rotation speed 200 r·min -1 Incubate for 8-12 hours.

[0038] (2) Cultivation and fermentation of Pseudomonas filamentosa: 2.47 g·L⁻¹ yeast extract -1 Tryptone 5.09 g·L -1 MgSO4 5g·L -1 pH 7.26, culture temperature 30.6℃, rotation speed 200 r·min -1 Incubate for 8-12 hours;

[0039] (3) Culture and fermentation of *Rhizobium sinense* and *Bacillus subtilis* in alfalfa: LB medium, culture temperature 30℃, rotation speed 180 r·min -1 Incubate for 8-12 hours.

[0040] *Arthrobacter spheroidae* was deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 26629 (patent number 202310246967.7); *Pseudomonas filamentosa* GSICC 31645 and *Rhizobium sinense* GSICC 31930 were purchased from the Gansu Branch of the China Industrial Microbiological Culture Collection Center; *Bacillus subtilis* CGMCC No. 24484 was purchased from the China General Microbiological Culture Collection Center.

[0041] Each of the above bacteria was cultured and fermented separately to obtain its own bacterial solution.

[0042] Example 2

[0043] The experiment was conducted in the artificial climate chamber of the Institute of Biology, Gansu Academy of Sciences. Licorice seeds were first treated with concentrated H₂SO₄ for 10 min, rinsed with tap water for 20 min, washed five times with sterile water, and then soaked in single-strain and mixed-strain fermentation broth for 4 h before being air-dried. The test soil was taken from the top 20 cm of soil in Xicha Town, Gaolan County, Lanzhou City, Gansu Province (saline-alkali soil, pH 7.80, total salt content 4.08 g·kg⁻¹). -1 After being sieved through a 2mm sieve and mixed thoroughly, the mixture was then subjected to dry heat sterilization and treated according to different treatment groups (the treatment groups included single-cell treatment and mixed-cell treatment, with a single-cell or mixed-cell concentration of 1×10⁻⁶). 7 The bacterial solution (CFU / mL) was added at a rate of 50 mL per liter of soil, mixed thoroughly, and then filled into separate 1L pots. Ten soaked licorice seeds were evenly sown in each pot and covered with 1cm of soil. Each treatment was replicated four times. Germination rate and germination potential were recorded after 7 days. Subsequently, every 7 days, 50 mL of a 100-fold diluted bacterial solution was evenly applied to the roots of the seedlings, for a total of three applications. The control group received sterile water. Water was replenished periodically using a weighing method, ensuring no water overflowed from the pots. After 30 days, once the true leaves of the licorice emerged, seedling length (vernier caliper), fresh weight, and levels of endogenous hormones IAA and GA were recorded. S And total flavonoids of licorice (at this time, glycyrrhizic acid has not yet been produced in licorice). Among them, germination rate = actual number of germinated seeds / number of tested seeds × 100%, germination rate = ∑G / t; G refers to the daily germination rate, and t is the total number of days of germination.

[0044] Endogenous hormones IAA, GA S Assay method: Ten fresh licorice seedlings (weighed before storage) were taken from a liquid nitrogen container and quickly aliquoted into 2 mL centrifuge tubes (to ensure thorough grinding and minimize loss). Steel balls and a small amount of pre-frozen 80% methanol solution were added, followed by grinding on a ball mill at 28 rpm for 1 min. The process could be repeated multiple times based on the grinding results. After grinding, the sample was rinsed with 80% methanol solution to a final volume of approximately 12 mL and extracted at 4°C for at least 8 hours.

[0045] Afterwards, centrifuge at 4℃ and 7000rpm for 15min. Filter the supernatant into a 10mL centrifuge tube (wet the filter paper with 80% methanol solution). Blow the supernatant to approximately 4mL using a nitrogen blower in a 40℃ water bath. Transfer the supernatant to a 25mL beaker and adjust the pH to 8. Rinse the 10mL centrifuge tube with petroleum ether and pour the rinsings into the corresponding beaker. Transfer the rinsings to a 60mL separatory funnel for petroleum ether extraction. Collect the lower phase into the corresponding beaker and discard the upper phase. Repeat the extraction process three times. After extraction, add 0.100g PVPP to the beaker and shake at 180rpm for 40min. Filter (wet the filter paper with deionized water) into a clean 25mL beaker and adjust the pH to 3. Add ethyl acetate to the beaker at a 1:1 volume ratio and transfer the rinsings to a separatory funnel for ethyl acetate extraction. Transfer the lower phase into the corresponding beaker and repeat the extraction process. Pour the upper phase into a 10mL centrifuge tube. The ethyl acetate extraction process was repeated three times, and the final volume of the three eluents was adjusted to approximately 11 mL. The eluent was then dried with nitrogen until no droplets remained and the column was no longer flowing. Next, 4 mL of pH 3 phosphate solution was added and the dry matter was dissolved in a vortex mixer. The C18 column was cleaned and activated by rinsing it three times with methanol solution, three times with 80% methanol solution, and once with deionized water. 4 mL of the sample solution dissolved in the previous phosphate solution was added to the column. The remaining sample solution in the centrifuge tube was rinsed with 1 mL of phosphate solution and added to the column. Washing buffer (18.5 mL of deionized water and 1.5 mL of 80% methanol to prepare 20 mL) was added, and the column was rinsed again. After rinsing, 80% methanol solution was added three times, and the filtrates were collected three times. The filtered sample solution was dried with nitrogen until no water remained, then rinsed twice with 500 mL of chromatographic alcohol. The volume was adjusted to 1 mL in a 2 mL centrifuge tube, and the solution was analyzed by high-performance liquid chromatography (HPLC). The total flavonoids of licorice were determined using the conventional AlCl3 colorimetric method.

[0046] The effects of different treatments on the germination rate and germination rate of licorice seeds are shown in the figure. Figure 1 The effects of different treatments on the whole plant length and fresh weight of licorice seedlings are shown in the figure. Figure 2 Different treatments affect the levels of endogenous hormones IAA and GA in licorice seedlings. S See the impact Figure 3 The effects of different treatments on the total flavonoid content of licorice seedlings are shown in the figure. Figure 4 The preparation of bacterial suspensions of *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis* is described in Example 1. Figures 1-4 In the diagram, A, B, C, and D represent individual treatments with *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense*, and *Bacillus subtilis* bacterial solutions, respectively, with a single bacterial solution concentration of 1 × 10⁻⁶. 7 CFU / mL; Group A+C consisted of a mixed bacterial culture obtained by mixing *Arthrobacter spheroidae* and *Rhizobium sinense* in equal proportions, with a bacterial concentration of 1×10⁻⁶. 7CFU / mL; Groups A and D consist of a mixed bacterial culture obtained by mixing *Arthrobacter spheroidae* and *Bacillus subtilis* in equal proportions, with a bacterial concentration of 1×10⁻⁶. 7 CFU / mL; A+C+D is a mixed bacterial culture obtained by mixing *Arthrobacter spheroides*, *Rhizobium sinense* from alfalfa, and *Bacillus subtilis* in equal proportions, with a bacterial concentration of 1×10⁻⁶. 7 CFU / mL; A+B+C+D is a mixed bacterial culture obtained by mixing *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* from alfalfa, and *Bacillus subtilis* in equal proportions, with a bacterial concentration of 1×10⁻⁶. 7 CFU / mL; negative control group: irrigation water (CK). The effects of different treatments on licorice seed germination rate, germination rate, whole plant length of licorice seedlings, fresh weight of licorice seedlings, and endogenous hormones IAA and GA in licorice seedlings were investigated. S In terms of the total flavonoid content of licorice seedlings, the A+B+C+D treatment group showed the best results.

[0047] Example 3

[0048] 1. Preparation of bacterial agents for each treatment group

[0049] (1) The compound microbial agent of the present invention: the bacterial suspensions of *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense*, and *Bacillus subtilis* prepared in Example 1 are mixed in an equal volume ratio of 1:1:1:1, with a viable count concentration of 2×10⁻⁶. 10 CFU·mL -1 ;

[0050] (2) Positive control group 1: See the compound microbial agent disclosed in Chinese invention patent CN118165877A. The compound microbial agent includes five bacterial solutions of Pseudomonas filamentosa BG4, Rhizobium sinense of alfalfa DG1, Arthrobacter glucosinolates GCG2, Pseudomonas filamentosa SG3, and Arthrobacter glucosinolates GCG3 mixed in equal proportions;

[0051] (3) Positive control group 2: Bacterial suspensions of *Arthrobacter spheroidae* GSICC30147, *Pseudomonas filamentosa* GSICC31645, *Bacillus pilaris* GSICC31638, and *Bacillus subtilis* GSICC32855 were mixed in equal volume ratios, with a viable count concentration of 2 × 10⁻⁶. 10 CFU·mL -1 ;

[0052] (4) Positive control group 3: Bacterial suspensions of *Arthrobacter spheroidae* GSICC30147, *Pseudomonas filamentosa* GSICC31645, *Rhizobium sinense* GSICC31930, and *Bacillus subtilis* GSICC32855 were mixed in equal volume ratios, with a viable count concentration of 2 × 10⁻⁶. 10 CFU·mL -1 ;

[0053] (5) Negative control group: Irrigation water (CK).

[0054] 2. Use the inoculants from each treatment group as seed coating agents or as seed dressing agents for one-year-old seedlings.

[0055] Before sowing seeds or planting seedlings, mix the seeds with mud containing the bacterial agents from each treatment group to ensure that each seed or seedling is evenly coated with mud, and then let it dry. The mud is made by mixing the prepared negative control group compound bacterial solution with local soil at a volume ratio of 1:2.

[0056] 2. Root irrigation during the growing season

[0057] After the seeds germinate and sprout true leaves, or after the seedlings sprout, dilute the inoculant for each treatment group 100 times with local irrigation water and apply it to the roots. Seedlings that have germinated from seeds should be irrigated 4 times during their first year of growth; seedlings that have grown for 1 year should be harvested and irrigated 4 times during their first year of growth; seedlings that have grown for 2 years should be harvested and irrigated 4 times each year, for a total of 8 irrigations over 2 years.

[0058] Experimental site: Located in Xicha Town, Gaolan County, Lanzhou City (36°42′N, 103°79′E), belonging to the temperate semi-arid continental climate zone. Soil type: loess. Soil nutrient content in the 0–20 cm soil layer: total nitrogen 0.22 g·kg⁻¹ -1 Available phosphorus 41.80 mg·kg -1 227.22 mg / kg of readily available potassium -1 Organic matter 6.84 g·kg -1 pH 7.80, total salt content 4.08 g·kg -1 The tested plants were Ural licorice seeds and one-year-old seedlings. Glycyrrhizin and glycyrrhizic acid contents were determined according to the methods published in the 2020 edition of the Chinese Pharmacopoeia (National Pharmacopoeia Commission of the People's Republic of China: Part I). The effects of different treatment groups of seed coating with inoculants on the germination rate of licorice seeds were statistically analyzed (Table 1); the effects of seed dressing and root irrigation treatments on licorice yield and active ingredient content one year after planting (Table 2); and the effects of licorice seedlings on licorice yield and active ingredient content two years after planting (Table 3).

[0059] Table 1. Effects of different seed coating treatments on the germination rate of licorice seeds.

[0060] Processing groups Germination rate (%) This invention's compound microbial agent 70.56±2.65a Positive control 1 55.28±2.88b Positive control 2 59.23±2.26b Positive control 3 64.11±4.60ab negative control 37.09±3.47c

[0061] Table 2 shows the effects of seed dressing and root irrigation treatments on licorice yield and active ingredient content one year after planting.

[0062]

[0063]

[0064] Table 3. Effects of licorice seedlings on licorice yield and active ingredient content 2 years after planting.

[0065]

[0066] The growth of licorice seedlings treated with the compound microbial agent of this invention and the negative control group was compared one year after planting. The comparison results are shown in the figure. Figure 5 .

[0067] Rhizosphere soil samples were collected from licorice plants after germination without root irrigation, after two root irrigations (August 2023), and after five root irrigations (October 2024). Specific methods: For each treatment, 10 plants were collected from each plot of licorice rhizosphere soil. The soil around the roots was gently shaken off, and the soil adhering to the root surface was gently brushed off with a brush. The soil was sieved to remove any residual roots, and then air-dried naturally before being stored at 4℃ for later use. Organic matter content was determined using the potassium dichromate oxidation method; soil nitrate nitrogen, available potassium, and available phosphorus contents, as well as urease and alkaline phosphatase activities, were detected using reagent kits purchased from Beijing Solarbio Science & Technology Co., Ltd. The effects of different treatments on the nutrients and enzyme activity of the rhizosphere soil of Glycyrrhiza uralensis after seedling germination are shown in Table 4. The effects of different treatments on the nutrients and enzyme activity of the rhizosphere soil of Glycyrrhiza uralensis in saline-alkali soil after two treatments of seed dressing and root irrigation are shown in Table 5. The effects of different treatments on the nutrients and enzyme activity of the rhizosphere soil of Glycyrrhiza uralensis in saline-alkali soil after five treatments of seed dressing and root irrigation are shown in Table 6.

[0068] Table 4. Effects of different treatments after seedling germination on nutrient composition and enzyme activity in the rhizosphere soil of licorice.

[0069]

[0070]

[0071] Table 5. Effects of different treatments on the nutrient composition and enzyme activity of licorice rhizosphere soil in saline-alkali soil after two treatments: seed dressing and root irrigation.

[0072]

[0073] Table 6. Effects of different treatments on the nutrient composition and enzyme activity of licorice rhizosphere soil in saline-alkali soil after five rounds of seed dressing and root irrigation treatment.

[0074]

[0075] The results in Tables 4-6 show that the contents of available phosphorus and available potassium in the three treatment periods after treatment with the compound microbial agent of this invention were significantly higher than those in the control group, indicating the best activation effect on phosphorus and potassium nutrients. After two and five root drenchings following seed treatment, the activities of urease and alkaline phosphatase were significantly increased, promoting nitrogen and phosphorus cycling and improving soil fertility. The nitrate nitrogen content showed no significant change compared to the control group after seed treatment and two root drenchings, but after five root drenchings, the nitrate nitrogen content decreased compared to the previous period and was significantly lower than the negative control, improving the utilization rate of nitrogen in the rhizosphere of licorice. The contents of available phosphorus, available potassium, and organic matter in the roots of licorice were significantly increased after seed treatment and two root drenchings compared to the control group. However, after five root drenchings, the contents of these components decreased compared to two root drenchings, indicating that while increasing the content of these nutrients, they were also utilized and transformed.

[0076] Example 4

[0077] Collection of plant root exudates: Licorice seeds were first treated with concentrated H2SO4 for 10 min, rinsed with tap water for 20 min, and washed 5 times with sterile water. Then, they were sown in seedling trays containing sterile vermiculite. The trays were approximately 20 cm in diameter at the top, 16.5 cm in height, and 14.5 cm in diameter at the bottom, with 3 seeds per tray, for a total of 10 trays. The trays were then cultured in a greenhouse (25℃, 16h light / 8h darkness). When the seedlings had 3 true leaves, they were all transferred to a hydroponic system (containing 1 / 2 Hoagland nutrient solution) with an aeration pump and cultured for 14 days. The culture solution was removed, and the roots were rinsed 3 times with sterile deionized water. 50 mL of sterile deionized water was added, and the plants were cultured in the dark with shaking (100 rpm, 25℃) for 24 h. The culture solution was collected and filtered through a 0.22 μm filter membrane to obtain the original root exudate solution. The exudate was concentrated using a rotary evaporator at 40℃ and a vacuum of 0.08 MPa. After concentration to a certain volume, it was freeze-dried into powder at low temperature and stored at -80℃. Control group: Maize seedlings were cultured simultaneously, and root exudates were collected from maize seeds (sweet and glutinous 182, nationally approved maize variety 2016004, purchased from Beijing Huanai Agricultural Development Co., Ltd.) using the same method. The above culture process was repeated 3 times.

[0078] Take a glass capillary tube and siphon in 2 μL of root exudate solution (concentration of 10 mg / mL, prepared with M9 medium), and seal the other end with paraffin.

[0079] Preparation of bacterial suspensions of the compound bacterial agent of the present invention and positive control 1: Each strain in the compound bacterial agent of the present invention and each strain in the positive control 1 bacterial agent (see Example 3 for details) were cultured separately to the logarithmic growth phase (OD). 600 =0.6), and after mixing in equal proportions to prepare a compound bacterial agent, each was centrifuged separately (4℃, 5000rpm, 10min), the supernatant was discarded, and the mixture was washed twice with M9 medium and resuspended to OD. 600 =1.0.

[0080] Add 1 mL of the bacterial suspension of the compound bacterial agent of this invention and the bacterial suspension of positive control 1 to 1.5 mL centrifuge tubes respectively, and insert the capillary tip into each bacterial suspension. The control group capillary tubes were injected with corn secretions and M9 medium (blank control) respectively. Incubate horizontally at 28℃ for 2 h, remove each capillary tube, break off the tip with sterile forceps, and blow the contents into 1 mL of M9 medium. After serial dilution, spread on LB agar plates, incubate at 30℃ for 24 h, and count colonies. The chemotactic index results of different treatment groups are shown in Table 7. Calculate the chemotactic index CI according to the following formula: CI = CFU of treatment group / CFU of blank control group.

[0081] Table 7 Chemotaxis index of different treatment groups

[0082] deal with Licorice rhizosphere secretions / CI maize root exudates / CI This invention's compound microbial agent 4.21±0.03Aa 2.30±0.03Ba Positive control 1 3.65±0.05Ab 1.65±0.06Bc Positive control 2 3.44±0.02Cc 1.86±0.03Cb

[0083] Different uppercase letters indicate that the CI values ​​in each row are significantly different (P<0.01), and different lowercase letters indicate that the CI values ​​in each column are significantly different (P<0.01).

[0084] Table 7 verifies the migration ability of the bacterial suspensions in each treatment group to rhizosphere exudates. The results show that the chemotaxis of the compound bacterial agent solution of this invention to licorice rhizosphere exudates is significantly stronger than that of the control group. Furthermore, compared with the chemotaxis of maize rhizosphere exudates, the chemotaxis index of this invention is 1.83 times that of maize rhizosphere exudates. Therefore, the bacterial strain combination provided by this invention is more conducive to colonization in the rhizosphere of licorice.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound microbial agent, characterized in that, The microbial agent includes *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense*, and *Bacillus subtilis*; the viable count concentration of the compound microbial agent is 1-2 × 10⁻⁶. 10 CFU / mL; The *Arthrobacter* species in question is *Arthrobacter* species with accession number CGMCC No. 26629; The filamentous Pseudomonas is filamentous Pseudomonas GSICC 31645; The *Rhizobium sinense* spp. mentioned is *Rhizobium sinense* GSICC 31930; The Bacillus subtilis in question is the Bacillus subtilis with accession number CGMCC No. 24484.

2. The compound microbial agent according to claim 1, characterized in that, In the compound microbial agent, the volume ratio of *Arthrobacter spheroidae*, *Pseudomonas filamentosa*, *Rhizobium sinense* of alfalfa, and *Bacillus subtilis* is 1:1:1:

1.

3. The application of the compound microbial agent according to claim 1 or 2 in licorice cultivation.

4. The application according to claim 3, characterized in that, The licorice was grown in saline-alkali land.

5. The application of the compound microbial agent according to claim 1 or 2 in the improvement of saline-alkali soil.

6. The application of the microbial inoculant according to claim 1 or 2 in promoting plant growth, characterized in that, The plant in question is licorice.

7. The application of the microbial inoculant according to claim 1 or 2 in improving plant yield and quality, characterized in that, The plant in question is licorice.

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