Acid-resistant growth-promoting composite microbial agent containing bacteria and fungi and application thereof

Abstract

This invention discloses an acid-resistant growth-promoting compound bacterial agent containing bacteria and fungi, wherein the bacteria is *Sinomonas sinensis* S188, and its classification name is *Sinomonas sinensis*. Sinomonas sp., with accession number CGMCC NO.34307; the fungus described is Aspergillus liugensis BF21, and its taxonomic name is Aspergillus liugensis. Aspergillus tamarii The preservation number is CGMCC NO.40048. This invention also discloses the application of the aforementioned compound microbial agent in promoting peanut growth in acidic soil. The *Sinomonas* S188 screened in this invention not only possesses acid-resistant properties but also exhibits acid-resistant IAA production and acid-resistant phosphorus-solubilizing functions, effectively promoting peanut growth in acidic soil. This invention prepares an acid-resistant, growth-promoting compound microbial agent containing bacteria and fungi, using *Sinomonas* S188 and *Aspergillus* BF21, which has a further enhanced promoting effect on peanut growth in acidic soil.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to an acid-resistant growth-promoting compound microbial agent containing bacteria and fungi and its application. Background Technology

[0002] Acidic soils account for approximately 40%-50% of the world's potential arable land area. They are mainly distributed in tropical and subtropical humid climate zones (average annual temperature >18°C, annual precipitation >1200mm), with four core distribution areas: southern China, Southeast Asia, the Congo Basin in Africa, and the edge of the Amazon rainforest in South America. In southern China, acidic soils cover several provinces south of the Yangtze River, particularly Jiangxi, Hunan, Fujian, and Guangdong, and the area affected by acidic soils is showing a continuous increasing trend. Therefore, improving and remediating acidic soils is of great significance for protecting the ecological environment and ensuring sustainable agricultural development. Currently, most methods for improving acidic soils involve physical and chemical methods. Physical methods primarily modify the soil's physical structure (such as aeration, water retention, and texture) to indirectly regulate the acidic environment. These methods offer advantages such as improved overall soil performance, high safety, no secondary pollution, and long-lasting effects. However, they also have disadvantages such as slow results, high costs, high labor intensity, and limited applicability. Chemical soil amendment directly neutralizes soil acidity by applying alkaline substances (such as lime, dolomite powder, and wood ash), rapidly adjusting pH levels. It boasts advantages such as quick results, ease of operation, and significant effectiveness in strongly acidic soils. However, it also has disadvantages including short-lasting effects, potential environmental pollution, soil structure deterioration, and nutrient imbalance. Microbial amendment, on the other hand, offers advantages such as eco-friendliness, long-lasting effects, wide applicability, and consideration for both soil fertility and crop health. However, current research on microbial amendment is limited, and readily available microbial resources for practical production remain scarce.

[0003] In acidic soils, beneficial microorganisms with functions such as IAA production, phosphorus solubilization, and potassium solubilization can directly or indirectly promote plant growth, yield increase, and soil improvement by colonizing the plant rhizosphere and soil, improving soil physicochemical properties, enhancing soil fertility, and inhibiting pathogen growth. However, pH is the limiting factor for the effectiveness of microorganisms in acidic soils. The pH of acidic soils in southern my country ranges from 4.5 to 6.5, and some strongly acidic soils even have a pH of 4.0 to 4.5. Therefore, screening for microorganisms that can promote growth under low pH conditions can support the improvement of productivity in acidic soils.

[0004] Peanuts are annual herbaceous plants belonging to the legume family and the genus Arachis. They are an important source of vegetable oil and protein in my country. Peanut oil contains over 50% unsaturated fatty acids, which are extremely beneficial to the human body. Furthermore, peanuts are high in resveratrol, phenolic acids, flavonoids, and phytosterols, which can inhibit cholesterol absorption, prevent cardiovascular and cerebrovascular diseases, and improve blood lipid levels. Therefore, peanuts play an important role in food, industrial production, and drug development, and are one of the main economic crops grown in acidic, dry land in southern my country. Therefore, this invention uses peanuts as the test crop. Summary of the Invention

[0005] To overcome the above problems, the present invention provides an acid-resistant growth-promoting compound microbial agent containing bacteria and fungi and its application.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The first aspect of this invention provides an acid-resistant growth-promoting compound bacterial agent containing bacteria and fungi, wherein the bacteria is *Sinomonas sinensis* S188, and its classification name is *Sinomonas sinensis*. Sinomonas The fungus, *Aspergillus oryzae* sp., is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 23, 2025, with accession number CGMCC NO.34307; the fungus described is *Aspergillus oryzae* BF21, and its taxonomic name is *Aspergillus oryzae*. Aspergillus tamarii The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2022, with accession number CGMCC NO.40048. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the compound microbial agent is prepared by the following steps:

[0009] (1) Preparation of Sinomonas S188 inoculum: Sinomonas S188 with preservation number CGMCC NO.34307 was subjected to liquid fermentation to obtain fermentation broth; the fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the Sinomonas S188 inoculum;

[0010] (2) Preparation of Aspergillus liugensis BF21 inoculum: Aspergillus liugensis BF21 with preservation number CGMCC NO.40048 was subjected to liquid fermentation to obtain fermentation broth; the fermentation broth was filtered to remove mycelia to obtain filtered fermentation broth; the filtered fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the Aspergillus liugensis BF21 inoculum;

[0011] (3) Preparation of acid-resistant growth-promoting compound microbial agent containing bacteria and fungi: The acid-resistant growth-promoting compound microbial agent containing bacteria and fungi is obtained by mixing the S188 microbial agent of *Sinomonas sinensis* and the BF21 microbial agent of *Aspergillus niger*.

[0012] Furthermore, in step (1), the concentration of the *Sinomonas sinensis* S188 bacterial agent is ≥1×10⁻⁶. 7 cfu / mL; in step (2), the spore concentration of the Aspergillus BF21 inoculum is ≥1×10⁻⁶. 7 / mL; In step (3), the S188 bacterial agent of *Sinomonas sinensis* and the BF21 bacterial agent of *Aspergillus niger* are mixed in equal volumes to obtain the acid-resistant growth-promoting compound bacterial agent containing bacteria and fungi.

[0013] Furthermore, the compound microbial agent is prepared by the following steps:

[0014] (1) Preparation of *Sinomonas sinensis* S188 inoculum: *Sinomonas sinensis* S188 with preservation number CGMCC NO.34307 was inoculated into a liquid culture medium for liquid fermentation. The liquid culture medium included TSB liquid medium or LB liquid medium. The liquid fermentation conditions were: temperature 28-30℃, rotation speed 170-200 rpm, fermentation time 1-2 days, to obtain fermentation broth. The fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the *Sinomonas sinensis* S188 inoculum. The bacterial concentration of the *Sinomonas sinensis* S188 inoculum was ≥1×10⁻⁶. 7 cfu / mL;

[0015] (2) Preparation of Aspergillus lilacinus BF21 inoculum: Aspergillus lilacinus BF21 with preservation number CGMCC NO.40048 was inoculated into a liquid culture medium for liquid fermentation, the liquid culture medium including PDB medium. The liquid fermentation conditions were: fermentation at 28-30℃ and 140-160 rpm, first under darkness for 2-3 days, then under light for 2-3 days, to obtain fermentation broth; the fermentation broth was filtered through multiple layers of gauze to remove mycelia, and the filtered fermentation broth was obtained; the filtered fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the Aspergillus lilacinus BF21 inoculum, the spore concentration of the Aspergillus lilacinus BF21 inoculum ≥1×10 7 cells / mL;

[0016] (3) Preparation of acid-resistant growth-promoting compound microbial agent containing bacteria and fungi: The acid-resistant growth-promoting compound microbial agent containing bacteria and fungi is obtained by mixing the S188 microbial agent of *Sinomonas sinensis* and the BF21 microbial agent of *Aspergillus niger* in equal volumes.

[0017] The second aspect of this invention provides the application of the above-mentioned acid-resistant growth-promoting compound microbial agent containing bacteria and fungi in promoting peanut growth in acidic soil.

[0018] Furthermore, the pH of the acidic soil is between 4.0 and 6.5.

[0019] Furthermore, during application, the compound microbial agent is applied some time after the peanut seedlings are transplanted, with an application rate of 50-70 mL per peanut seedling.

[0020] Furthermore, the compound microbial agent is applied to the soil near the roots of the peanut seedlings 5-7 days after transplanting.

[0021] The beneficial effects of this invention are:

[0022] The *Sinomonas* S188 strain screened in this invention not only possesses acid-resistant properties but also exhibits acid-resistant IAA production and acid-resistant phosphorus-solubilizing functions, effectively promoting peanut growth in acidic soils. This invention further develops an acid-resistant, growth-promoting compound microbial agent containing bacteria and fungi, formulated from *Sinomonas* S188 and *Aspergillus* BF21. This agent has an even stronger promoting effect on peanut growth in acidic soils, supporting increased agricultural productivity in acidic soils. It is of great significance for promoting green and sustainable agricultural development and enhancing the synergistic ecological and economic benefits of acidic soil regions. Attached Figure Description

[0023] Figure 1 The acid resistance of the selected strains was investigated to determine their ability to produce IAA and their acid resistance to phosphorus solubility.

[0024] Figure 2 The effect of inoculation with S188 microbial agent on the plant height of peanut seedlings in the first season of pot experiment.

[0025] Figure 3 The effect of inoculation with S188 microbial agent on the stem diameter of peanut seedlings in the first season of pot experiment.

[0026] Figure 4 The effect of inoculation with S188 microbial agent on the aboveground fresh weight of peanut seedlings in the first season of pot experiment.

[0027] Figure 5 The effect of inoculation with S188 microbial agent on the aboveground dry weight of peanut seedlings in the first season of pot experiment.

[0028] Figure 6 The effect of inoculation with S188 microbial agent on the chlorophyll content (SPAD) of peanut seedlings in the first season of pot experiment.

[0029] Figure 7 The effects of different inoculants on the plant height of peanut seedlings in a second-season pot experiment were investigated.

[0030] Figure 8 The effect of inoculating different inoculants on the stem diameter of peanut seedlings in a second-season pot experiment.

[0031] Figure 9 The effects of inoculating different inoculants on the aboveground fresh weight of peanut seedlings in a second-season pot experiment.

[0032] Figure 10The effect of inoculating different inoculants on the aboveground dry weight of peanut seedlings in the second season of pot experiment.

[0033] Figure 11 The effect of inoculating different microbial agents on the chlorophyll content (SPAD) of peanut seedlings in a second-season pot experiment.

[0034] Figure 12 The effects of different inoculants on the plant height of peanut seedlings in a pot experiment during the third season.

[0035] Figure 13 The effect of inoculating different microbial agents on the stem diameter of peanut seedlings in a pot experiment during the third season.

[0036] Figure 14 The effects of inoculating different inoculants on the aboveground fresh weight of peanut seedlings in a pot experiment during the third season.

[0037] Figure 15 The effect of inoculating different inoculants on the aboveground dry weight of peanut seedlings in a pot experiment during the third season.

[0038] Figure 16 The effects of inoculating different microbial agents on the chlorophyll content (SPAD) of peanut seedlings in a pot experiment during the third season.

[0039] Figure 17 Photograph of a plate of strain S188.

[0040] Figure 18 Phylogenetic tree of 16S rRNA of strain S188.

[0041] In the above bar chart, * indicates P<0.05, which is statistically significant; ** indicates P<0.01, which is extremely statistically significant; the different letters above the bars are statistically significant (P<0.05) differences as defined by one-way ANOVA.

[0042] Information on the preservation of biological materials

[0043] S188, classified and named *Sinomonas sinensis* Sinomonas sp., deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 23, 2025, with accession number CGMCC NO.34307.

[0044] BF21, classified as Aspergillus oryzae Aspergillus tamarii It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is January 19, 2022, and the accession number is CGMCC NO.40048. Detailed Implementation

[0045] The following embodiments and accompanying drawings are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0046] Unless otherwise specified, the raw materials used in the following examples are as follows:

[0047] The seedling substrate is Xingxing Xiangnong brand seedling substrate produced by Jiangsu Xingnong Substrate Technology Co., Ltd., with product number 161102G0097N.

[0048] TSB solid medium (1L): 30g tryptone soybean broth, bring to a final volume of 1L with deionized water, add 20g agar powder, and sterilize at 115℃ for 30min. Remove the agar powder from the TSB liquid medium.

[0049] LB liquid medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, bring the volume to 1L with deionized water, and sterilize at 115℃ for 30min.

[0050] PVK liquid medium (1L): glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, FeSO4·7H2O 0.03g, MgSO4·7H2O 0.3g, MnSO4·4H2O 0.03g, yeast extract 0.4g, AlPO4 3.93g, deionized water to a final volume of 1L, sterilize at 115℃ for 30min.

[0051] PDB medium (1L): 6g potato extract powder, 20g glucose, deionized water to a final volume of 1L, sterilize at 115℃ for 30min.

[0052] PDA medium (1L): 6g potato extract powder, 20g glucose, deionized water to a final volume of 1L, 20g agar powder, sterilized at 115℃ for 30min.

[0053] Unless otherwise specified, the petri dishes (plates) involved in the following examples are 90 mm in diameter.

[0054] Example 1 Screening of acid-resistant strains

[0055] Step 1: Shaking the culture

[0056] The Jiangsu Provincial Key Laboratory for High-Tech Research on Activation of Solid Organic Waste Resource Utilization isolated 206 bacterial strains from soil or rhizosphere of crops such as peanuts, rapeseed, and bananas, and incubated them in 3 mL LB liquid medium at 30°C and 170 rpm for 2 days to obtain fermentation broth.

[0057] Step 2: Determination of the growth performance of the strain under acidic conditions

[0058] ① Centrifuge the fermentation broth prepared in the first step at 4000 rpm for 6 minutes, remove the supernatant and resuspend it in sterile water to obtain a bacterial suspension. Adjust the OD of the bacterial suspension from 600 to 1 with sterile water.

[0059] ② Prepare LB liquid culture media with different pH values. Adjust the pH of the LB liquid culture media to 4.0 and 5.0 respectively using 0.04 mol / L dilute hydrochloric acid, and set up an unadjusted LB liquid culture medium (pH=6.86) as a control. Add 180 μL of the prepared LB liquid culture media with different pH values ​​to different 96-well plates for later use. Inoculate 20 μL of the diluted bacterial suspension from ① into the LB liquid culture media with different pH values ​​in sequence. Set up 4 replicates for each strain. Incubate in a constant temperature incubator at 30℃ for 1 day and 2 days. Then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD of each strain after 1 day and 2 days of incubation. 600 A higher OD value indicates stronger bacterial growth performance. All 206 tested bacterial strains were able to grow in LB liquid medium without pH adjustment; 178 strains were able to grow in LB liquid medium at pH 5.0; and 14 strains were able to grow in LB liquid medium at pH 4.0 (9 of these strains, although able to grow in LB liquid medium at pH 4.0, were identified as not having the ability to produce IAA and / or solubilize phosphorus (method as in Example 2), therefore these 9 strains were not involved in subsequent experiments). The strain S188 described in this invention can grow at pH 4.0. The subsequently used control strains N32, OF89, CK4, and CK22 were also able to grow at pH 4.0.

[0060] Example 2: Determination of the Functionality of the Strains

[0061] The functionality of the 178 acid-tolerant strains screened in Example 1 that could grow in LB liquid medium at pH 5.0 was further determined.

[0062] (1) Screening of acid-resistant IAA-producing strains

[0063] The pH of the LB liquid medium was adjusted to 5.0 with 0.04 mol / L dilute hydrochloric acid, and L-tryptophan (0.1 mg / mL) was added. The prepared LB liquid medium containing L-tryptophan at pH 5.0 was dispensed into shaking tubes, 3 mL per tube. The 178 acid-resistant strains screened in Example 1 were inoculated into the LB liquid medium containing L-tryptophan at pH 5.0. After culturing at 30°C and 170 rpm for 2 days, the bacterial culture was obtained.

[0064] Take 100µL of bacterial suspension and drop it onto a white ceramic plate. Simultaneously add an equal volume of colorimetric solution (1g of FeCl3·6H2O dissolved in 21.485mL of concentrated H2SO4, slowly diluted in distilled water, and then brought to a final volume of 50mL with distilled water). Use a mixture of 100µL of uninoculated LB broth containing L-tryptophan (pH 5.0) and an equal volume of colorimetric solution as a control. Place the white ceramic plate at room temperature in the dark for 30 minutes and observe. A pink color indicates the ability to secrete IAA; a deeper color indicates greater secretion intensity. No color change indicates the inability to secrete IAA.

[0065] Quantitative determination was performed on strains with IAA secretion ability obtained from the initial screening. 1 mL of bacterial culture was centrifuged at 1000 rpm for 10 min, the supernatant was collected, an equal volume of colorimetric solution was added, and the mixture was incubated in the dark for 30 min. Three replicates were set for each strain, and its OD was measured. 530 Value. A standard curve was plotted using the analytical grade IAA serial dilution method, and the IAA content per unit volume of bacterial culture was calculated by referring to the standard curve. Five highly efficient acid-resistant IAA-producing strains were screened. Figure 1 (Left figure), in which the strain S188 described in this invention has the best acid-resistant IAA production ability.

[0066] (2) Screening of acid-tolerant phosphate-solubilizing strains

[0067] To quantitatively assess the aluminum phosphate solubility of the screened strains, 178 acid-tolerant strains screened in Example 1 were first inoculated into LB liquid medium and cultured in a constant-temperature shaker (30℃, 180 rpm) until the logarithmic phase (OD600nm=1.0) to prepare seed culture for each strain. Then, the seed culture of each strain was transferred at a ratio of 0.5 v / v% to Erlenmeyer flasks pre-filled with 100 mL of PVK liquid medium (pH adjusted to 5.0 with 0.04 mol / L dilute hydrochloric acid). The Erlenmeyer flasks were cultured at 30℃ and 180 rpm for 5 days. After centrifugation at 4℃ and 10000 rpm for 15 min, the supernatant was collected. The soluble phosphorus content in the supernatant was determined using the molybdenum blue colorimetric method (Bao Shidan. Soil Agricultural Chemical Analysis Methods [M]. Beijing: China Agriculture Press, 2000). Five strains with highly efficient acid-tolerant phosphorus-solubilizing ability were screened. Figure 1 (See right figure), among which strain S188 of the present invention has the best acid resistance to phosphorus solubility.

[0068] Since strains CK13, CK38, and CK77 can survive at pH 5.0 but not at pH 4.0, these three strains were not included in the pot experiment.

[0069] Based on the acid tolerance, IAA production capacity, and phosphorus solubility of the strains, strain S188 was selected for the first season of pot experiment verification. Both strain S188 and strain N32 can efficiently produce IAA and soluble phosphorus in acid, so strains S188 and N32 were selected for the second season of pot experiment verification. The third season will be a pot experiment for screening compound microbial agents.

[0070] Three functional strains were cultured in liquid medium and the inoculum was prepared.

[0071] Single bacterial inoculum: Bacterial strains (strain S188, strain N32, strain CK22, strain CK4, strain OF89, and Escherichia coli OP50) were inoculated onto TSB solid medium and activated by incubation at 37°C for 2 days. Once colonies had grown, the selected single colonies were inoculated into liquid medium (including TSB liquid medium or LB liquid medium; LB liquid medium was used in this example) and incubated at 30°C and 170 rpm on a shaker for 2 days to obtain the fermentation broth. The broth was diluted and plated for cell counting. The cell concentration of the fermentation broth was ≥1×10⁻⁶. 8 CFU / mL. Centrifuge the fermentation broth at 4000 rpm for 6 min, discard the supernatant, and resuspend the precipitate in 10 times the volume of sterile water to prepare a single bacterial inoculum. The bacterial cell concentration of the single inoculum should be ≥1×10⁻⁶. 7 cfu / mL (in this example, sterile water was used to uniformly adjust the concentration to 1×10⁻⁶). 7 (cfu / mL).

[0072] Single fungal inoculum: A fungal strain (strain BF21) was inoculated onto a PDA medium plate and cultured at 28°C for 7 days until the mycelium completely covered the plate and produced abundant spores. The plate was washed with sterile water to obtain a spore suspension. 0.1 mL of the spore suspension was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PDB medium and placed in a shaker at 28°C and 150 rpm. Fermentation was carried out first in the dark for 3 days, followed by fermentation under light for 3 days to obtain the fermentation broth. The fermentation broth was filtered through four layers of sterile gauze to remove the mycelium, obtaining the filtered fermentation broth. The number of spores in the filtered fermentation broth was determined using the hemocytocyte count method. The spore concentration of the filtered fermentation broth was ≥0.5 × 10⁻⁶. 8 The filtered fermentation broth was centrifuged at 4000 rpm for 6 min, the supernatant was discarded, and the precipitate was resuspended in 5 times the volume of sterile water to prepare a single fungal inoculum. The spore concentration of the single fungal inoculum was ≥1×10⁻⁶. 7 Cells / mL (in this example, sterile water was uniformly adjusted to 1×10⁻⁶) 7 (units / mL).

[0073] Compound microbial agents: The compound microbial agents involved in this invention are dual-strain compound microbial agents, and the single-strain microbial agents involved (bacterial single-strain agents with a bacterial cell concentration of 1×10⁻⁶) are also included. 7 CFU / mL, the concentration of a single fungal inoculum is 1×10⁻⁶. 7 The compound bacterial agent is obtained by mixing equal volumes of (number per mL) of bacteria.

[0074] Example 4: Experiment on the growth-promoting effect of functional bacteria on peanut seedlings in acidic soil during the first season.

[0075] Peanut seeds (Ganhua No. 5) were soaked in warm water and dried before use. Seedling substrate, seedling trays, and plastic pots were prepared. Jiangxi soil (pH 4.5) and quartz sand were mixed at a volume ratio of 9:1 to obtain mixed soil. 600g of mixed soil was placed in each plastic pot (bottom diameter 8.8cm, height 10.7cm, mouth diameter 12m) for later use.

[0076] This experiment was conducted in a greenhouse at a temperature of 25°C and a relative humidity of 60%-70%. The conditions were dark before the peanuts sprouted. After the peanuts sprouted, the plant grow lights in the greenhouse were turned on and automatically turned off from 22:00 at night to 6:00 the next morning.

[0077] Sow peanut seeds in seedling trays filled with seedling substrate, one seed per cell. Water thoroughly and allow the seeds to germinate in 4-5 days. Water regularly during the seedling stage (every 2 days). Once the peanuts have germinated, select seedlings with uniform growth and transplant them into plastic pots filled with mixed soil (one seedling per pot). After transplanting, water the plastic pots and continue watering regularly thereafter (every 2 days).

[0078] The peanut seedlings transplanted into plastic pots were divided into two treatments: CK (equal volume of sterile water, control) and S188 (inoculated with S188 inoculant). Each treatment had 6 replicates. After the transplanted peanut seedlings had been growing steadily for one week, they were inoculated with S188 inoculant (prepared in Example 3, the S188 inoculant was poured into the mixed soil in the plastic pots, close to the roots of the peanut seedlings). The inoculation ratio was 10% (100µL inoculant / g dry weight of mixed soil, i.e., 60mL inoculant per plastic pot). After inoculation, the seedlings were cultured for another 3 weeks. The plant height, stem diameter, aboveground fresh weight, aboveground dry weight, and chlorophyll content (SPAD) of the peanut seedlings were measured to compare the growth status of the peanut seedlings in each treatment.

[0079] The methods for measuring each indicator are as follows:

[0080] Plant height: Measure the length of the above-ground part of the peanut seedling with a tape measure (i.e., the distance from the junction of the above-ground and underground parts to the highest point of the above-ground part of the main stem).

[0081] Stem thickness: Measure the thickness of the peanut seedling's stem (the stem at the junction of the above-ground and underground parts) using vernier calipers;

[0082] Determination of fresh weight of above-ground parts: Cut the peanut seedling at the junction of the above-ground and underground parts and weigh the above-ground parts;

[0083] Determination of aboveground dry weight: After weighing the fresh weight, the aboveground parts of the peanut seedlings were placed in an envelope and blanched at 105℃ for 15 minutes, then dried at 70℃ to constant weight and weighed.

[0084] Chlorophyll content (SPAD) determination: The chlorophyll content of leaves from the upper, middle and lower parts of peanut seedlings was measured using a SPAD meter, and the average value was taken.

[0085] Depend on Figures 2 to 6 It can be seen that, in terms of plant height, stem diameter, above-ground fresh weight, above-ground dry weight, and chlorophyll content, peanut seedlings in the S188 inoculant treatment group were superior to those in the uninoculated control group. Combining the results of plant height, stem diameter, above-ground fresh weight, above-ground dry weight, and chlorophyll content, it is clear that the peanut seedlings in the S188 inoculant treatment group generally grew better. In conclusion, compared to the uninoculated control group, inoculation with the S188 inoculant showed a good growth-promoting effect on potted peanut seedlings in acidic soil.

[0086] Example 5: Experiment on the growth-promoting effect of functional bacteria in the second season on peanut seedlings in acidic soil.

[0087] Peanut seeds (Ganhua No. 5) were soaked in warm water and dried before use. Seedling substrate, seedling trays, and plastic pots were prepared. Jiangxi soil (pH 4.5) and quartz sand were mixed at a volume ratio of 9:1 to obtain mixed soil. 600g of mixed soil was placed in each plastic pot (bottom diameter 8.8cm, height 10.7cm, mouth diameter 12cm) for later use.

[0088] This experiment was conducted in a greenhouse at a temperature of 25°C and a relative humidity of 60%-70%. The conditions were dark before the peanuts sprouted. After the peanuts sprouted, the plant grow lights in the greenhouse were turned on and automatically turned off from 22:00 at night to 6:00 the next morning.

[0089] Sow peanut seeds in seedling trays filled with seedling substrate, one seed per cell. Water thoroughly and allow the seeds to germinate in 4-5 days. Water regularly during the seedling stage (every 2 days). Once the peanuts have germinated, select seedlings with uniform growth and transplant them into plastic pots filled with mixed soil (one seedling per pot). After transplanting, water the plastic pots and continue watering regularly thereafter (every 2 days).

[0090] The peanut seedlings transplanted into plastic pots were divided into three treatments: CK (equal volume of sterile water, control), N32 (inoculated with N32 inoculant), and S188 (inoculated with S188 inoculant). Each treatment had 6 replicates. After the transplanted peanut seedlings had been growing steadily for one week, they were inoculated with either S188 or N32 inoculant (prepared in Example 3, by pouring S188 or N32 inoculant into the mixed soil in the plastic pots, close to the roots of the peanut seedlings). The inoculation ratio was 10% (100µL inoculant / g dry weight of mixed soil, i.e., 60mL inoculant per plastic pot). After inoculation, the seedlings were cultured for another 3 weeks. The plant height, stem diameter, aboveground fresh weight, aboveground dry weight, and chlorophyll content (SPAD) of the peanut seedlings were measured to compare the growth status of the peanut seedlings in each treatment.

[0091] The methods for measuring each indicator are the same as in Example 4.

[0092] Depend on Figures 7 to 11 It can be seen that, in terms of plant height, stem diameter, above-ground fresh weight, above-ground dry weight, and chlorophyll content, peanut seedlings in the S188 inoculant treatment group were superior to those in the uninoculated control group and the N32 inoculant treatment group. This result is consistent with the results of the first season pot experiment (Example 4), further verifying that inoculation with S188 inoculant has a good growth-promoting effect on potted peanut seedlings in acidic soil.

[0093] Example 6: Experiment on the growth-promoting effect of functional bacteria in the third season on peanut seedlings in acidic soil.

[0094] Peanut seeds (Ganhua No. 8) were soaked in warm water and dried for later use. Seedling substrate, seedling trays, and plastic pots were prepared. Jiangxi soil (pH 4.0) and quartz sand were mixed at a volume ratio of 9:1 to obtain mixed soil. 600g of mixed soil was placed in each plastic pot (bottom diameter 8.8cm, height 10.7cm, mouth diameter 12m) for later use.

[0095] This experiment was conducted in a greenhouse at a temperature of 25°C and a relative humidity of 60%-70%. The conditions were dark before the peanuts sprouted. After the peanuts sprouted, the plant grow lights in the greenhouse were turned on and automatically turned off from 22:00 at night to 6:00 the next morning.

[0096] Sow peanut seeds in seedling trays filled with seedling substrate, one seed per cell. Water thoroughly and allow the seeds to germinate in 4-5 days. Water regularly during the seedling stage (every 2 days). Once the peanuts have germinated, select seedlings with uniform growth and transplant them into plastic pots filled with mixed soil (one seedling per pot). After transplanting, water the plastic pots and continue watering regularly thereafter (every 2 days).

[0097] As shown in Table 1, a total of 9 treatments were set up for the peanut seedlings transplanted into plastic pots, including 6 single bacterial agents, 2 compound bacterial agents and 1 sterile water control, with 6 replicates for each treatment. Among them, strain S188, which can grow at pH 4.0 and has the strongest acid tolerance for IAA production and acid tolerance for phosphorus solubility, was used to prepare a single bacterial agent and to prepare compound bacterial agents with other strains; strain N32, which can grow at pH 4.0 and has acid tolerance for IAA production and acid tolerance for phosphorus solubility, was used to prepare a compound bacterial agent with strain S188. In the second season of pot experiment, it was verified that the acid tolerance and growth-promoting ability of strain N32 was not as good as that of strain S188, so strain N32 was no longer involved in the single bacterial agent; strains CK22, strain OF89 and strain CK4 can all grow at pH 4.0 and have acid tolerance for IAA production or acid tolerance for phosphorus solubility, so these strains were prepared as single bacterial agents respectively; strain BF21 is a fungal strain previously screened by our research group. The inventors found that it can grow at pH 4.0 and has acid tolerance for IAA production, so it was prepared as a single bacterial agent and to prepare a compound bacterial agent with strain S188; and a single bacterial agent of Escherichia coli OP50 and a sterile water control were set up.

[0098]

[0099] After the transplanted peanut seedlings have been growing steadily for one week, they are inoculated with a microbial agent (prepared in Example 3, the microbial agent is poured into the mixed soil in plastic pots, near the roots of the peanut seedlings). The inoculation ratio is 10% (100µL of microbial agent / g dry weight of mixed soil, i.e., 60mL of microbial agent is poured into each plastic pot). After the inoculation is completed, the seedlings are cultured for another 3 weeks. The plant height, stem diameter, above-ground fresh weight, above-ground dry weight and chlorophyll content (SPAD) of the peanut seedlings are measured, and the growth status of peanut seedlings in each treatment is compared.

[0100] The methods for measuring each indicator are the same as in Example 4.

[0101] Depend on Figures 12 to 16 It can be seen that, in terms of plant height, stem diameter, above-ground fresh weight, above-ground dry weight, and chlorophyll content, peanut seedlings in the group inoculated with compound microbial agent S188-BF21 were superior to those in the control group without inoculation and those inoculated with other microbial agents. In conclusion, compared to without inoculation and inoculation with other microbial agents, inoculation with compound microbial agent S188-BF21 showed a good growth-promoting effect on potted peanut seedlings in acidic soil.

[0102] Example 7 Identification of strain S188

[0103] The biological identification of strain S188 was performed using morphological and 16S rRNA gene sequence analysis.

[0104] like Figure 17As shown, strain S188 forms yellow, regularly round, opaque, moist colonies with relatively smooth edges when cultured on TSB solid medium plates at 30°C for 1-2 days.

[0105] By comparing the 16S rRNA gene sequence of strain S188 with similar sequences and constructing a phylogenetic tree, such as... Figure 18 As shown. The results indicate that strain S188 has a high degree of homology with *Sinomonas*, and its... Sinomonas flava strain CW 108 is in the same branch, with a homology of 98.93%.

[0106] Based on the colony morphology characteristics of strain S188 and the results of 16S rRNA phylogenetic tree comparison analysis, it was identified as *Sinomonas sinensis*. Sinomonas sp . Strain S188 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34307.

Claims

1. An acid-resistant, growth-promoting compound microbial agent containing bacteria and fungi, characterized in that, The bacteria in question is *Sinomonas sinensis* S188, and its classification name is *Sinomonas sinensis*. Sinomonas The fungus, *Aspergillus oryzae* sp., is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 23, 2025, with accession number CGMCC NO.34307; the fungus described is *Aspergillus oryzae* BF21, and its taxonomic name is *Aspergillus oryzae*. Aspergillus tamarii It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2022, with accession number CGMCC NO.40048; The compound microbial agent is prepared by the following steps: (1) Preparation of *Sinomonas sinensis* S188 inoculum: *Sinomonas sinensis* S188 with preservation number CGMCC NO.34307 was subjected to liquid fermentation to obtain fermentation broth; the fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the *Sinomonas sinensis* S188 inoculum; the bacterial concentration of the *Sinomonas sinensis* S188 inoculum was ≥1×10⁻⁶. 7 cfu / mL; (2) Preparation of Aspergillus lilacinus BF21 inoculum: Aspergillus lilacinus BF21 with preservation number CGMCC NO.40048 was subjected to liquid fermentation to obtain fermentation broth; the fermentation broth was filtered to remove mycelia, and the filtered fermentation broth was obtained; the filtered fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the Aspergillus lilacinus BF21 inoculum; the spore concentration of the Aspergillus lilacinus BF21 inoculum was ≥1×10⁻⁶. 7 cells / mL; (3) Preparation of acid-resistant growth-promoting compound microbial agent containing bacteria and fungi: The acid-resistant growth-promoting compound microbial agent containing bacteria and fungi is obtained by mixing the S188 microbial agent of *Sinomonas sinensis* and the BF21 microbial agent of *Aspergillus niger* in equal volumes.

2. The acid-resistant growth-promoting compound microbial agent containing bacteria and fungi according to claim 1, characterized in that, The compound microbial agent is prepared by the following steps: (1) Preparation of *Sinomonas sinensis* S188 inoculum: *Sinomonas sinensis* S188 with preservation number CGMCC NO.34307 was inoculated into a liquid culture medium for liquid fermentation. The liquid culture medium included TSB liquid medium or LB liquid medium. The liquid fermentation conditions were: temperature 28-30℃, rotation speed 170-200 rpm, fermentation time 1-2 days, to obtain fermentation broth. The fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the *Sinomonas sinensis* S188 inoculum. The bacterial concentration of the *Sinomonas sinensis* S188 inoculum was ≥1×10⁻⁶. 7 cfu / mL; (2) Preparation of Aspergillus lilacinus BF21 inoculum: Aspergillus lilacinus BF21 with preservation number CGMCC NO.40048 was inoculated into a liquid culture medium for liquid fermentation, the liquid culture medium including PDB medium. The liquid fermentation conditions were: fermentation at 28-30℃ and 140-160 rpm, first under darkness for 2-3 days, then under light for 2-3 days, to obtain fermentation broth; the fermentation broth was filtered through multiple layers of gauze to remove mycelia, and the filtered fermentation broth was obtained; the filtered fermentation broth was centrifuged, the supernatant was removed, and the precipitate was resuspended in sterile water to obtain the Aspergillus lilacinus BF21 inoculum, the spore concentration of the Aspergillus lilacinus BF21 inoculum ≥1×10 7 cells / mL; (3) Preparation of acid-resistant growth-promoting compound microbial agent containing bacteria and fungi: The acid-resistant growth-promoting compound microbial agent containing bacteria and fungi is obtained by mixing the S188 microbial agent of *Sinomonas sinensis* and the BF21 microbial agent of *Aspergillus niger* in equal volumes.

3. The application of the acid-resistant growth-promoting compound microbial agent containing bacteria and fungi as described in any one of claims 1-2 in promoting peanut growth in acidic soil.

4. The application according to claim 3, characterized in that, The pH of the acidic soil is between 4.0 and 6.

5.

5. The application according to claim 3, characterized in that, When applying the compound microbial agent, apply it some time after the peanut seedlings are transplanted, at a rate of 50-70 mL per peanut seedling.

6. The application according to claim 5, characterized in that, Apply the compound microbial agent to the soil near the roots of the peanut seedlings 5-7 days after transplanting.

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