A compound microbial agent for crop drought resistance, its preparation method and application

Abstract

This invention relates to the field of microbial technology, specifically to a compound microbial agent for crop drought resistance, its preparation method, and its application. The active ingredient is *Pseudomonas urinaria* (…). Pseudomonas fish CH-RS23, *Clostridium cepacia* ( Curtobacterium allii ZM-RS40, Acinetobacter rice ( Acinetobacter oryzae ZM-RS39 and cold-resistant short-spores ( Peribacillus frigoritolerans CH-RS15. The compound microbial agent provided by this invention promotes the growth and development of wheat under drought stress.

Description

Technical Field

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

[0002] Drought is one of the most severe natural disasters affecting agricultural production. It not only directly impacts crop water absorption and utilization, weakening plant growth, but also further influences regional agricultural productivity, water resource utilization efficiency, and ecosystem function by altering soil moisture, thermodynamic properties, and nutrient availability. With climate change and the increasing frequency of extreme weather events, drought's constraints on sustainable agricultural development are becoming increasingly prominent, making it a significant abiotic stress factor affecting stable crop yields. Wheat, as one of my country's major food crops, holds a crucial strategic position in ensuring national food security and promoting sustainable agricultural development, especially in the arid and semi-arid rainfed agricultural areas of northern my country. Throughout its growth cycle, wheat is susceptible to various biotic and abiotic stresses, with drought stress being a key abiotic factor limiting its growth, development, and yield. Under drought conditions, wheat's physiological metabolic processes, root development, and water use efficiency are all affected to varying degrees, leading to decreased yield and quality. Therefore, improving wheat's drought resistance is of great significance for ensuring stable wheat production and national food security. Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial microorganisms that can colonize the rhizosphere of plants and promote growth. They can promote plant growth and development through various pathways, including nitrogen fixation, nutrient dissolution or activation, synthesis of plant hormones, and inhibition of pathogens. Studies have shown that PGPR can also alleviate the adverse effects of drought stress on crops to some extent by regulating host plant metabolic processes, improving the rhizosphere environment, and enhancing plant water use efficiency.

[0003] However, existing technologies for single-strain inoculation still have certain limitations in field applications. For example, they exhibit weak colonization ability in complex soil microbial communities, insufficient functional expression stability, and complex interactions with native microbial communities, making it difficult to consistently achieve their stress-promoting effects. Combining multiple strains with complementary functions in specific proportions can promote the reconstruction of beneficial rhizosphere microbial communities, enhance the adaptability and functional stability of the inoculant in complex environments, and thus strengthen plant stress resistance. Therefore, developing a compound inoculant microbial product that can effectively improve wheat drought resistance has high application value and practical significance. Summary of the Invention

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

[0005] This invention provides a compound microbial agent for crop drought resistance, the active ingredient of which is *Pseudomonas urinaria* (…). Pseudomonas piscicola CH-RS23, *Clostridium cepacia* ( Curtobacterium allii ZM-RS40, Acinetobacter rice ( Acinetobacter oryzae ZM-RS39 and cold-resistant short-spores ( Peribacillus cold-tolerant CH-RS15; The *Pseudomonas urinaria* CH-RS23 strain is deposited at the China Center for Type Culture Collection (CCTCCNO: M 2026412) on March 10, 2026. The *Corynebacterium cepacia* ZM-RS40 is deposited at the China Center for Type Culture Collection (CCTCCNO: M 2026416) on March 10, 2026. The Acinetobacter rice ZM-RS39 was deposited at the China Center for Type Culture Collection (CCTCCNO: M 2026413) on March 10, 2026. The cold-resistant Bacillus brevis CH-RS15 is deposited at the China Center for Type Culture Collection (CCTCCNO: M 2026417) on March 10, 2026.

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

[0007] Furthermore, the viable counts of *Pseudomonas urinaria* CH-RS23, *Clostridium cepacia* ZM-RS40, *Acinetobacter oryzae* ZM-RS39, and *Bacillus pyriformis* ZM-RS6 in the compound microbial agent are all 10. 8 CFU / mL ~10 9 CFU / mL.

[0008] This invention provides a method for preparing a compound microbial agent for crop drought resistance, comprising the following steps: *Pseudomonas urinaria* CH-RS23, *Clostridium cephalosporin* ZM-RS40, *Acinetobacter oryzae* ZM-RS39, and *Bacillus brevis* ZM-RS6 were inoculated into TSB liquid medium and cultured at 28℃–29℃ and 180–200 rpm until the cells reached the logarithmic growth phase. The cells were collected by centrifugation, washed with sterile water, and diluted to a concentration of 10-1. 8 CFU / mL ~10 9CFU / mL was used to obtain suspensions of four strains, and the suspensions of the four strains were mixed to obtain a compound bacterial agent.

[0009] Furthermore, the suspensions of the four strains were in equal volume ratios.

[0010] This invention provides an application of a 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 *Pseudomonas yucca* CH-RS23 in the compound bacterial agent possesses the following functions: nitrogen fixation, phosphorus solubilization, siderophore production, IAA production, and ACC deaminase production; the *Corynebacterium cepacia* ZM-RS40 possesses the following functions: nitrogen fixation, phosphorus solubilization, siderophore production, IAA production, and ACC deaminase production; the *Acinetobacter oryzae* ZM-RS39 possesses the following functions: nitrogen fixation, siderophore production, IAA production, and ACC deaminase production; and the *Bacillus thuringiensis* CH-RS15 possesses 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 to improve the drought resistance of wheat, which is composed of... Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter oryzae ZM-RS39 and Peribacillus cold-tolerant This compound microbial agent is composed of CH-RS15. Treatment with this agent can promote wheat growth under different drought gradients.

[0015] Information on the Preservation of Biological Materials CH-RS23, referred to herein as *Pseudomonas urinaria* CH-RS23, was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026412. The address of the depository is Wuhan, Hubei Province, 430072, China. It is classified as follows: Pseudomonas piscicola CH-RS23.

[0016] ZM-RS40, referred to herein as *Corynebacterium cepacia* ZM-RS40, was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026416. The address of the depository is Wuhan, Hubei Province, 430072, China. It is classified as follows: Curtobacterium alliiZM-RS40.

[0017] ZM-RS39, referred to herein as Acinetobacter rice ZM-RS39, was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026413. The address of the depository is Wuhan, Hubei Province, 430072, China. It is classified and named as follows: Acinetobacter oryzae ZM-RS39.

[0018] CH-RS15, referred to herein as *Bacillus frostridae* CH-RS15, was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026417. The address of the depository is Wuhan, Hubei Province, 430072, China. It is classified as follows: Peribacillus frigoritolerans CH-RS15. Attached Figure Description

[0019] 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.

[0020] Figure 1 The morphology of the four strains in this invention on solid TSB medium; In the figure, A shows the morphology of strain CH-RS23 on solid TSB medium; B shows the morphology of strain ZM-RS40 on solid TSB medium; C shows the morphology of strain ZM-RS39 on solid TSB medium; and D shows the morphology of strain CH-RS15 on solid TSB medium.

[0021] Figure 2 The nitrogen fixation capabilities of the four strains in this invention are shown. In the figure, A represents the nitrogen fixation capacity of strain CH-RS23; B represents the nitrogen fixation capacity of strain ZM-RS40; C represents the nitrogen fixation capacity of strain ZM-RS39; and D represents the nitrogen fixation capacity of strain CH-RS15.

[0022] Figure 3 This invention relates to the determination of the phosphate-solubilizing ability of two strains. In the figure, A represents the phosphate solubility of strain CH-RS23; B represents the phosphate solubility of strain ZM-RS40; C represents the phosphate solubility of strain ZM-RS39; and D represents the phosphate solubility of strain CH-RS15.

[0023] Figure 4 This is the IAA standard curve.

[0024] Figure 5 This is the standard curve for α-butanone.

[0025] Figure 6 The four strains provided by this invention exhibit antagonistic effects; In the figure, A shows the antagonistic effect between strains detected using CH-RS15 as the indicator bacterium and CH-RS23, ZM-RS40, ZM-RS39, and ZM-RS3 as antagonistic bacteria; B shows the antagonistic effect between strains detected using CH-RS23 as the indicator bacterium and CH-RS15, ZM-RS40, ZM-RS39, and ZM-RS3 as antagonistic bacteria; C shows the antagonistic effect between strains detected using ZM-RS39 as the indicator bacterium and CH-RS15, ZM-RS40, ZM-RS39, and ZM-RS3 as antagonistic bacteria. 40. CH-RS23 and ZM-RS3 represent the antagonistic effects between antagonistic bacteria detected; D represents the antagonistic effects between antagonistic bacteria detected using ZM-RS40 as the indicator bacterium and CH-RS15, CH-RS23, ZM-RS39, and ZM-RS3 as antagonistic bacteria detected; E represents the antagonistic effects between antagonistic bacteria detected using ZM-RS3 as the indicator bacterium and CH-RS15, ZM-RS40, ZM-RS39, and CH-RS23 as antagonistic bacteria detected.

[0026] 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

[0027] 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.

[0028] The strains CH-RS23, ZM-RS40, ZM-RS39, and CH-RS15 described in this invention belong to the core rhizosphere microbial group of dryland farmland. They were obtained from wheat rhizosphere soil collected in Yangling area, Shaanxi, China, through high-throughput sequencing analysis and isolation culture, and were identified as belonging to the genus *Pseudomonas*. Pseudomonas ), genus *Brucea* ( Curtobacterium Acinetobacter spp. Acinetobacter ) and Bacillus spp. ( Peribacillus ).

[0029] Methods for identifying core strains: The strains CH-RS23, ZM-RS40, ZM-RS39, and CH-RS15 described in this invention were derived from the rhizosphere soil of wheat in dryland farmland. Different nitrogen additions (0 kg N ha) were used to differentiate them. - ¹、90 kg N ha - ¹, 180 kg N ha - ¹) High-throughput sequencing analysis was performed on the rhizosphere bacterial communities of two wheat varieties (Changhan 58 (dryland-growing variety with strong drought resistance) and Zhengmai 9023 (ZM, irrigated variety with weak drought resistance)) under different treatment conditions. Operational taxonomic units (ASVs) with relatively high abundance and stable presence in the community were screened to construct a rhizosphere bacterial community co-occurrence network. The interactions between microorganisms were analyzed based on the network topology characteristics. Based on the connectivity characteristics of each node within and between modules in the network, microbial groups that play a key role in the stability of the community structure were identified. Simultaneously, rhizosphere samples of wheat from dryland farmland were isolated and cultured to obtain multiple culturable bacterial strains. The 16S rRNA gene sequences of these strains were compared with the bacterial sequences obtained from high-throughput sequencing using BLAST to determine the source of the corresponding culturable strains. Furthermore, by combining the shared microbial groups that are stable under different treatment conditions, microbial groups with key network positions, and successfully isolated and cultured strains, the intersection of these three factors was comprehensively screened to define the core microbial groups of the rhizosphere in dryland farmland. Based on this, core microbial strains that are relatively enriched in the rhizosphere of drought-resistant wheat varieties were further screened as the source of core strains for the compound microbial agent of this invention, and used for subsequent verification and application research on drought resistance and growth promotion functions.

[0030] Example 1: Isolation, screening and identification of strains.

[0031] 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 -6Suspension. 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 5 days. Single colonies are picked and repeatedly streaked onto fresh TSB solid medium to purify the strains. The resulting strains are named CH-RS23, ZM-RS40, ZM-RS39, and CH-RS15.

[0032] The isolated 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 the total genomic DNA of the strains was extracted. Using genomic DNA as a template, the 16S rRNA gene fragment was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGACTT-3'). Sequencing yielded the 16S rRNA gene sequences of strains CH-RS23, ZM-RS40, ZM-RS39, and CH-RS15, as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The 16S rRNA gene sequence was compared with the EZ Biocloud bioinformatics database to identify the strains as follows: Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter rice ZM-RS39 and Peribacillus frigoritolerans CH-RS15.

[0033] SEQ ID NO.1: SEQ ID NO.2: SEQ ID NO.3: SEQ ID NO.4: Example 2: Performance determination of the isolated strain.

[0034] 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. The diameter D of the transparent halo was measured to determine the nitrogen-fixing capacity 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. Through measurement, Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter rice ZM-RS39 and Peribacillus frigoritolerans The nitrogen fixation capabilities of CH-RS15 are denoted as: 2, 2, 1, 2.

[0035] 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 7.5) plates, with four replicates for each strain; incubated at 28℃ for 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. Through measurement, Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter oryzae ZM-RS39 and Peribacillus frigoritolerans The phosphorus solubility of CH-RS15 was recorded as 3, 3, 0, and 0, respectively.

[0036] 3. Determination of iron production capacity The strain was 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). After reacting in the dark for 60 min, 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 Through measurement, Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter rice ZM-RS39 and Peribacillus frigoritolerans The siderophore expression levels of CH-RS15 were 0.287, 0.134, 0.525, and 0.282, respectively.

[0037] 4. IAA production capacity determination The strain was inoculated 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. Its OD was then measured. 530 The IAA content was calculated using a standard curve obtained by serial dilution of IAA. The CH-RS23 value was determined. Curtobacterium garlic ZM-RS40 Acinetobacter oryzae ZM-RS39 and Peribacillus frigoritolerans The IAA yield of CH-RS15 was 6.136 mg / L. -1 19.324 mg L -1 4.821 mg / L -1 4.515 mg L -1 ...

[0038] 5. Assay for ACC deaminase production The strain was 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): 2.8 mg H3BO3, 1.4 mg ZnSO4•7H2O, 1.8 mg MnCl2•4H2O, 0.04 mg CuSO4•5H2O, 0.02 mg MoO3), and incubated on a shaker at 28°C at 125 rpm for 2 days. The cells were washed three times by centrifugation in DF medium (nitrogen-free), each time at 10,000 rpm for 10 min. 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 at 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 reacted at 37°C for 30 min, followed by the addition of 50 μL 0.5 M HCl. 300 μL DNPH (0.2% in 2 M HCl) was added, and the mixture was incubated at 30°C for 15 min. After adding 2 mL 2 M NaOH, the absorbance was measured at 540 nm. Enzyme activity was calculated using a standard curve based on a gradient dilution of α-ketobutyrate. The results showed that… Pseudomonas fish CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter oryzae ZM-RS39 and Peribacillus frigoritolerans The ACC deaminase production capacity of CH-RS15 is 0.704 μmol / (min mg). -1 ), 0.052 μmol / (min mg -1 ), 0.087 μmol / (min mg -1 ), 0.032 μmol / (min mg -1 ).

[0039] Example 3: Compound microbial agent, its preparation method and application.

[0040] I. Determination of antagonistic effects of bacterial strains The following are the core bacteria screened by the data analysis of this invention. Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter oryzae ZM-RS39 Peribacillus cold-tolerantCH-RS15 and Streptomyces roseifaciens Results of ZM-RS3 bacterial antagonistic activity assay: CH-RS23 bacterial suspension: Pseudomonas piscicola Single colonies were inoculated into 10 mL of TSB liquid medium. The medium was placed on a shaker at 28°C and 125 rpm and incubated until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL.

[0041] ZM-RS40 bacterial solution: Curtobacterium allii Single colonies were inoculated into 10 mL of TSB liquid medium. The medium was placed on a shaker at 28°C and 125 rpm and incubated until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL.

[0042] ZM-RS39 bacterial solution: Acinetobacter oryzae Single colonies were inoculated into 10 mL of TSB liquid medium. The medium was placed on a shaker at 28°C and 125 rpm and incubated until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL.

[0043] CH-RS15 bacterial culture: Peribacillus frigoritolerans Single colonies were inoculated into 10 mL of TSB liquid medium. The medium was placed on a shaker at 28°C and 125 rpm and incubated until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL.

[0044] ZM-RS3 bacterial solution: Streptomyces roseifaciens Single colonies were inoculated into 10 mL of TSB liquid medium. The medium was placed on a shaker at 28°C and 125 rpm and incubated until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL.

[0045] 30 µL of CH-RS23 bacterial suspension was inoculated onto TSB solid medium and spread. Seven-mm diameter mycelial discs of ZM-RS3, ZM-RS40, ZM-RS39, and CH-RS15 were picked and inoculated into the center of the plate. The plates were incubated for 3 days, observed, and photographed. The appearance of inhibition zones indicates antagonistic activity.

[0046] 30 µL of ZM-RS40 bacterial suspension was inoculated onto TSB solid medium and spread. Seven-mm diameter mycelial discs of ZM-RS3, CH-RS23, ZM-RS39, and CH-RS15 were picked and inoculated into the center of the plate. The plates were incubated for 3 days, observed, and photographed. The appearance of inhibition zones indicates antagonistic activity.

[0047] 30 µL of ZM-RS39 bacterial suspension was inoculated onto TSB solid medium and spread. Seven-mm diameter mycelial discs of ZM-RS3, ZM-RS40, CH-RS23, and CH-RS15 were picked and inoculated into the center of the plate. The plates were incubated for 3 days, observed, and photographed. The appearance of inhibition zones indicates antagonistic activity.

[0048] 30 µL of CH-RS15 bacterial suspension was inoculated onto TSB solid medium and spread. Seven-mm diameter mycelial discs of ZM-RS3, ZM-RS40, ZM-RS39, and CH-RS23 were picked and inoculated into the center of the plate. The plates were incubated for 3 days, observed, and photographed. The appearance of inhibition zones indicates antagonistic activity.

[0049] 30 µL of ZM-RS3 bacterial suspension was inoculated onto TSB solid medium and spread. Seven-mm diameter mycelial discs of CH-RS23, ZM-RS40, ZM-RS39, and CH-RS15 were picked and inoculated into the center of the plate. The plates were incubated for 3 days, observed, and photographed. The appearance of inhibition zones indicates antagonistic activity.

[0050] The results are as follows Figure 4 As shown, Streptomyces roseifaciens ZM-RS3 and Curtobacterium garlic ZM-RS40 exhibits significant antagonistic behavior; therefore, strains with excellent growth-promoting properties should be selected. Curtobacterium allii ZM-RS40 and Pseudomonas piscicola CH-RS23, Acinetobacter oryzae ZM-RS39 Peribacillus cold-tolerant CH-RS15 was used to construct a compound microbial agent.

[0051] II. Compound microbial agents and their preparation methods Selected from those cultured in TSB solid medium for 24 hours Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter oryzae ZM-RS39 Peribacillus cold-tolerant Single colonies of CH-RS15 were inoculated onto TSB liquid medium and cultured at 28°C and 200 rpm for 24 h. Once the cells reached the logarithmic growth phase, they were collected by centrifugation at 4°C and 10,000 rpm. The cells were washed twice with sterile tap water and then diluted to a 10⁻⁶ oz / mL. 8 CFU / mL was used to obtain bacterial suspensions. The suspensions of the four strains were mixed thoroughly at a volume ratio of 1:1 (the viable cell count in the bacterial 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.

[0052] III. Application of Compound Microbial Agents in Improving Wheat Drought Resistance Perlite and vermiculite were mixed in a 3:2 mass ratio and packed into mushroom bags, 800 mL per bag. 350 mL of Hoagland's nutrient solution (containing 1417 mg / L of macronutrients including 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 1.81mg L -1 ZnSO4·7H2O 0.22 mg L -1 CuSO4·5H2O 0.08 mg L -1 H₂MnO₄·H₂O 0.02 mg / L -1 FeSO4 50 mg L -1 (pH 7.0 ± 0.2). After sealing, sterilize at 121℃ for 25 min.

[0053] 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:

[0054] The prepared mushroom 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 wheat using a disposable syringe, 2 mL per plant, with only one treatment. Seedlings without bacterial agent but with an equal amount of sterile water served as a blank control. Ten replicates were performed 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:

[0055] 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.

[0056] (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.

[0057] (3) Plant height: Measure the natural height of each wheat plant with a ruler.

[0058] (4) Root length: Measure the length of the main root of each wheat plant with a ruler.

[0059] 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).

[0060] 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.

[0061] As shown in Table 1, under different drought gradient treatments, inoculation with the SynCom microbial community significantly promoted wheat seedling growth, showing a clear advantage compared to the corresponding uninoculated control (CK). P＜0.05Under 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 37.34 cm, 0.677 g, 0.767 g, 1.443 g, and 70.75 cm, respectively, with underground fresh weight, total fresh weight, and root length significantly increasing by 118.39%, 53.51%, and 113.30%, respectively. Under 10% drought stress, the wheat plant height, underground fresh weight, aboveground fresh weight, total fresh weight, and root length of the SynCom treatment increased by 15.62%, 148.83%, 16.44%, 51.56%, and 136.04% compared to the CK, respectively, demonstrating a significant growth-promoting effect. Under 20% drought stress, the SynCom treatment 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 17.14%, 93.16%, 15.00%, 37.44%, and 111.95%, respectively, compared to the control (CK). Furthermore, under different drought gradients, the underground dry weight, aboveground dry weight, and total dry weight of wheat treated with SynCom were all higher than those treated with CK. Compared to CK, the increase in underground dry weight was 22.50%–52.27%, the increase in aboveground dry weight was 27.12%–42.34%, and the increase in total dry weight was 31.75%–45.16%. These results indicate that this complex microbial community can significantly promote wheat growth and improve its drought resistance under different moisture conditions.

[0062] The above data shows that the bacterial agent of the present invention has the following advantages: The four strains constituting the compound microbial agent of this invention are all core microbial groups in the rhizosphere of dryland farmland. They are microbial groups that exist stably under different treatment conditions, occupy key network positions, and can be isolated and cultured. At the same time, they are relatively enriched in the rhizosphere of drought-resistant wheat varieties.

[0063] The four bacterial strains constituting the compound bacterial agent of this invention all possess good growth-promoting properties. Pseudomonas fish CH-RS23, Curtobacterium allii ZM-RS40 Acinetobacter oryzae ZM-RS39 Peribacillus frigoritolerans CH-RS15 all possess the abilities of nitrogen fixation, phosphorus solubilization, IAA production, siderophore production, and ACC deaminase production. Pseudomonas piscicola CH-RS23 and Curtobacterium allii ZM-RS40 bacteria have a high phosphorus-solubilizing ability; Pseudomonas piscicola CH-RS23, Curtobacterium allii ZM-RS40 and Peribacillus frigoritolerans CH-RS15 bacteria have a strong nitrogen-fixing ability; Curtobacterium alliiZM-RS40 has the strongest IAA production capacity; Pseudomonas piscicola CH-RS23 has the strongest ability to produce ACC deaminase and siderophores.

[0064] 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.

[0065] 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 total fresh weight (53.51%), total dry weight (45.16%), plant height (17.14%), and root length (136.04%). Simultaneously, under different drought treatments, the fresh and dry weights of both the underground and aboveground parts of wheat were significantly higher than those in the CK. 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 has the potential to improve crop yield stability and production efficiency under drought conditions, demonstrating promising prospects for agricultural application and development.

[0066] 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.

[0067] 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 for crop drought resistance, characterized in that, The active ingredient is *Pseudomonas urticaria* (…). Pseudomonas piscicola CH-RS23, *Clostridium cepacia* ( Curtobacterium allii ZM-RS40, Acinetobacter rice ( Acinetobacter oryzae ZM-RS39 and cold-resistant short-spores ( Peribacillus frigoritolerans CH-RS15; The *Pseudomonas urinaria* CH-RS23 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026412 on March 10, 2026. The *Corynebacterium cepacia* ZM-RS40 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026416, on March 10, 2026. The Acinetobacter rice ZM-RS39 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026413 on March 10, 2026. The cold-resistant Bacillus brevis CH-RS15 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026417, on March 10, 2026.

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

3. The compound microbial agent for crop drought resistance according to claim 2, characterized in that, The viable counts of *Pseudomonas urinaria* CH-RS23, *Clostridium cepacia* ZM-RS40, *Acinetobacter oryzae* ZM-RS39, and *Bacillus frostbite* ZM-RS6 in the compound bacterial agent were all 10. 8 CFU / mL ~10 9 CFU / mL.

4. A method for preparing a compound microbial agent for crop drought resistance as described in any one of claims 1 to 3, characterized in that, Includes the following steps: *Pseudomonas urinaria* CH-RS23, *Clostridium cephalosporin* ZM-RS40, *Acinetobacter oryzae* ZM-RS39, and *Bacillus brevis* ZM-RS6 were inoculated into TSB liquid medium and cultured at 28℃–29℃ and 180–200 rpm until the cells reached the logarithmic growth phase. The cells were collected by centrifugation, washed with sterile water, and diluted to a concentration of 10-1. 8 CFU / mL ~10 9 CFU / mL was used to obtain suspensions of four strains, and the suspensions of the four strains were mixed to obtain a compound bacterial agent.

5. The preparation method according to claim 4, characterized in that, The suspensions of the four strains were in equal volume ratios.

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 *Pseudomonas yucca* CH-RS23 in the compound bacterial agent has the following functions: nitrogen fixation, phosphorus solubilization, siderophore production, IAA production, and ACC deaminase production; the *Clostridium cepacia* ZM-RS40 has the following functions: nitrogen fixation, phosphorus solubilization, siderophore production, IAA production, and ACC deaminase production; the *Acinetobacter oryzae* ZM-RS39 has the following functions: nitrogen fixation, siderophore production, IAA production, and ACC deaminase production; and the *Bacillus thuringiensis* CH-RS15 has the following functions: nitrogen fixation, siderophore production, IAA production, and ACC deaminase production.

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