Composite flora for preventing and treating cotton verticillium wilt and microbial agent and application thereof

Abstract

The invention provides a composite flora for preventing and treating cotton verticillium wilt and a microbial agent and application thereof, and belongs to the technical field of agricultural microorganisms. The composite flora and the microbial agent provided by the invention comprise pseudoarthrobacter oxydans CRB-17 and bacillus velezensis CRB-46; the preservation numbers of the pseudoarthrobacter oxydans CRB-17 and the bacillus velezensis CRB-46 are respectively CCTCC (China Center for Type Culture Collection) NO: M20252254 and CCTCC NO: M20252255, and the pseudoarthrobacter oxydans CRB-17 and the bacillus velezensis CRB-46 are both preserved in the China Center for Type Culture Collection on October 20, 2025. The pseudoarthrobacter oxydans CRB-17 and the bacillus velezensis CRB-46 are stably colonized in the rhizosphere of the cotton, have a strong direct antagonism effect on verticillium dahliae, can effectively and continuously prevent and treat the verticillium wilt of the cotton, effectively promote the germination of cotton seeds, the elongation of radicles and hypocotyls, promote the growth of the cotton and improve the defensive power of the cotton.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, and in particular to a compound microbial community for controlling cotton Verticillium wilt, its microbial agents, and their applications. Background Technology

[0002] cotton( Gossypium hirsutum (This is a description of a plant, possibly related to Verticillium dahliae, which is an important economic crop in my country.) Verticillium dahliae Verticillium wilt, caused by the fungus *Verticillium wilt*, is a devastating soil-borne vascular disease. This pathogen can survive in the soil for 10-15 years, with a complex infection process and difficult control, often causing large-scale yield reductions or even crop failure. It is often referred to as the "cancer" of cotton and severely restricts the sustainable development of my country's cotton industry. Currently, Verticillium wilt control still mainly relies on chemical pesticides, but these have multiple problems, including unstable efficacy, difficulty in degradation, and the risk of high pathogen resistance. Many researchers have also cultivated and utilized disease-resistant cotton varieties to control Verticillium wilt, but the scarcity of highly resistant germplasm resources and the diversity of pathogen physiological races make the development of resistant varieties difficult, and their widespread application also faces severe challenges. Against this backdrop, biological control has become a research hotspot due to its environmentally friendly and sustainable characteristics.

[0003] Currently, some domestic and international research has been conducted based on the genus Bacillus (Bacillus). Bacillus ), Pseudomonas spp. Pseudomonas Biocontrol agents developed from single-function strains such as [list of strains] generally suffer from drawbacks such as limited functionality, low rhizosphere colonization efficiency, and unstable field efficacy. Theoretically, compound microbial agents composed of multiple strains can overcome bottlenecks through functional complementarity and synergistic effects. However, simple random combinations often result in poor efficacy or even failure due to antagonism or ineffective competition between strains. Therefore, how to accurately screen core strain combinations that can stably coexist and synergistically enhance efficacy from massive microbial resources remains a long-standing unsolved technical challenge in this field. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a compound microbial flora and its microbial agent for the prevention and control of cotton Verticillium wilt, and its application, to solve the technical bottlenecks of existing biocontrol agents such as single function, poor rhizosphere colonization ability, and unstable field control efficacy. The compound microbial flora and its microbial agent provided by this invention have a strong direct antagonistic effect on Verticillium dahliae, which can effectively and continuously control cotton Verticillium wilt, effectively promote cotton seed germination, radicle and hypocotyl elongation, promote cotton growth, and improve cotton's defense capabilities.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A compound microbial community for controlling cotton Verticillium wilt, said compound microbial community includes *Pseudomonas oxysporum* (… Pseudarthrobacter oxydans CRB-17 and Bacillus belesiensis ( Bacillus velezensis) CRB-46; the preservation numbers of the Pseudoclavibacter oxidants CRB-17 and the Bacillus velezensis CRB-46 are CCTCC NO: M20252254 and CCTCC NO: M20252255 respectively, which are preserved in the China Center for Type Culture Collection on October 20, 2025.

[0006] In some embodiments, the ratio of the viable cell number of the Pseudoclavibacter oxidants CRB-17 and the Bacillus velezensis CRB-46 is preferably (0.5-2): 1, more preferably 1: 1.

[0007] The present application also provides a microbial inoculant comprising the complex microbial flora described in the above technical solution.

[0008] In some embodiments, the microbial inoculant preferably includes but is not limited to liquid inoculant, solid inoculant, wettable powder.

[0009] In some embodiments, the microbial inoculant preferably further comprises an agriculturally acceptable carrier.

[0010] The present application also provides the use of the complex microbial flora described in the above technical solution or the microbial inoculant described in the above technical solution in inhibiting or antagonizing the Verticillium dahliae (V. dahliae). Verticillium dahliae ).

[0011] The present application also provides the use of the complex microbial flora described in the above technical solution or the microbial inoculant described in the above technical solution in preventing and / or treating cotton Verticillium wilt.

[0012] In some embodiments, the pathogenic bacteria of cotton Verticillium wilt is Verticillium dahliae.

[0013] The present application also provides the use of the complex microbial flora described in the above technical solution or the microbial inoculant described in the above technical solution in promoting the germination of cotton seeds.

[0014] The present application also provides the use of the complex microbial flora described in the above technical solution or the microbial inoculant described in the above technical solution in promoting the growth of cotton.

[0015] The present application also provides the use of the complex microbial flora described in the above technical solution or the microbial inoculant described in the above technical solution in improving the defense of cotton plants.

[0016] Beneficial technical effects: the present application provides a complex microbial flora for preventing and treating cotton Verticillium wilt, a microbial inoculant thereof and applications, the complex microbial flora and the microbial inoculant thereof provided by the present application have the following beneficial technical effects: (1) Synergistic biocontrol effect: The complex microbial consortium / microbial inoculant showed a significant synergistic effect in the prevention and control of cotton Verticillium wilt. Greenhouse pot experiments confirmed that its control effect was as high as 90.87%, significantly better than that of two strains alone, achieving a control effect of "1+1>2".

[0017] (2) Multiple functional complementation: ①Direct inhibition of pathogens: Bacillus velezensis CRB-46 has strong direct antagonism against L. theoma. ②Highly efficient induction of resistance: The complex microbial consortium can systematically activate the defense system of cotton, significantly improve the activities of superoxide dismutase (SOD) and catalase (CAT), and effectively reduce the activities of peroxidase (POD) and the accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA), thereby alleviating oxidative damage.

[0018] (3) Significant promotion of growth: Strain CRB-17 has the ability to efficiently synthesize indole acetic acid (IAA), which can effectively promote cotton seed germination, radicle and hypocotyl elongation.

[0019] (4) Stable rhizosphere colonization: There is positive metabolic interaction between the strains. The metabolic products of CRB-17 can specifically promote the biofilm formation of CRB-46, and the two strains show cross-feeding and mutual growth-promoting characteristics. This interaction greatly enhances the colonization competitiveness and population stability of the complex microbial consortium in the rhizosphere of cotton, providing a fundamental guarantee for the persistent exertion of biocontrol function.

[0020] (5) Environment-friendly and convenient production: The raw materials of the microbial inoculant are easy to obtain, the preparation process is simple, the application is convenient, and it is safe to the environment and crops, meeting the development needs of green and sustainable agriculture, and having good industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the Verticillium wilt symptom and disease index of cotton seedlings treated with different treatments; wherein, Figure 1 A is the Verticillium wilt symptom of cotton seedlings treated with different treatments, Figure 1 B is the disease index of cotton seedlings treated with different treatments; Figure 2 is the phylogenetic tree of strains isolated based on the specific enrichment of root exudates of disease-resistant varieties; wherein, the red labeled strains are derived from the 4HR treatment group, and the blue labeled strains are derived from the DMSO control group; Figure 3 is the effect of specific enrichment strains on L. theoma; wherein, Figure 3 A is a plate confrontation photo of the effect of specific enrichment strains on L. theoma, Figure 3 B is the effect of specific strains on the growth of L. theoma colonies, Figure 3 C is the inhibition effect of specific strains on L. theoma; Figure 4 Heatmap of the results of the determination of the ability of the strains to specifically enrich IAA production; Figure 5 Heatmap of the cross-feeding effect of the strains specifically enriched; Figure 6 Effect of cell-free filtrate of different strains on biofilm formation of B. velezensis CRB-46; Figure 7 Effect of different treatments on cotton seed germination; wherein, Figure 7 A is the effect of the complex microbial community and its member single bacteria on seed germination rate, Figure 7 B is the photo of seed germination on the seventh day of different treatments, Figure 7 C is the germination index, Figure 7 D is the hypocotyl length, Figure 7 E is the radicle length; Figure 8 Effect of different treatments on cotton seed germination; wherein, Figure 8 A is the photo of the cotton seedling Verticillium wilt phenotype treated by the complex microbial community and its member single bacteria, Figure 8 B is the effect of the complex microbial community and its member single bacteria on cotton Verticillium wilt occurrence, Figure 8 C-G are the graphs of POD, MDA, SOD, CAT enzyme activity, and H2O2 content, respectively. DETAILED DESCRIPTION

[0022] In order to better understand the present application, the content of the present application will be further illustrated below in combination with examples, but the content of the present application is not limited only to the following examples. The materials, reagents and the like used in the examples and test examples of the present application can be obtained from commercial channels unless otherwise specified; the methods used in the examples and test examples of the present application are conventional methods unless otherwise specified.

[0023] Example 1: Effect of rhizosphere microorganisms of resistant and susceptible varieties on cotton Verticillium wilt occurrence 1.1 Preparation of rhizosphere microbial suspension of resistant and susceptible varieties On August 16, 2022 (flowering stage), rhizosphere soil of resistant variety (Zhongmiansuo 100) and susceptible variety (Jimian 11) was collected in the test field of the Cotton Institute of Chinese Academy of Agricultural Sciences (Anyang, Henan) by five-point sampling method, and was quickly placed into an ice box for storage and transportation back to the laboratory. 10 g of the collected rhizosphere soil of resistant and susceptible varieties was weighed and mixed with 100 mL of tryptone soy broth (TSB) medium. Each rhizosphere soil treatment was set up with 15 independent biological replicates. The mixed liquid was placed in a 28℃, 60 rpm condition for 2d of shaking culture, and then the supernatant was collected. The collected supernatant was continued to be shaken and cultured for 3d under the same conditions (28℃, 60 rpm) to enrich the rhizosphere microorganisms. After the culture was completed, the obtained rhizosphere microbial suspension was stored at 4℃ for standby use.

[0024] 1.2 Preparation of Verticillium dahliae spore suspension Mycelial discs of the highly pathogenic *Verticillium dahliae* strain Vd080 were selected and inoculated into Erlenmeyer flasks containing 100 mL of Czapek's medium. The flasks were incubated at 25°C with shaking at 180 rpm for 4–5 days. After incubation, the culture was filtered through eight layers of sterile gauze to remove mycelia. The resulting filtrate was the spore suspension. Finally, the spore concentration was adjusted to 1 × 10⁻⁶ using a hemocytometer. 7 Quantity / mL, and store at 4℃ for later use.

[0025] 1.3 The Influence of Rhizosphere Microorganisms on the Occurrence of Cotton Verticillium Wilt in Resistant and Susceptible Varieties The experiment included four treatments: (1) rhizosphere microbial suspension of susceptible variety + resistant variety (SC+RS); (2) rhizosphere microbial suspension of susceptible variety + susceptible variety (SC+SS); (3) rhizosphere microbial suspension of resistant variety + resistant variety (RC+RS); and (4) rhizosphere microbial suspension of resistant variety + susceptible variety (RC+SS).

[0026] The specific steps are as follows: 200g of natural soil sterilized by gamma rays (irradiation dose >50kGray) was divided into 9-ounce paper pots. Each treatment group had at least 6 replicates, with each replicate consisting of 6 paper pots (a total of 36 paper pots). 20mL of the corresponding rhizosphere microbial suspension was added to each group of paper pots, followed by wetting the substrate with 50mL of sterile water. The pots were then covered with mulch and incubated for 5 days to establish the rhizosphere microbial community. After incubation, 10 surface-sterilized seeds of the corresponding cotton variety were placed in each paper pot. Seven days after sowing, thinning was performed, leaving 5 seedlings per pot, and the seedlings were again inoculated with 20mL of the same rhizosphere microbial suspension to strengthen colonization. When the first true leaf of the cotton seedlings unfolded, 10mL of a 10% concentration was inoculated using the root-dipping method. 7 Dahliae Verticillium spore suspension at a concentration of 1 spore / mL. The Verticillium wilt disease index was calculated for each treatment 21 days after inoculation. The disease index grading criteria followed the "Technical Specifications for Cotton Verticillium Wilt Monitoring and Forecasting".

[0027] The results are as follows Figure 1 As shown. By Figure 1 A and Figure 1 B shows that the occurrence of cotton Verticillium wilt is significantly regulated by the rhizosphere microbial community. Specifically, the disease index of susceptible varieties inoculated with resistant rhizosphere microorganisms (SC-RS) (26.88±0.06) was significantly lower than that of varieties inoculated with their own rhizosphere microorganisms (SC-SS) (55.49±0.03). pThe treatment of the susceptible variety inoculated with the rhizosphere microorganism (RC-SS) of the resistant variety (28.75 ± 0.05) had no significant difference with the treatment of the susceptible variety inoculated with the rhizosphere microorganism (RC-SS) of the resistant variety (28.75 ± 0.05). This shows that the rhizosphere microorganism community of the resistant variety can effectively endow the susceptible variety with resistance to Verticillium wilt, which provides a theoretical basis for subsequent directional screening of beneficial strains from the rhizosphere of the resistant variety.

[0028] Example 2: Isolation and identification of key root exudate-specific enrichment strains of resistant varieties 2.1 Establishment of specific enrichment system 4-Hexylresorcinol (4-Hexylresorcinol, hereinafter referred to as 4HR) is a key root exudate signal molecule that is specifically enriched in the rhizosphere of the resistant cotton variety (Zhongzhi cotton 100) identified by our team through previous metabolomics analysis. Based on the hypothesis that "4HR may act as a signal molecule to mediate the assembly of beneficial rhizosphere microorganisms", this invention first establishes it as a chemotactic target for the directional enrichment of strains with biocontrol potential against cotton Verticillium wilt.

[0029] 10 g of farmland soil without cotton planting history (Baibi Town, Anyang, Henan Province) was weighed and added to a conical flask containing 100 mL of chemotactic buffer (K2HPO4 4.4 g / L, KH2PO4 4.2 g / L, EDTA 20 mM) and 8 glass beads with a diameter of 4 mm, and shaken at 180 rpm and room temperature for 1 h. Then the mixture was centrifuged at 500 g for 5 min, and the supernatant was collected to obtain a soil microbial suspension. Using a 1 mL syringe equipped with a 25G needle, 100 μL of 4HR solution (working concentration 6 μg / mL) dissolved in 1% DMSO was taken as the test group, and an equal volume of 1% DMSO solution was taken as the blank control. The needle was immersed in a PCR tube containing 200 μL of the above soil microbial suspension and left at room temperature for 1 h to allow the microorganisms with chemotaxis to 4HR to enrich in the needle and syringe.

[0030] 2.2 Isolation and identification of enriched strains After the end of the standing, the syringe was removed, the outer wall of the needle was rinsed with sterile water and wiped with a sterile cotton swab to remove non-specifically attached microorganisms. The liquid in the syringe was injected into a sterile 1.5 mL centrifuge tube, gradient diluted with sterile water, and plated on TSA plates. Invert culture at 37°C for 48 h. Single colonies were picked for at least three rounds of streaking to ensure pure, single-morphology bacterial colonies. The purified single colonies were picked and transferred to 5 mL TSB medium, which was incubated at 180 rpm, 37°C constant temperature shaker overnight. 1 μL of bacterial culture was used as a DNA template for 16S rRNA gene identification using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', Seq_1) and 1492R (5'-TACGACTTAACCCCAATCGC-3', Seq_2) for amplification. The PCR product was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing, and the obtained sequence was subjected to homology comparison and classification identification by NCBI database.

[0031] The results showed that a total of 72 single colonies were isolated, and 8 non-redundant strains were obtained through 16S rRNA gene sequence alignment identification—CRB-05, CRB-07, CRB-08, CRB-17, CRB-26, CRB-28, CRB-46 and CRB-68, whose nucleotide sequences were Seq_3-Seq_10, respectively. As shown in the phylogenetic tree, 5 strains of Pseudoclavibacter, 1 strain of Bacillus and 1 strain of Weihuaia were all 4HR-specific enrichment strains, while the strain (CRB-07) isolated in the blank control was not significantly enriched. Figure 2

[0032] Example 3: Disease inhibition and growth promotion ability determination of specific enrichment strains 3.1 Determination of the ability of strains to antagonize P. dianthicola The direct antagonistic effect of the strains on P. dianthicola was determined by the plate confrontation method. The specific steps are as follows: a 9 mm diameter P. dianthicola fungus cake was inoculated on one side of a PDA plate (3 cm from the edge), and the 7 strains to be tested were streaked on the other side of the PDA plate (3 cm from the edge). A PDA plate inoculated with only the P. dianthicola fungus cake was used as a blank control. All treatments were set up in three independent biological replicates. The plates were placed in a 25°C constant temperature incubator for confrontation culture for 14 d. After the incubation, the growth radius of the pathogenic fungal colonies towards the bacteria was measured, and the inhibition rate was calculated for each treatment with 3 replicates.

[0033] Inhibition rate (%) = [(control colony radius - test colony radius) / (control colony radius)] x 100.

[0034] The results are as follows: Figure 3 ​As shown in Table 1, among the 7 strains to be tested, only CRB-05, CRB-08 and CRB-46 had direct antagonistic effect on the growth of Verticillium dahliae, and CRB-46 had the strongest antagonistic ability Figure 3 A and Figure 3 B), with a high inhibition rate of 97% Figure 3 C), and was determined as the core biocontrol strain.

[0035] 3.2 Determination of the ability of strains to produce IAA Salkowski colorimetric method was used to determine the ability of strains to produce IAA. The specific steps are as follows: single colonies of the 7 strains to be tested were inoculated in 5 mL TSB medium containing 100 mg / L L-tryptophan, and incubated at 37°C, 180 rpm for 48 h. After incubation, the culture solution was centrifuged at 5000 rpm for 10 min to collect the supernatant. 100 μL of the supernatant was mixed with an equal volume of Salkowski color developing solution (35% HCIO4 and 0.5 M FeCl3 mixed at a volume ratio of 50:1) in a 96-well plate, and the same medium without inoculation was used as a blank control. After 30 min of reaction at room temperature in the dark, the absorbance value (OD 530nm ) of the solution was measured at 530 nm. The IAA production of each strain was calculated by a previously prepared standard concentration curve of IAA (y=0.03414x+0.1039, R 2 =0.9958). The results are shown in Table 2. Figure 4 As shown in Table 2, CRB-05, CRB-17, CRB-26, CRB-28, CRB-46 and CRB-68 all had the ability to produce IAA, among which CRB-17 had the strongest ability to produce IAA (31±0.12 mg / L·OD 600nm -1 ).

[0036] Example 4: Verification of the synergistic interaction between strains 4.1 Preparation of cell-free filtrate and bacterial suspension of strains Single colonies of the 7 strains of specifically enriched bacteria obtained in Example 2 were inoculated in 5 mL TSB medium, and incubated at 37°C, 180 rpm for 48 h. After incubation, the bacterial solution was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm sterile filter to obtain the cell-free filtrate. The bacterial pellet was resuspended with sterile TSB medium, and the concentration was adjusted to OD 600nm =1.0 to obtain the bacterial suspension.

[0037] 4.2 Determination of the cross-feeding effect between strains To evaluate the interactions between strains through metabolic products, cross-feeding experiments were performed. The seven cell-free filtrates were mixed with equal volume of fresh sterile TSB medium as selective medium, respectively.

[0038] The specific operation is as follows: Three treatments were set in the experiment: (1) experimental group: 900 μL of selective medium + 100 μL of recipient bacterial suspension; (2) positive control group: 900 μL of fresh TSB + 100 μL of recipient bacterial suspension; (3) blank control group: 1 mL of sterile TSB medium. Three biological replicates were set for each treatment. The above system was placed in 180 rpm, 37°C shaking culture for 48 h, and the OD 600nm value was measured, and the relative growth rate was calculated according to the following formula to quantify the interaction effect between strains.

[0039] Relative growth rate (%) = [(experimental group OD 600nm - blank group OD 600nm ) / (positive control group OD 600nm - blank group OD 600nm ) - 1] x 100.

[0040] Results Figure 5 As shown in the table, the interaction between strains has high strain specificity. It is worth noting that the cell-free filtrate of CRB-46 significantly promotes the growth of CRB-17 (relative growth rate 21.4%, P p = 0.04), while the cell-free filtrate of CRB-17 also significantly promotes the growth of CRB-46 (relative growth rate 16.7%, P p = 0.03), indicating a mutual promotion cross-feeding relationship between the two. However, the autologous filtrate of CRB-17 significantly inhibits its own growth (relative growth rate -22.7%, P p = 0.02), indicating that the mutual beneficial relationship between the two is not due to simple self-stimulation, but may depend on metabolic complementation or specific signal molecule exchange between the two strains. Overall, in the background of strains interaction that is generally antagonistic or neutral, the mutual beneficial combination of CRB-46 and CRB-17 shows significant synergistic potential, and therefore is screened as the core strain for constructing a complex bacterial community.

[0041] CRB-17 and CRB-46 were deposited at the China Center for Type Culture Collection on October 20, 2025, with accession numbers CCTCC NO: M20252254 and CCTCC NO: M20252255, respectively, and the address of deposit is: China. Wuhan. Wuhan University.

[0042] 4.3 Effect of strains on biofilm formation of Bacillus velezensis CRB-46 The effect of different cell-free filtrates on biofilm formation of Bacillus belyssus CRB-46 was evaluated using the crystal violet staining method.

[0043] The specific steps are as follows: Add 900 μL of the cell-free filtrate, 900 μL of fresh TSB culture medium, and 200 μL of the CRB-46 bacterial suspension (OD) to a 10 mL transparent glass test tube. 600nm =0.1), mix well, seal, and incubate at 30℃ for 48 h to promote biofilm formation. 1.8 mL of fresh TSB medium + 200 μL of CRB-46 bacterial suspension was used as a positive control, and 2 mL of fresh TSB medium was used as a blank control.

[0044] After incubation, discard the culture medium and gently wash twice with 2 mL of PBS buffer to thoroughly remove airborne bacteria. Fix with 2 mL of methanol for 15 min, discard the methanol, and air dry at room temperature for 30 min. Then, add 2 mL of 1% (w / v) crystal violet solution and allow to stain for 20 min. Wash away unbound stain with sterile deionized water. Finally, transfer to a test tube containing 2 mL of 96% ethanol and elute for 30 min to dissolve the bound dye. After vortexing and mixing, measure the OD. 570nm Values. Each process has 3 repetitions.

[0045] The results are as follows Figure 6 As shown, compared with cell-free filtrates of other strains and positive controls, the addition of CRB-17 to cell-free filtrate significantly enhanced the biofilm formation ability of CRB-46. (OD) 570nm Quantitative analysis showed that the biofilm biomass of CRB-46 in cell-free filtrate containing CRB-17 was significantly increased by 13.3% compared to the positive control. This result indicates that metabolites secreted by CRB-17 can specifically promote biofilm formation by CRB-46. This finding mechanistically explains why the two can stably co-colonize in the rhizosphere and exert a synergistic effect.

[0046] Example 5: Effects of inoculation of a complex microbial community and its members on cotton seed germination 5.1 Preparation of inoculant and seed treatment Fermentation broths of CRB-17 and CRB-46 were centrifuged to collect bacterial cells, which were then resuspended in sterile PBS buffer and adjusted to OD values. 600nm =1.0, as a single bacterium. Two single-bacterial agents were mixed at a volume ratio of 1:1 to prepare a compound bacterial agent. Sterile PBS buffer was used as a blank control. Plump cotton seeds of the Ji Mian 11 variety were selected, surface-sterilized, and then soaked for 2 hours in the compound bacterial agent, CRB-17 bacterial agent, CRB-46 bacterial agent, and PBS buffer blank control at a seed-to-inoculum volume ratio of 1:2.

[0047] 5.2 Germination test and index determination Two layers of sterile filter paper were laid in a 9 cm diameter Petri dish as the germination bed, and 20 treated seeds were evenly placed in each dish. Each treatment was set with 3 replicates. The Petri dishes were placed in a phytotron with the conditions set as follows: temperature 25℃, light intensity 12,000 lx, light cycle 12 h / 12 h (day / night), and humidity 50%. The filter paper was replaced every 2 days and kept moist with sterile water during the experiment. The germination of seeds was observed and recorded every day, and the emergence of the embryo was recorded as germination when the embryo broke through the seed coat and exceeded half the length of the seed itself. The germination rate was counted on the 7th day, and the germination index was calculated. At the same time, photographs were taken under a stereomicroscope, and the radicle and hypocotyl lengths of the germinated seeds were measured using ImageJ software. The calculation formulas for germination rate and germination index are as follows: Germination rate (%) = total number of germinated seeds / total number of tested seeds x 100 GR Germination index = ∑(t / t) GI Gt Dt Gt wherein t is the number of germinated seeds at time t days, and t is the corresponding germination time. Dt

[0048] The results are shown in Figure 7 A, all the microbial inoculants promoted the germination of cotton seeds to some extent. At the early stage of germination (3-5 days), the germination rate of seeds inoculated with CRB-46 was significantly higher than that of the control, and it continued to rise. By the 7th day, the germination rates of the three microbial inoculant treatments all exceeded 60%, with the highest germination rate (70.37 ± 0.98%) in the complex microbial population treatment, which was about 2.11 times that of the control (33.33 ± 3.21%). The germination rates of the CRB-46 and CRB-17 single bacterial treatments were lower, but still significantly better than the control. Figure 7 B) further confirmed that the seeds in the microbial inoculant treatment groups germinated uniformly and the seedlings were healthy, while the seeds in the control group germinated sparsely and grew slowly. The statistical analysis results of germination index, radicle and hypocotyl length showed that Figure 7 ​​​​​C–7E), the germination index, hypocotyl length and radicle length of cotton were significantly improved by bacterial inoculation. The germination index of CRB-46 treatment group was the highest (15.18±0.12), and the composite microbial population (13.36±0.02) and CRB-17 (13.14±0.55) were significantly higher than the blank control. The hypocotyl and radicle length of each bacterial treatment group were significantly better than the blank control, indicating that bacterial treatment effectively promoted the early morphological development of seedlings. Overall, CRB-46 and CRB-17 can promote cotton seed germination and early growth of seedlings, and the composite application shows a synergistic effect, the seed germination rate and seedling vigor reach the best, which confirms the growth potential of the composite microbial population in agricultural application.

[0049] Example 6: Prevention and treatment effect of composite microbial population and its member single bacteria on cotton verticillium wilt 6.1 Preparation of culture medium and application of bacterial agents The vermiculite, sand and nutrient soil were mixed in a volume ratio of 3:2:1, and after high-pressure steam sterilization, they were used as the cultivation medium. 200g of the medium was placed in a 9-ounce paper pot. The following five treatment groups were set up: (1) composite microbial agent group: 20mL of the composite microbial agent (CRB-17:CRB-46=1:1) prepared in Example 5 was added; (2) CRB-17 single bacterial group: 20mL of CRB-17 single bacterial agent was added; (3) CRB-46 single bacterial group: 20mL of CRB-46 single bacterial agent was added; (4) inactivated control group: 20mL of the composite microbial agent inactivated by heat (121℃, 20min) was added; (5) blank control group: 20mL of sterile TSB medium was added as a blank control. Each treatment was set up with 6 replicates, and each replicate consisted of 6 paper pots (i.e. a total of 36 experimental units). Then, 50mL of sterile water was added to each pot to moisten the medium, and the pots were covered with mulch and incubated in a greenhouse for 5d to facilitate microbial colonization.

[0050] 6.2 Greenhouse verification of composite microbial population and its member single bacteria for prevention and treatment of cotton verticillium wilt After incubation, 10 seeds of Jicun 11 cotton were placed in each pot, and 1cm of sterilized sand was covered. After germination, 3 seedlings were left in each pot. When the first true leaf of the cotton seedling was flat, 10mL of 10 7 After 21d of inoculation, the growth and disease of the cotton seedlings were observed, and the disease index of each treatment was investigated according to the "Cotton Verticillium Wilt Field Investigation Grading Standard". At the same time, cotton leaf samples were collected, and the SOD, POD, CAT enzyme activities and MDA and H2O2 contents in the leaves were determined immediately using the nitrogen blue tetrazolium method, guaiacol method, ultraviolet absorption method, thiobarbituric acid method and titanium salt colorimetric method, respectively, according to the "Principles and Techniques of Plant Physiology and Biochemistry".

[0051] The results are shown in Table 6 and Figure 6.Figure 8 As shown in A, B and 8B, different bacterial agent treatments significantly affect the occurrence of cotton Verticillium wilt. Compared with the blank control, the treatment group of compound microbial flora has the most prominent control effect, and the disease index is only 6.94±4.52, which is significantly lower than that of the blank control group (76.04±3.87), and the relative control effect is as high as 90.87%. CRB-46 and CRB-17 can also effectively reduce the disease index, but the control effect is significantly lower than that of the compound bacterial agent treatment, which proves that the compound microbial flora has a synergistic effect. There is no significant difference between the inactivated control group and the blank control group, which shows that the biocontrol effect of the application depends on the colonization and metabolic activity of living bacteria, and not on the transient immunity caused by bacterial components. For example Figure 8 As shown in C-G, the compound microbial flora treatment can systematically regulate the cotton defense. Specifically, the compound microbial flora treatment induces the rapid increase of SOD and CAT enzyme activities in cotton leaves, which are (410.9±13.07) U / mL and (2399±55.53) μmol / min / g, respectively, which are about 0.81 and 4.56 times higher than those of the blank control. At the same time, the compound microbial flora treatment significantly reduces the accumulation of POD (1980±166.2 OD 470 / min / g), MDA (57.6±0.28 nmol / g) and H2O2 (13.66±0.15 μmol / g), which are about 82.64%, 19.19% and 36.08% lower than those of the blank control, respectively. In summary, the compound microbial flora provided by the application not only directly inhibits the growth of L. theobromae, but also effectively removes active oxygen by activating the core defense enzyme system of SOD, CAT and other cotton plants, reduces the damage of membrane lipid peroxidation, and thus systematically enhances the resistance of plants to Verticillium wilt. This "bacteriostatic-inducing" synergistic mechanism is the basis for realizing high and stable control effect.

[0052] The above only describes the preferred embodiments of the application, and it should be noted that for those skilled in the art, without departing from the principles of the application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A compound microbial community for controlling cotton Verticillium wilt, characterized in that, The complex microbial community includes *Pseudomonas oxidans* (… Pseudarthrobacter oxydans CRB-17 and Bacillus belesiensis ( Bacillus velezensis The accession numbers for *Pseudomonas oxysporum* CRB-17 and *Bacillus belyssus* CRB-46 are CCTCC NO: M20252254 and CCTCC NO: M20252255, respectively, and both were deposited at the China Center for Type Culture Collection on October 20, 2025.

2. The compound bacteria according to claim 1, characterized in that, The ratio of viable bacteria of *Pseudomonas oxysporum* CRB-17 to *Bacillus belyssae* CRB-46 was (0.5~2):

1.

3. A microbial inoculant, characterized in that, It includes the complex microbial community as described in claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that, The microbial agents include, but are not limited to, liquid agents, solid agents, and wettable powders.

5. The composite microbial community according to claims 1-2 or the microbial agent according to claims 3-4 in inhibiting or antagonizing Verticillium dahliae ( Verticillium dahliae Applications in ).

6. The use of the compound microbial community according to claims 1-2 or the microbial agent according to claims 3-4 in the prevention and / or treatment of cotton Verticillium wilt.

7. The application according to claim 6, characterized in that, The pathogen causing the cotton Verticillium wilt is Verticillium dahliae.

8. The application of the compound microbial community according to claims 1-2 or the microbial agent according to claims 3-4 in promoting cotton seed germination.

9. The application of the compound microbial community according to claims 1-2 or the microbial agent according to claims 3-4 in promoting cotton growth.

10. The application of the compound microbial community according to claims 1-2 or the microbial agent according to claims 3-4 in improving the defense of cotton plants.

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