A compound microbial agent, its preparation method and application

Abstract

This invention relates to the field of microbial technology, specifically to a compound bacterial agent, its preparation method, and its application, wherein the active ingredient is Bacillus arachidica (B. arachidica). Peanut Bacillus CH-RS16 and cold-resistant *Bacillus subtilis* ( Peribacillus frigoritolerans ZM-RS6. The compound microbial agent provided by this invention promotes the growth and development of wheat and improves its drought resistance.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound bacterial agent, its preparation method, and its application. Background Technology

[0002] Drought is one of the major meteorological disasters leading to crop yield reduction, and the losses and impacts of agricultural drought have increased in recent years. Drought stress is considered one of the abiotic stresses that affects crop growth and development, limits agricultural production, erodes global food security, and threatens sustainable crop development. Wheat is a valuable crop, ranking third among the world's major crops after rice and corn, accounting for about 30% of global grain production. It is an important source of carbohydrates and protein, meeting the needs of 21% of the world's population. Drought stress affects wheat growth and development in multiple ways, not only inhibiting wheat growth to some extent but also directly affecting wheat's physiological metabolism, leading to a decline in wheat yield and quality. Improving wheat's drought resistance is one of the key issues that urgently needs to be addressed to ensure national food security. Plant growth promoting rhizobacteria (PGPR) are a class of beneficial bacteria that can promote plant growth and the absorption and utilization of mineral nutrients, while inhibiting harmful organisms. PGPR can not only secrete growth-promoting hormones but also activate rhizosphere nutrients, promote seed germination under adverse conditions, and enhance crop drought resistance.

[0003] Microorganisms have been widely validated in the field of crop drought resistance. They can improve crop water use efficiency and physiological stability under drought stress through multiple mechanisms, such as promoting root growth, regulating plant hormone balance, enhancing osmotic regulation and antioxidant defense capabilities, and optimizing the rhizosphere microenvironment. However, current products, mainly single-strain agents, have insufficient stress resistance. It is necessary to develop novel microbial products to promote drought resistance in wheat. Summary of the Invention

[0004] To develop microbial products that promote drought resistance in wheat, this invention provides a compound microbial agent, its preparation method, and its application. The compound microbial agent provided by this invention promotes the growth and development of wheat and improves its drought resistance.

[0005] This invention provides a compound microbial agent, the active ingredient of which is Bacillus arachidica (B. arachidica). Bacillus arachidis CH-RS16 and cold-resistant *Bacillus subtilis* ( Peribacillus frigoritolerans ZM-RS6; The described Bacillus arachidica CH-RS16 is deposited at the China Center for Type Culture Collection (CCTCCNO: M 2026415) on March 10, 2026. The cold-resistant *Bacillus subtilis* ZM-RS6 is deposited at the China Center for Type Culture Collection (CCTCCNO: M 2026414) on March 10, 2026.

[0006] Furthermore, the compound microbial agent is a solution.

[0007] Furthermore, the viable counts of both *Bacillus arachidica* CH-RS16 and *Bacillus thuringiensis* ZM-RS6 in the compound microbial agent are 10. 8 CFU / mL ~10 9 CFU / mL.

[0008] The present invention also provides a method for preparing the aforementioned compound microbial agent, comprising the following steps: Bacillus arachidica CH-RS16 and Bacillus thuringiensis ZM-RS6 were inoculated into TSB liquid medium and cultured at 28℃~29℃ and 180rpm~200rpm until the cells reached the logarithmic growth phase. The cells were collected by centrifugation, washed with sterile water, and diluted to a concentration of 10. 8 CFU / mL ~10 9 CFU / mL was used to obtain a suspension of Bacillus arachidica CH-RS16 and a suspension of Bacillus thuringiensis ZM-RS6, which were then mixed to obtain a compound bacterial agent.

[0009] Furthermore, the *Bacillus arachidica* CH-RS16 suspension and the *Bacillus thuringiensis* ZM-RS6 suspension are in equal volume ratio.

[0010] This invention also provides the application of the above-mentioned compound microbial agent in improving the drought resistance of wheat.

[0011] Furthermore, the compound microbial agent is used to promote the growth and development of wheat plants under drought stress.

[0012] Furthermore, the promotion of wheat plant growth and development includes promoting plant height, underground fresh weight, above-ground fresh weight, total fresh weight, and root length.

[0013] Furthermore, the *Bacillus arachidica* CH-RS16 in the compound bacterial agent has the following functions: nitrogen fixation, phosphorus solubilization, siderophore production, IAA production, and ACC deaminase production; the *Bacillus thuringiensis* ZM-RS6 in the compound bacterial agent has the following functions: nitrogen fixation, siderophore production, IAA production, and ACC deaminase production.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a compound microbial agent that promotes wheat growth under drought stress, which is composed of... Bacillusarachidis CH-RS16 and Peribacillus frigoritoleransThis compound microbial agent is composed of ZM-RS6. Treatment with this compound microbial agent can promote wheat growth under different drought gradients.

[0015] Information on the Preservation of Biological Materials CH-RS16, referred to herein as Bacillus arachidica CH-RS16, was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026415. The address of the depository is Wuhan, Hubei Province, 430072, China. It is classified as follows: Bacillus arachidis CH-RS16.

[0016] ZM-RS6, referred to herein as *Bacillus thuringiensis* ZM-RS6, was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026414. The address of the depository is Wuhan, Hubei Province, 430072, China. It is classified as follows: Peribacillus frigoritolerans ZM-RS6. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0018] Figure 1 The morphology of the two strains in this invention on solid TSB medium; In the figure, A shows the morphology of strain CH-RS16 on solid TSB medium; B shows the morphology of strain ZM-RS6 on solid TSB medium.

[0019] Figure 2 The nitrogen fixation capacity of the two strains in this invention is compared. In the figure, A represents the nitrogen fixation capacity of strain CH-RS16; B represents the nitrogen fixation capacity of strain ZM-RS6.

[0020] Figure 3 This invention relates to the determination of the phosphate-solubilizing ability of two strains. In the figure, A represents the phosphorus solubility assay of strain CH-RS16; B represents the phosphorus solubility assay of strain ZM-RS6.

[0021] Figure 4 This is the IAA standard curve.

[0022] Figure 5 This is the standard curve for α-butanone.

[0023] Figure 6 The two strains provided by this invention exhibit antagonistic effects; In the figure, A represents the detection of the antagonistic effect between two strains, using strain CH-RS16 as the indicator bacterium and strain ZM-RS6 as the antagonistic bacterium. B is a strain ZM-RS6 as an indicator bacterium and strain CH-RS16 as an antagonistic bacterium, used to detect the antagonistic effect between the two strains.

[0024] Figure 7 The effect of inoculation with compound microbial agent on different drought gradient stresses compared with the control (CK). In the figure, A represents the effect of inoculation with compound microbial agent on wheat plants under no drought stress conditions compared to the CK control; B represents the effect of inoculation with compound microbial agent and the CK control on wheat plants under drought stress conditions of 10% PEG6000; The effect of inoculation with compound microbial agent and CK control on wheat plants under drought stress conditions (C = 20% PEG6000). Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Isolation, screening and identification of strains.

[0027] Wheat rhizosphere soil was collected. 0.5 g of soil was added to 4.5 ml of sterile water and placed in a sterilized 5 ml centrifuge tube. The tube was shaken to disperse the soil particles, and the mixture was prepared into 10... -1 The suspension was distilled down to 4.5 ml of sterile water to prepare 10 ml of solution. -2 Suspension, diluted sequentially to 10 -6 Suspension. Transfer 80 µL of the suspension to TSB solid medium (17.0 g tryptone, 3.0 g soybean peptone, 5.0 g NaCl, 2.5 g K₂HPO₄, 2.5 g glucose, 15.0 g agar, 1000 mL distilled water, pH 7.0) and spread on a plate. Incubate at 28°C for 3–5 days. Pile single colonies onto fresh medium and streak repeatedly to purify the strains, ultimately isolating strains CH-RS16 and ZM-RS6.

[0028] Strains CH-RS16 and ZM-RS6 were inoculated into 5 mL of liquid TSB medium (17.0 g tryptone, 3.0 g soybean peptone, 5.0 g NaCl, 2.5 g K₂HPO₄, 2.5 g glucose, 1000 mL distilled water, pH 7.0) and cultured at 28 °C with shaking at 150 rpm for 48 h. The bacterial cells were collected by centrifugation at 4 °C and 8000 rpm, and total genomic DNA was extracted. Using the genomic DNA as a template, the 16S rRNA gene fragment was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGT TACGACTT-3'). The 16S rRNA gene sequence of strain CH-RS16 is shown in SEQ DIN NO.1, and the 16S rRNA gene sequence of strain ZM-RS6 is shown in SEQ DI NO.2. The 16S rRNA gene sequence was compared with the EZ Biocloud bioinformatics database, confirming that strain CH-RS16 is... Bacillus arachidis The strain ZM-RS6 is Peribacillus frigoritolerans .

[0029] SEQ ID NO.1: SEQ ID NO.2: Example 2: Performance determination of Bacillus arachidica CH-RS16 and cold-resistant Bacillus thuringiensis ZM-RS6.

[0030] 1. Nitrogen fixation capacity determination After activation, the isolated single strains were cultured in nitrogen-fixing medium (KH₂PO₄ 0.2 g, MgSO₄ 0.2 g, NaCl 0.2 g, CaCO₃ 5.0 g, mannitol 10.0 g, CaSO₄ 0.1 g, agar 15 g, 0.5% Congo red solution 10 mL, distilled water 1 L, pH 7.0±0.1) plates, with four replicates for each strain; incubated at 28℃ for 6 days. The presence of a transparent halo on the plate indicated nitrogen-fixing bacteria, and the nitrogen-fixing capacity was determined by measuring the diameter D of the transparent halo. No halo was recorded as 0; halo thickness 2.5 mm was recorded as 1; halo thickness between 2.5 and 5 mm was recorded as 2; and halo thickness > 5 mm was recorded as 3. The nitrogen-fixing capacity of *Bacillus arachidica* CH-RS16 and *Bacillus thuringiensis* ZM-RS6 was both recorded as 1.

[0031] 2. Phosphorus solubility test After activation, the isolated single strains were cultured in inorganic phosphorus medium (10.0 g glucose, 0.5 g (NH4)2SO4, 0.5 g yeast extract, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4, 0.03 g FeSO4, 0.03 g MnSO4, 5.0 g Ca3(PO4)2, 20.0 g agar, 1 L distilled water, pH 7.0-7.5) plates, with four replicates for each strain. The plates were incubated at 28°C for 5-7 days. The presence of a transparent phosphate-solubilizing halo on the plate indicated phosphate-solubilizing bacteria. The diameter D of the transparent halo was measured to determine the phosphate-solubilizing ability of the bacteria. No halo was recorded as 0; halo thickness 2.5 mm was recorded as 1; halo thickness between 2.5 and 5 mm was recorded as 2; and halo thickness > 5 mm was recorded as 3. The phosphorus-solubility of Bacillus arachidica CH-RS16 was recorded as 2, and the phosphorus-solubility of Bacillus thuringiensis ZM-RS6 was recorded as 0.

[0032] 3. Determination of iron production capacity CH-RS16 and ZM-RS6 were inoculated into TSB liquid medium and cultured on a shaker at 28°C for 2 days at 125 rpm. After centrifugation at 10,000 rpm for 10 min, 100 µL of the supernatant was taken and mixed vigorously with 100 µL of CAS detection solution (6 mL of 10 mM CTAB, 1.5 mL of 1 mM FeCl3 solution, 7.5 mL of 2 mM CAS solution, 4.307 g of anhydrous piperazine, pH 5.6, and deionized water to a final volume of 100 mL). The mixture was incubated in the dark for 60 min, and the absorbance at 630 nm was measured using a microplate reader (A). 630 Separately mix 100 μL of CAS detection solution with 100 μL of uninoculated TSB liquid culture supernatant, incubate in the dark for 60 min, and then measure the absorbance at 630 nm using a microplate reader (A). 630 ,r), Calculate siderophore expression level: Siderophore expression level = A 630 ,rA 630 The siderophore expression levels of strains CH-RS16 and ZM-RS6 were measured to be 0.498 and 0.354, respectively.

[0033] 4. IAA production capacity determination CH-RS16 and ZM-RS6 were inoculated separately into TSB liquid medium and cultured on a shaker at 28°C and 125 rpm for 2 days. After centrifugation at 10,000 rpm for 10 min, 80 µL of the supernatant was vigorously mixed with 160 µL of Salkowski's reagent (2 ml of 0.5 mol / L FeCl3•6H2O and 98 ml of 35% HClO4) and incubated at room temperature for 30 min. OD values ​​were then measured. 530 The IAA content was calculated using a standard curve obtained by serial dilution of IAA. The IAA yields of strains CH-RS16 and ZM-RS6 were determined to be 4.33 mg / L. -1 and 5.13 mg L -1 .

[0034] 5. Assay for ACC deaminase production CH-RS16 and ZM-RS6 were inoculated into DF medium (2.0 g glucose, 0.2 g KH2PO4, 0.8 g Na2HPO4, 0.2 g MgSO4•7H2O, 0.01 g FeSO4•7H2O, 0.02 g CaCl2, 1 mL trace element solution, 1000 mL distilled water, pH 7.2); trace element solution (per liter): H3BO3 2.8 mg, ZnSO4•7H2O 1.4 mg, MnCl2•4H2O 1.8 mg, CuSO4•5H2O 0.04 mg, MoO3 0.02 mg) and incubated on a shaker at 28°C at 125 rpm for 2 days. The cells were washed three times by centrifugation in nitrogen-free DF medium, 10,000 rpm for 10 min each time. They were then inoculated into ADF liquid medium (DF medium with 0.5 g / L filtered sterilized ACC added) and cultured on a shaker at 28°C for 125 rpm for 2 days. Cells were collected by centrifugation (8,000 rpm, 10 min), resuspended in 0.1 M Tris-HCl (pH 8.5), and sonicated to disrupt the cells. The supernatant was collected as the crude enzyme solution. 1 mL Tris-HCl buffer + 5 mM ACC + crude enzyme solution were added and reacted at 37°C for 30 min. 50 μL of 0.5 M HCl was added. 300 μL of DNPH (0.2% in 2 M HCl) was added, and the mixture was incubated at 30°C for 15 min. After adding 2 mL of 2 M NaOH, the absorbance was measured at 540 nm. Enzyme activity was calculated using a standard curve obtained by gradient dilution of α-ketobutyrate. The ACC deaminase production capacity of strains CH-RS16 and ZM-RS6 was determined to be 0.074 μmol / (min mg). -1 ) and 0.207 μmol / (min mg -1 ).

[0035] Example 3: Compound microbial agent, its preparation method and application.

[0036] I. Determination of antagonistic effects of bacterial strains CH-RS16 bacterial culture: Inoculate a single colony of CH-RS16 into 10 mL of TSB liquid medium and incubate on a shaker at 28°C and 125 rpm until the bacterial concentration reaches 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain CH-RS16 bacterial culture.

[0037] ZM-RS6 bacterial culture: A single colony of ZM-RS6 was inoculated into 10 mL of TSB liquid medium and incubated on a shaker at 28°C and 125 rpm until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain ZM-RS6 bacterial culture.

[0038] 30 µL of CH-RS16 bacterial suspension was inoculated onto TSB solid medium and spread. A 7 mm diameter ZM-RS6 bacterial disc was picked and inoculated into the center of the plate. After culturing for 3 days, observation and photography were performed. The appearance of inhibition zones indicates antagonistic activity.

[0039] 30 µL of ZM-RS6 bacterial suspension was inoculated onto TSB solid medium and spread. A 7 mm diameter CH-RS16 bacterial disc was picked and inoculated into the center of the plate. The plate was incubated for 3 days, observed, and photographed. The appearance of inhibition zones indicates antagonistic activity.

[0040] The results are as follows Figure 4 As shown, no inhibition zone was observed, indicating that the two strains do not exhibit antagonistic activity. This suggests that the two strains can be combined to prepare a compound bacterial agent.

[0041] II. Compound microbial agents and their preparation methods Single colonies of *Bacillus arachidica* CH-RS16 and *Bacillus thuringiensis* ZM-RS6, cultured on TSB solid medium for 24 h, were picked and inoculated separately into TSB liquid medium. The colonies were incubated at 28°C and 200 rpm for 24 h until they reached the logarithmic growth phase. The colonies were then collected by centrifugation at 4°C and 10,000 rpm. The colonies were washed twice with sterile water and diluted to a final concentration of 10⁻⁶. 8 CFU / mL was used to obtain CH-RS16 and ZM-RS6 strain suspensions. The two strain suspensions were mixed thoroughly at a volume ratio of 1:1 (the viable cell count in the suspension was 10-1). 8 (CFU / mL) to obtain a compound bacterial agent. Then seal and store in a -4°C freezer for later use.

[0042] III. Application of Compound Microbial Agents in Improving Wheat Drought Resistance Perlite and vermiculite were mixed at a mass ratio of 3:2 and packed into mushroom bags, 800 mL per bag. 350 mL of Hoagland's nutrient solution (containing 1417 mg / L Ca(NO3)2·4H2O) was added to each bag. -1 ;KNO3 607 mg L -1 MgSO4 493 mg L -1 (NH4)3PO4 115 mg L -1 Trace elements include H3BO3 2.86 mg / L -1 MnCl2·4H2O 1.81 mg L -1 ZnSO4·7H2O 0.22 mg L -1 CuSO4·5H2O 0.08 mg L -1H₂MnO₄·H₂O 0.02 mg / L -1 FeSO4 50 mg / L -1 (pH 7.0 ± 0.2). After sealing, sterilize at 121℃ for 25 min.

[0043] Select plump, uniformly sized Zhengmai seeds. Sterilize the seed surface with sodium hypochlorite and alcohol, soak in sterile water to allow the seeds to fully absorb water and swell, then place them in a clean bench for later use. Transfer the seeds to 1.4% water agar medium until germination occurs. Select wheat seeds with uniform sprout length and transplant them into mushroom bags. Plant 4 seedlings in each bag. After two weeks of growth, thin out the seedlings, leaving 2 healthy seedlings per bag, and apply the prepared compound microbial agent. The specific steps include the following:

[0044] The prepared planting bags were placed in a 26℃ greenhouse for cultivation, with 14 hours of light per day. After 7 days, the compound bacterial agent was applied to the rhizosphere of the wheat using a disposable syringe, 2 mL per plant, with only one treatment. Seedlings without bacterial solution but with an equal amount of sterile water served as a blank control. Ten replicates were made for each treatment. After approximately one week of cultivation, when the wheat reached the two-leaf stage, 10% PEG6000 and 20% PEG6000 were added to both the inoculated and uninoculated treatments, respectively, followed by a 12-day drought treatment. The growth of the wheat was observed and recorded. After 12 days, the wheat was removed from the bags, the soil on the surface was washed off, and the root length, plant height, fresh weight, and dry weight of each treatment were measured and recorded. The specific measurement methods are as follows:

[0045] 1. Determination of seedling indicators (1) Fresh weight of the plant: Pull the plant out of the bag with its roots, wash the perlite and vermiculite attached to the roots with clean water, wipe off the water, and weigh the above-ground, underground and whole weight of the plant with a balance.

[0046] (2) Dry weight of plants: Wheat was packed into envelopes according to the number and dried in an 80℃ oven. After 2 days, the weight of the above-ground, underground and whole plants was weighed again using a balance.

[0047] (3) Plant height: Measure the natural height of each wheat plant with a ruler.

[0048] (4) Root length: Measure the length of the main root of each wheat plant with a ruler.

[0049] The results are shown in Table 1, which compares wheat pot experiments with different drought gradients (0%, 10%, 20% PEG6000) treatments, with control groups of those inoculated with compound microbial agent (SynCom) and those not inoculated with compound microbial agent (CK).

[0050] Table 1. Effects of SynCom inoculation on wheat plant growth Note: CK is the control, which is the uninoculated treatment; SynCom is the treatment inoculated with the compound bacterial agent. In the table, identical lowercase letters indicate no significant difference between the control and inoculated treatments, while different letters indicate significant differences between the control and treatments.

[0051] As shown in Figure 1, under different drought gradient treatments, inoculation with the compound microbial agent (SynCom) significantly promoted wheat growth, demonstrating a clear advantage compared to the corresponding sterile control (CK). P＜0.05 Under normal water supply conditions (0% drought), the plant height, underground fresh weight, aboveground fresh weight, total fresh weight, and root length of the CK treatment were 32.00 cm, 0.310 g, 0.633 g, 0.940 g, and 33.17 cm, respectively. After SynCom treatment, these indicators increased to 37.31 cm, 0.560 g, 0.803 g, 1.363 g, and 71.47 cm, respectively, with underground fresh weight, total fresh weight, and root length significantly increasing by 80.65%, 45.00%, and 115.47%, respectively. Under 10% drought stress, the plant height, underground fresh weight, aboveground fresh weight, total fresh weight, and root length of the SynCom treatment increased by 14.05%, 92.49%, 26.61%, 44.08%, and 109.35% compared to the CK treatment, respectively. Under 20% drought stress, SynCom significantly alleviated the inhibitory effect of drought on wheat growth, with plant height, underground fresh weight, aboveground fresh weight, total fresh weight, and root length increasing by 14.71%, 54.21%, 26.98%, 35.01%, and 118.51% respectively compared to the control (CK). Furthermore, under different drought gradients, the underground dry weight, aboveground dry weight, and total dry weight of the SynCom treatment were significantly higher than those of the CK, with increases ranging from 17.50%–26.92%, 21.62%–30.51%, and 21.94%–29.41%, respectively.

[0052] The above results show that, Bacillus arachidis CH-RS16 has a high phosphorus solubility, and also has the ability to fix nitrogen, produce iron carriers, produce IAA, and produce ACC deaminase. Peribacillus frigoritolerans ZM-RS6 has a strong ability to produce IAA and ACC deaminases, as well as nitrogen fixation and siderophore production. Therefore, both strains constituting the compound bacterial agent of this invention have good growth-promoting characteristics.

[0053] Strains with highly efficient growth-promoting properties were screened out to ensure that there were no antagonistic reactions between them, and these strains could be used to produce compound microbial agents.

[0054] Pot experiments showed that under different drought gradients, the compound microbial agent (SynCom) treatment significantly promoted wheat growth, exhibiting a stable and significant growth-promoting effect compared to the sterile control (CK). After inoculation with the compound microbial agent, wheat plant height, root growth, and biomass accumulation were significantly increased, with the highest increase in fresh weight (45.00%), dry weight (29.41%), plant height (16.59%), and root length (118.51%). Simultaneously, the fresh and dry weights of both underground and aboveground parts were significantly higher than those in the CK under all drought treatments. These results indicate that the compound microbial agent can effectively alleviate the inhibitory effect of drought stress on wheat growth, significantly promote root development and biomass accumulation, and possesses the potential to improve crop yield stability and production efficiency under drought conditions, demonstrating promising prospects for agricultural application and development.

[0055] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0056] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A compound microbial agent, characterized in that, The active ingredient is Bacillus arachidica (B. arachidica) Bacillus arachidis CH-RS16 and cold-resistant *Bacillus subtilis* ( Peribacillus frigoritolerans ZM-RS6; The described Bacillus arachidica CH-RS16 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026415, and the deposit date is March 10, 2026. The cold-resistant *Bacillus subtilis* ZM-RS6 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026414 on March 10, 2026.

2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is a solution.

3. The compound microbial agent according to claim 2, characterized in that, The viable cell number of Bacillus phaseicus CH-RS16 and Bacillus pauciloculatus ZM-RS6 in the complex microbial agent is 10 8 CFU / mL ~ 10 9 CFU / mL.

4. A method for preparing the compound microbial agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: Respectively inoculate Bacillus aridilupini CH-RS16 and Bacillus pauciloculatus ZM-RS6 into TSB liquid medium, culture at 28-29℃, 180-200 rmp until the bacteria reach logarithmic growth phase, centrifugal collect the bacteria, wash with sterile water, and then dilute to 10 8 CFU / mL-10 9 CFU / mL, obtain Bacillus aridilupini CH-RS16 suspension and Bacillus pauciloculatus ZM-RS6 suspension, and mix to obtain the compound microbial agent.

5. The preparation method according to claim 4, characterized in that, The *Bacillus arachidica* CH-RS16 suspension and the *Bacillus thuringiensis* ZM-RS6 suspension were in equal volume ratio.

6. The application of the compound microbial agent according to any one of claims 1 to 3 in improving the drought resistance of wheat.

7. The application according to claim 6, characterized in that, The compound microbial agent is used to promote the growth and development of wheat plants under drought stress.

8. The application according to claim 7, characterized in that, The promotion of wheat plant growth and development refers to promoting plant height, underground fresh weight, above-ground fresh weight, total fresh weight, and root length.

9. The application according to claim 8, characterized in that, The *Bacillus arachidica* CH-RS16 in the compound bacterial agent has the following functions: nitrogen fixation, phosphorus solubilization, siderophore production, IAA production, and ACC deaminase production; the *Bacillus thuringiensis* ZM-RS6 in the compound bacterial agent has the following functions: nitrogen fixation, siderophore production, IAA production, and ACC deaminase production.

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