A composite microbial agent, a preparation method and application thereof

Abstract

This invention relates to the field of microbial technology. To address the problem that the current growing environment for onions is mostly saline-alkali, and there is no effective compound microbial agent for controlling onion basal rot under such conditions, this invention discloses a compound microbial agent, its preparation method, and its application. This compound microbial agent is composed of *Trichoderma harzianum* M3, *Bacillus bereaves* MP6, *Bacillus halophilus* SYP202, *Staphylococcus squirrelii* LAB2, and *Microbacterium acetylaceum* LAB3-2. Among them, *Trichoderma harzianum* M3, *Staphylococcus squirrelii* LAB2, and *Microbacterium acetylaceum* LAB3-2 are screened from onion rhizosphere soil and combined with the superior growth-promoting strains *Bacillus bereaves* MP6 and *Bacillus halophilus* SYP202 from a microbial strain resource bank to form a compound microbial agent. This compound microbial agent can achieve disease prevention and growth promotion effects, as well as soil improvement effects. It aims to provide technical support for the green control of onion basal rot and the improvement of onion yield and varieties.

Description

Technical Field

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

[0002] In recent years, due to intensive planting and continuous cropping, onion basal rot has become a serious problem, affecting onion yield and quality. Onion basal rot is a fungal disease with a field incidence rate typically between 5% and 10%, but can reach 40% to 50% in severe cases. The pathogen causing onion basal rot is *Fusarium oxysporum* (Fusarium oxysporum). Fusarium oxysporum Fusarium solani ( ) F. solani ) and Fusarium moniliformes ( F. moniliforme Currently, there is a lack of green and effective control measures, while microbial control is safe, non-toxic, long-lasting, and environmentally compatible. WIN et al. found that *Trichoderma echinosporum* (…) Trichoderma asperellum ) against the pathogen Fusarium trifida ( F. tricinctum The antibacterial rate was 74.51%. The *Bacillus belye* strain obtained by Shi Bin et al. (…) Bacillus velezensis The compound microbial agent showed good inhibitory effects against Fusarium solani, the pathogen causing cowpea root rot, with an inhibition rate of 72.98%. However, compared with single strains, compound microbial agents not only increase the variety of metabolites but also improve disease control efficacy and community stability through synergistic effects among strains. For soil-borne diseases such as root rot, Li Jianjun et al. found that the compound microbial agent "Tuweiwei No. 4" had a control efficacy of approximately 60% against soil-borne root rot, while also having growth-promoting effects and improving soil nutrients. Li Xueping et al. found that three strains of Bacillus belyi (… B. velezensis The compound bacterial system composed of *Bacillus amyloliquefaciens* showed a control efficacy of over 70% against root rot diseases in highland barley, and also exhibited a significant growth-promoting effect. Studies by Ma Jiayong et al. have shown that *Bacillus amyloliquefaciens* (…)… B. amyloliquefaciens ), Highland Bacillus ( B. altitudinis ) and Bacillus subtilis ( B. subtilis The compound microbial agent composed of 63% showed a 63% control effect against Fusarium root rot in tomatoes, while also improving soil nutrients and significantly promoting growth.

[0003] Soil salinization has severely impacted agricultural development and the ecological environment. Statistics show that the global area of ​​saline-alkali soil has reached 9.54 × 10⁻⁶. 8 hm 2Saline-alkali soils have poor aeration and permeability, making them prone to waterlogging. This leads to slow soil warming, inhibited soil enzyme activity, reduced soil organic matter content, and fertility decline, thus affecting crop growth and yield. Currently, saline-alkali soil improvement typically employs physical, biological, and chemical methods. Physical methods for improving saline-alkali soil involve large-scale engineering projects, have a long lead time, and high maintenance costs. Chemical methods offer faster results, but are costly and prone to environmental pollution. Microbial methods, due to their economic, safe, and sustainable advantages, have become the most effective biological measure for improving saline-alkali soils. Onions also grow primarily in saline-alkali conditions, and currently, there are no compound microbial agents that can effectively control onion basal rot under these conditions. Summary of the Invention

[0004] This invention aims to address the problems existing in the prior art by providing a compound microbial agent, its preparation method, and its application. The invention involves screening salt-alkali-reducing bacterial strains from onion rhizosphere soil and clarifying their characteristics. These strains are then combined with previously screened superior disease-preventing and growth-promoting bacteria in the laboratory to form a compound microbial system. Comprehensive analysis and screening yield a superior compound microbial system. Finally, the formulation of this superior compound microbial system is optimized, clarifying the taxonomic position of the strains, their disease-preventing and growth-promoting effects, and their soil-improving effects. This aims to provide technical support for the green control of onion basal rot disease and the improvement of onion yield and varieties.

[0005] The technical solution adopted in this invention is as follows: In a first aspect, the present invention protects a composite microbial agent, which is composed of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylaceum LAB3-2. Among them, the accession number of Trichoderma harzianum M3 is CCTCC NO: M2026146, the depositary institution is China Center for Type Culture Collection, and the deposit date is January 16, 2026. The accession number of Bacillus velezensis MP6 is CCTCC NO: M20242192, the depositary institution is China Center for Type Culture Collection, and the deposit date is October 14, 2024. The accession number of Bacillus halotolerans SYP202 is CCTCC NO: M2022358, the depositary institution is China Center for Type Culture Collection, and the deposit date is March 31, 2022. The accession number of Staphylococcus sciuri LAB2 is CCTCC NO: M2026144, the depositary institution is China Center for Type Culture Collection, and the deposit date is January 16, 2026. The accession number of Exiguobacterium acetylicum LAB3-2 is CCTCCNO: M 2026145, the depositary institution is China Center for Type Culture Collection, and the deposit date is January 23, 2026.

[0006] Secondly, this invention protects a method for preparing a composite microbial agent, the method comprising: Step 1: Take rhizosphere soil from onions and isolate and screen Trichoderma harzianum M3; Step 2: Take onion rhizosphere soil and isolate and screen salt- and alkali-tolerant bacterial strains; Step 3: The salt-tolerant and alkali-tolerant strains screened in Step 2, as well as the excellent disease-preventing and growth-promoting strains in the strain resource bank, are subjected to salt-lowering and alkali-lowering identification to screen out salt-lowering and alkali-lowering strains. Step 4: Construct a compound bacterial system by combining the salt-lowering and alkali-lowering strains screened in Step 3 with Trichoderma harzianum M3 screened in Step 1 and Bacillus belyss MP6, a superior disease-preventing and growth-promoting strain from the bacterial strain resource bank, and screen out the superior compound bacterial system formula. Step 5: Optimize the proportions of the superior compound microbial strain formulas selected in Step 4 to determine the optimal volume ratio of the superior compound microbial strain formulas.

[0007] Thirdly, the present invention protects the application of compound microbial agents, including any one of the following A1-A4: A1: Application in the control of onion basal rot; A2: Application in improving onion growth morphology indicators; A3: Application in improving the nutritional components of onion leaves; A4: Application in improving onion quality indicators; Among them, growth shape indicators include onion plant height, stem diameter, root length, and bulb; leaf nutrients include the content of IAA, POD, MDA, and chlorophyll in onion leaves; quality indicators include the content of soluble protein, soluble sugar, and flavonoids in onions.

[0008] Fourthly, the present invention protects the application of compound microbial agents, including any one of the following B1-B3: B1: Application in increasing microbial biomass nitrogen, microbial biomass phosphorus, and microbial biomass carbon in soil; B2: Application in reducing soluble salts and soil pH in soil; B3: Applications in increasing the number of microorganisms in soil.

[0009] Microorganisms include bacteria, actinomycetes, and fungi.

[0010] Beneficial effects: (1) This invention screens salt-alkali-reducing bacterial strains from onion rhizosphere soil and clarifies their characteristics. These strains are then combined with previously screened superior disease-preventing and growth-promoting bacteria in the laboratory to form a compound bacterial system. Comprehensive analysis and screening yields an excellent compound bacterial system, which is formed by mixing Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in a volume ratio of 9:9:3:7:5. This compound microbial agent can rapidly restore the number of beneficial soil bacteria, inhibit the proliferation of pathogenic microorganisms, improve soil nutrients, and effectively enhance the plant's resistance to stress and its growth and development capabilities, thereby significantly improving crop yield and quality.

[0011] (2) The compound microbial agent proposed in this invention has a significant effect on increasing the height of onion plants, stem diameter, root length, and bulb size. P <0.05), of which the plant height increased by 31.88%, the stem diameter increased by 50.68%, and the root length and bulb increased by 47.86% and 17.04%, respectively.

[0012] (3) The compound microbial agent proposed in this invention significantly increases the content of nutrients such as IAA, POD, MDA, and chlorophyll in onion leaves. P <0.05), of which IAA content was 120.27 nmol / g, an increase of 23.54 nmol / g compared with the control group; POD content was 9484 μg / g, an increase of 1923.33 μg / g; and chlorophyll content was 1.19 mg / g, an increase of 0.67 mg / g.

[0013] (4) The compound microbial agent proposed in this invention significantly increases the microbial biomass nitrogen, microbial biomass phosphorus, and microbial biomass carbon in the soil. P <0.05). Among them, the microbial biomass nitrogen content was 96.62 mg / kg, which was 68.93 mg / kg higher than the control group; the microbial biomass phosphorus content was 62.29 mg / kg, which was 18.25 mg / kg higher; and the microbial biomass carbon content was 512.45 mg / kg, which was 314.28 mg / kg higher than the control group.

[0014] (5) The compound microbial agent proposed in this invention reduced soil pH and soluble salt concentration. Soil pH decreased by 0.5 and soluble salt concentration decreased by 44.46 ms / cm.

[0015] (6) The compound microbial agent proposed in this invention significantly increases the number of bacteria, actinomycetes, and fungi in the soil. P <0.05), the number of fungi in the rhizosphere soil of onions was 9.5×10. 7 The CFU / g level was significantly higher than the control, with an increase of 30.52%. The bacterial count was 1.12 × 10⁻⁶. 14 The CFU / g level increased by 0.12 × 10⁻⁶ compared to the control CK. 14 CFU / g. The number of actinomycetes in the treatment group was 3.57 × 10⁻⁶. 11 The CFU / g count increased by 0.73 × 10⁻⁶ compared to the treatment group. 11 CFU / g.

[0016] (7) The compound microbial agent proposed in this invention significantly increases the content of soluble protein, soluble sugar and flavonoids in onions. P <0.05), the soluble sugar content increased from 31.09 mg / g to 29.88 mg / g, an increase of 1.21 mg / g, the soluble protein content increased by 0.048 mg / g, and the flavonoid content was 5.42 mg / g, an increase of 1.81 mg / g compared with the control group. 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] It should be noted that in the accompanying drawings and specific embodiments, *Trichoderma harzianum* M3, *Bacillus belye* MP6, *Bacillus halophilus* SYP202, *Staphylococcus squirrelii* LAB2, and *Microbacterium acetylacetonate* LAB3-2 appear as M3 (Trichoderma strain M3), MP6, SYP202, LAB2, and LAB3.2, respectively. The compound microbial agent is named "Tuweiwei No. 9".

[0019] Figure 1 Comparison results of 8 Trichoderma strains with target Fusarium oxysporum.

[0020] Figure 2 Morphological characteristics of Trichoderma: A, B: Colony morphology of Trichoderma cultured on PDA for 7 days; C, D, E, F: Spore morphology of Trichoderma.

[0021] Figure 3 Based on 8 Trichoderma strains TEF-1α , ITS and RPB2 The sequences were subjected to phylogenetic analysis using the maximum likelihood method.

[0022] Figure 4 Salt tolerance of superior strains.

[0023] Figure 5 Alkali tolerance of superior strains.

[0024] Figure 6 Salt reduction rate of superior strains.

[0025] Figure 7 Superior strains have a lower alkali reduction rate.

[0026] Figure 8 Interaction determination among some strains: A: MX38+LAB3.2; B: M3+MX38; C: MX38+MX50; D: LAB3.2+LAB2; E: M3+MP6.

[0027] Figure 9 The antibacterial effects of three compound bacterial strains on Fusarium oxysporum. A: CK; B: Antibacterial effect of compound bacterial strain A1 on Fusarium oxysporum; C: Antibacterial effect of compound bacterial strain A2 on Fusarium oxysporum; D: Antibacterial effect of compound bacterial strain A3 on Fusarium oxysporum.

[0028] Figure 10 Disease prevention and growth promotion characteristics of superior compound microbial strains. A: Inhibition rate; B: Salt reduction rate; C: Alkali reduction rate; D: Soluble inorganic phosphorus; E: Soluble organic phosphorus; F: Secreted IAA; G: Iron carrier production; H: Potassium solubilization; I: Nitrogen fixation; J: Comprehensive score index.

[0029] Figure 11 The strain is based on 16S rRNA, RpoB , gyrB Phylogenetic tree of gene sequences. A: Phylogenetic tree of LAB2 and LAB3.2 constructed based on 16S rRNA; B: Phylogenetic tree of LAB2 constructed based on 16S rRNA. RpoB Constructing a phylogenetic tree; C: LAB3.2 based gyrB Construct a phylogenetic tree.

[0030] Figure 12 Optimization of the optimal ratio of the compound bacterial strain.

[0031] Figure 13 Comparison of the control efficacy of compound microbial agent "Tuweiwei No. 9" against onion basal rot: CK: root drenching with pathogen; Treatment group D1: root drenching with pathogen + undiluted compound microbial agent "Tuweiwei No. 9"; D2: root drenching with pathogen + compound microbial agent "Tuweiwei No. 9" diluted 100 times; D3: root drenching with pathogen + compound microbial agent "Tuweiwei No. 9" diluted 1000 times.

[0032] Figure 14 Effects of compound microbial inoculant "Tuweiwei No. 9" on the nutrient composition of crop leaves: A: plant height; B: stem diameter; C: root length; D: corm; E: IAA content; F: POD content; G: SOD content; H: chlorophyll content; I: malondialdehyde content; CK: control group; F1: compound microbial inoculant treatment group.

[0033] Figure 15 Effects of compound microbial agent "Tuweiwei No. 9" on soil nutrients: A: available nitrogen; B: available phosphorus; C: available potassium; D: microbial biomass nitrogen; E: microbial biomass carbon; F: microbial biomass phosphorus; G: soluble salt concentration; H: pH; CK: control group; F1: compound microbial agent treatment group.

[0034] Figure 16 Effects of compound microbial agent "Tuweiwei No. 9" on the number of soil rhizosphere microorganisms: A: number of fungi; B: number of bacteria; C: number of actinomycetes; CK: control group; F1: compound microbial agent treatment group.

[0035] Figure 17 Effects of different treatments of compound microbial agent "Tuweiwei No. 9" on onion quality: A: soluble protein content; B: soluble sugar content; C: flavonoid content; CK: control group; F1: compound microbial agent treatment group. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0037] Example 1: Compound microbial inoculant This embodiment specifically provides a composite microbial agent, which is composed of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in a volume ratio of a:b:c:d:e; wherein a, b, c, d, and e are odd numbers from 3 to 9.

[0038] The concentrations of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in the inoculum were all greater than 10. 8 CFU / mL.

[0039] Preferably, the microbial agent is formed by compounding Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in a volume ratio of 9:9:3:7:5.

[0040] Among them, the preservation number of Trichoderma harzianum M3 is CCTCC NO: M 2026146; the preservation number of Bacillus belyssus MP6 is CCTCC NO: M 20242192; the preservation number of Bacillus halophilus SYP202 is CCTCC NO: M 2022358; the preservation number of Staphylococcus aureus LAB2 is CCTCC NO: M 2026144; and the preservation number of Microbacterium acetylacetonate LAB3-2 is CCTCC NO: M2026145.

[0041] Example 2: Preparation method of compound microbial inoculant This embodiment specifically provides a method for preparing the composite microbial agent in Example 1, which includes the following steps: Here, it is necessary to emphasize the sources of the materials involved in the steps as follows: Soil samples tested: collected from the rhizosphere of diseased onions at an onion planting base in Jingtai County, Gansu Province (37°10'28″N, 104°0'47″E).

[0042] The onion variety tested was Hongxi, produced by Henan Zhongxi Agricultural Technology Co., Ltd.

[0043] Test bacteria: MP6 Bacillus belye ( Bacillus velezensis ), SYP202 halophilic Bacillus ( B. halotolerans (This is a resource bank for disease prevention and growth promotion bacteria in the Economic Crop Laboratory of the Institute of Plant Protection, Gansu Academy of Agricultural Sciences.)

[0044] Test pathogen: Fusarium oxysporum ( Fusarium oxysporum The sample was previously isolated from the roots of onion plants infected with basal plate rot by our research group.

[0045] Test media: LB medium (g / L): tryptone 10.0, yeast extract 5.0, NaCl 10.0, agar powder 15.0~20.0, pH natural; PDA (Potato dextrose agar) medium (g / L): peeled potato 200.0, glucose 20.0, agar powder 15.0~20.0, pH natural; MRS medium (g / L): peptone 5.0, tryptone 10.0, MgSO4 0.58, MnSO4 0.25, glucose 20.0, K2HPO4·3H2O 2.0, beef extract 5.0, yeast extract 5.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, Tween-80 1.0, agar powder 15.0~20.0, pH natural; BCP medium (g / L): peptone 5.0, yeast extract 2.5, glucose 1.0, polysorbate 80 1.0, cysteine ​​0.1, bromocresol purple 0.06, agar powder 15.0~20.0, pH natural. PKO inorganic phosphorus medium; Monkina organic phosphorus medium; NFM nitrogen-free medium and potassium feldspar medium were prepared according to (Yang Hongguang, Wang Bing, Peng Baoliang, Hu Zhichao, Gao Xuemei. Current status of onion planting and research trend of mechanized harvesting at home and abroad [J]. China Vegetables, 2019, (9): 1-6). King's B medium was prepared according to the reference (Cheng Xinyu, Wang Jilian, Mai Riyangu, Yasheng, Li Mingyuan. Isolation and growth-promoting characteristics of IAA-producing strains in rhizosphere soil of *Salix babylonica* [J]. Acta Prataculturae Sinica, 2024, 33(4): 110-121.).

[0046] Test instruments: Microplate reader, Meigu CMax Plus; pH meter, pHS-37; Intelligent constant temperature and humidity incubator, Ningbo Jiangnan Instrument Factory CXZ model; PCR instrument, Bio-Rad T100; PCR agar gel electrophoresis system, Bio-Rad PowerPac Basic; Benchtop high-speed centrifuge, Ningbo Xinzhi HSC-3020L; Intelligent artificial climate chamber, Kete KT-G7; Conductivity meter, Leici DDS-307.

[0047] The preparation process of compound microbial agents is explained in detail below: Step 1: Isolation and screening of Trichoderma strain M3 1.1 Test Materials Onion basal rot disease-affected plants: collected in August 2024 from an onion planting base in Jingtai County, Gansu Province (37°10'28″N, 104°0'47″E).

[0048] Soil samples were collected from the rhizosphere of diseased onions from the same plantation as the diseased plants.

[0049] Onion variety tested: Green ( Allium cepa 'Gerui').

[0050] Test culture medium: Potato dextrose agar (PDA): 200 g peeled potatoes, 20 g glucose, 15-20 g agar powder, and distilled water to a final volume of 1000 mL, pH at natural.

[0051] Bengal Red Medium: 5.0 g peptone, 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 10 g glucose, 0.1 g chloramphenicol, 0.033 g Bengal Red, 15-20 g agar, distilled water to a final volume of 1000 mL, pH at rest.

[0052] CLA medium: 15-20 g agar, 1000 mL distilled water, add 3-4 sterile carnation leaves about 1 cm in length to each petri dish, pH at rest.

[0053] 1.2 Isolation and screening of Trichoderma strain M3 Take onion rhizosphere soil and prepare solutions with a concentration of 10. -2 10 -3 and 10 -4 Soil suspensions were prepared in triplicate for each concentration gradient, with 150 μL spread on each Bengal Red agar plate. After incubation at 25°C for 7 days, mycelial blocks were picked from the colony edge and transferred to fresh PDA medium for purification according to the Boamah method. Fusarium oxysporum, the pathogen of onion basal rot, was used. F. oxysporum Using the pathogen as the target, antagonistic Trichoderma strains were initially screened on PDA plates using a two-point method, with only pathogen inoculation as a control. Then, the antibacterial effect was determined by the four-point plate confrontation method. Each Trichoderma strain was replicated three times. After incubation at 25°C for 7 days, the radius of the pathogen was measured and the inhibition rate was calculated.

[0054] Inhibition rate = (Coronavirus colony radius in control plate - Colony radius in treatment group) / (Coronavirus colony radius in control group - Colony block radius) × 100% like Figure 1 As shown in Table 1, eight strains exhibited antagonistic effects against *Fusarium oxysporum*, the pathogen causing onion basal rot, with relative inhibition rates ranging from 66.86% to 77.69%. Among them, strain M3 antagonized *Fusarium oxysporum*, which had the smallest colony radius (only 0.95 cm) and an inhibition rate of 77.69%. Except for M5 and M7, the inhibition rates of the other strains were all above 70%. Strain M7 had the lowest inhibition rate at 66.86%.

[0055] Table 1. Inhibitory effects of eight Trichoderma strains on Fusarium oxysporum. ; 1.3 Identification of Trichoderma 1.3.1 Morphological identification The Trichoderma strains selected in step 1.2 were inoculated onto PDA plates and cultured at a constant temperature of 25°C. The colony morphology and pigment production of each strain were observed and recorded daily. The hyphae and conidia morphology were observed under a Scope A1 40x optical microscope.

[0056] 1.3.2 Molecular biological identification Mycelia of the strain activated for 7 days on PDA plates were collected, dried, and ground into powder. Genomic DNA was extracted from *Trichoderma* according to the procedures outlined in the Fungal DNA Kit (OMEGA). Primers TEF1-F / TEF1-R (5'-CCGTGAYTTCATCAAGAA-3' / 5'-TTGGCAGTGTCCATCTTGTTG-3'), RPB2-7cr / RPB2-5f2 (5'-CCCATRGCTTGYTTRCCCAT-3' / 5'-GGWGAYCAGAAGAAGGC-3'), and ITS1 / ITS4 (5'-TCCGTAGGTGAACCTGCGG-3' / 5'-TCCTCCGCTTATTGATTGC-3') were used to target the mycelia of the strain. TEF-1α , RPB2 , ITS Gene fragments were amplified. The PCR reaction system consisted of 25 µL: 1 µL DNA template, 1 µL each of the forward and reverse primers, 12.5 µL Taq mix, and 9.5 µL ddH2O. ITS PCR reaction program for the sequence: 94 °C pre-denaturation for 3 min, 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 45 s, 30 cycles, 72 °C extension for 5 min. RPB2 PCR reaction program for the sequence: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 90 s, and further extension at 72℃ for 10 min, for a total of 35 cycles; TEF-1α The PCR reaction program for the sequence was as follows: pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 30 s, and further extension at 72℃ for 10 min, for a total of 35 cycles. After the PCR products were verified by 1% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were assembled, impurities were removed, and BLAST comparison was performed using NCBI. Sequences with similarity greater than 99% and closely related genera were downloaded, and the strains were sorted according to their genus using SnapGenge software. TEF-1α , RPB2 , ITSThe sequences were concatenated into a dataset, and a phylogenetic tree was constructed using the adjacency join method in MEGA 11.0 with 1000 repetitions. Finally, each sequence was submitted to NCBI to obtain a GenBank accession number.

[0057] All strains grew rapidly on PDA medium. After 3 days of culture, white, fluffy mycelia appeared and covered the entire plate. Subsequently, the mycelia gradually turned yellowish-green, and after 7 days, the mycelia turned dark green. The sporulation clusters were distributed in a ring and expanded outward. Figure 2 -A, B). No obvious odor. Under a 40x microscope, the main branches of the conidiophores can be observed to be tree-like, forming phialides or secondary branches at the apex. Figure 2 -C), conidia are spherical to short obovate, spores are small (2.2~2.6) μm × (2.5~2.8) μm ( Figure 2 -D, E, F). It is related to Trichoderma harzianum ( Trichoderma harzianum Their morphological characteristics are relatively consistent.

[0058] 1.3.3 Molecular biological identification of Trichoderma strains The ITS, RPB2, and TEF-1α sequences of strains M1–M8 were compared using BLAST in NCBI. The results showed that all eight strains were similar to *Trichoderma harzianum*. Trichoderma harzianum The highest similarity was 3. Phylogenetic tree construction revealed ( Figure 3 ), strains M1~M8 and T. harzianum The eight strains clustered together, with a genetic distance of 0.01 and a self-spreading support rate of 100%. They were submitted to GenBank and obtained accession numbers, which were consistent with the preliminary morphological identification results. Therefore, the taxonomic status of these eight strains was determined to be Trichoderma harzianum.

[0059] Step 2: Take onion rhizosphere soil and isolate and screen salt- and alkali-tolerant bacterial strains; 2.1 Isolation and Determination of Salt-Alkali Tolerance Characteristics of Salt-Alkali-Decreasing Strains Take onion rhizosphere soil and prepare solutions with a concentration of 10. -2 10 -3 10 -4 10 -5 Soil suspensions were prepared in triplicate for each concentration gradient. 100 μL of each MRS solid medium and BCP medium plate was spread and incubated at 37 ℃ for 2–3 days. Colonies showing a calcium dissolution zone or turning the medium yellow were picked, streaked, and incubated at 30 ℃. The streaking was repeated, and the culture was subcultured three times to obtain single colonies. The activated inoculum was then inoculated at a 1% inoculum into LB liquid medium with a 10% NaCl concentration and a pH of 10, respectively, and incubated at 30 ℃ for 36 h. The colonies were then measured. OD 600 The values ​​were used to screen out strains with strong salt and alkali tolerance.

[0060] A total of 20 strains of salt-tolerant bacteria were isolated, and their salt-alkali tolerance characteristics are as follows: 2.1.1 Salt tolerance of the strain like Figure 4 As shown, at a salt (NaCl) concentration of 10%, the OD values ​​of strains Y1-2.1, Y2-9, Y2-7, and Y1-1 were... 600 The value was as low as 0.2, indicating that a 10% salt concentration completely inhibited the growth of strains Y1-2.1, Y2-9, Y2-7, and Y1-1. Meanwhile, the OD values ​​for strains Y2-10, LAB3.2, LAB2, and Y1-6 were significantly lower. 600 The value is still greater than 1. Therefore, strains Y2-10, LAB3.2, LAB2, and Y1-6, which grow well at a 10% salt concentration, were identified as salt-tolerant strains.

[0061] 2.1.2 Alkali resistance like Figure 5 As shown, when the pH of the culture medium is 10, except for Y2-8, R3, Y2-2.1, LAB3.2, LAB2, and Y1-2.2, the OD values ​​of the remaining strains are... 600 The values ​​were all less than 1.5, but the OD600 values ​​of three strains, Y2-8, LAB3.2, and LAB2, all reached above 2.5, which was significantly higher. P The concentration of alkali resistant bacteria (<0.05) was higher than that of other strains. Therefore, Y2-8, LAB3.2, and LAB2 were identified as alkali-resistant strains.

[0062] Step 3: The salt-tolerant and alkali-tolerant strains screened in Step 2, as well as the excellent disease-preventing and growth-promoting strains in the strain resource bank, are subjected to salt-lowering and alkali-lowering identification to screen out salt-lowering and alkali-lowering strains. 3.1 Determination of salt reduction rate In LB liquid medium with a NaCl concentration of 1.5% and a pH of 9, strains with strong salt and alkali tolerance selected during screening and superior disease-preventing and growth-promoting strains from the laboratory disease-preventing and growth-promoting bacterial resource bank were inoculated at a 1% inoculum rate. The cultures were then incubated with shaking at 30℃. After 48 h and 72 h, appropriate amounts of fermentation samples were taken, and the salt concentration of the culture medium before and after fermentation was determined according to the nitrate titration method of Yan Hong et al. The formula for calculating the salt reduction rate is as follows: Salt reduction efficiency of the strain = (salt concentration of liquid culture medium before fermentation - salt concentration of fermentation broth after fermentation) / salt concentration of liquid culture medium before fermentation × 100% like Figure 6 As shown, after culturing for 48 h at a NaCl concentration of 1.5% and a pH of 9.5, strains LAB2, SYP202, and MFL22 showed significantly better salt reduction effects than the other strains. P<0.05), the salt reduction rates were 13.64%, 13.64%, and 9.96%, respectively; after 72 h of culture, SYP202, LAB2, and MFL22 were still significantly ( P The salt degradation rates of LAB2, SYP202, MX50, and MFL22 were higher than those of the other strains (<0.05), with reduction rates of 19.26%, 17.75%, and 10.61%, respectively. The newly added MX50 showed a higher salt degradation rate of 11.26%. Therefore, LAB2, SYP202, MX50, and MFL22 were identified as salt-degrading strains.

[0063] 3.2 Determination of Alkali Reduction Rate The strain was cultured and sampled according to the method for determining the salt reduction rate of the strain in section 3.1, and the pH value of the culture medium before and after fermentation was measured using a pH meter. The formula for calculating the salt reduction rate is as follows: Alkalinity reduction efficiency of the strain = (pH of liquid culture medium before fermentation − pH of fermentation broth after fermentation) / pH of liquid culture medium before fermentation × 100% like Figure 7 As shown, after culturing for 48 h at a NaCl concentration of 1.5% and a pH of 9.5, strains LAB2, LAB3.2, SYP202, MX38, and MFL379 exhibited higher alkali reduction rates than the other strains, at 10.62%. At 72 h of culturing, LAB3.2 showed the highest alkali reduction rate, at 17.6%, compared to LAB2. LAB3.2, LAB2, and MX38 demonstrated significantly better alkali reduction effects than the other strains. P <0.05), with alkali reduction rates of 17.6%, 14.44%, and 12.3%, respectively. Therefore, LAB3.2, LAB2, and MX38 were identified as alkali-reducing strains.

[0064] Step 4: Construct a compound bacterial system by combining the salt-lowering and alkali-lowering strains screened in Step 3 with Trichoderma harzianum M3 screened in Step 1 and Bacillus belyss MP6, an excellent anti-growth and growth-promoting strain from the bacterial resource bank, and screen out an excellent compound bacterial system formula. 4.1 Construction and Screening of Compound Microbial Lines First, the salt-lowering strains obtained in step 3 were compared with the superior disease-preventing and growth-promoting strains M3 and MP6 from the strain resource bank using the pairwise streak method and the two-point method to determine whether there was any antagonistic effect. Next, strains with no antagonistic effect and different functions were selected and combined to construct different compound bacterial strain formulations. Then, 1 mL of activated strains was taken according to each formulation. OD 600 Bacterial suspensions with a pH ≥ 0.5 were inoculated into Erlenmeyer flasks at a volume ratio of 1:1 (100 mL / 250 mL). The flasks were incubated at 30 °C and 180 r / min for 48 h. The inhibition rate, pH, phosphorus solubility, nitrogen fixation, potassium solubility, IAA secretion, and siderophore secretion capacity of each composite strain were measured, with three replicates per strain. Finally, a comprehensive Topsis analysis was conducted to determine the optimal composite strains.

[0065] like Figure 8 As shown in A, 8B, and 8C, strain MX38 is clearly broken at the cross-section with LAB3.2 and MX50, indicating antagonistic effects. Therefore, they should not be used together when constructing a compound bacterial system. The other five strains grow well in pairs at the cross-section without breaking, indicating no antagonistic effects between them, and they can be co-cultured when constructing a compound bacterial system.

[0066] 4.2 Disease prevention, salt and alkali reduction and growth promotion characteristics of compound microbial strains Three composite bacterial strains were constructed: A1 (M3+MP6+SYP202+LAB2 strain combination), A2 (M3+MP6+SYP202+LAB3-2 strain combination), and A3 (M3+MP6+SYP202+LAB3-2+LAB2 strain combination). Their disease resistance characteristics were determined. Figure 9 , Figure 10 A), the inhibitory rates of compound bacterial strains A1 and A2 against the tested fungus Fusarium oxysporum were 93.36% and 90.33%, respectively, while compound bacterial strain A3 showed significantly higher inhibition rates than A1 and A2, reaching 95.15% against Fusarium oxysporum. Its salt-alkali reduction properties were determined to be ( Figure 10 (B, 10C) Among the compound bacterial strains, A2 had the lowest salt reduction rate at 14.07%, while A1 was stronger than A2 with a salt reduction rate of 16.24%, but its alkali reduction rate was weaker than A2 at only 7.21%. Compound bacterial strain A3 exhibited stronger salt and alkali reduction characteristics than both A1 and A2, with a salt reduction rate of 18.83% and an alkali reduction rate of 15.16%. Measurements of its growth-promoting properties revealed ( Figure 10 Among the bacterial strains, the A2 strain had the highest organic phosphorus solubility (325.4 μg / mL); the A1 strain had the highest nitrogen fixation (0.394 g / L); and the A3 strain had the highest inorganic phosphorus solubility, potassium solubility, siderophore production, and IAA secretion, with 2813 μg / mL of inorganic phosphorus solubility, 99.13 mg / L of potassium solubility, and siderophore production. su The value was 39.54, and the secreted IAA amount was 10.66 mg / L. Topsis comprehensive evaluation found that the A3 statistical value ranked first, at 0.872, which was significantly higher than the other two groups. Therefore, it was determined to be an excellent compound bacterial strain formula.

[0067] 4.3 Identification of Superior Strains The superior strains LAB3.2 and LAB2 involved in the optimal composite bacterial strain were extracted using a DNA extraction kit (Bacterial DNA Kit, Omega Bio-tek) according to the manufacturer's instructions. PCR amplification was performed using universal primers for the 16S rRNA gene (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′). The PCR reaction system (25 μL) consisted of 1 μL each of forward and reverse primers, 1 μL of DNA template, 12.5 μL of Taq mix, and 9.5 μL of ddH2O. The PCR reaction program was as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for a total of 35 cycles; final extension at 72 ℃ for 10 min.

[0068] LAB2 utilizes RpoB PCR amplification was performed using (1418f*: 5′-CAATTCATGGACCAAGC-3′; 3554r: 5′-CCGTCCCAAGTCATGAAAC-3′). The LAB2 PCR reaction system (25 μL) consisted of 1 μL each of forward and reverse primers, 1 μL of DNA template, 12.5 μL of Taq mix, and 9.5 μL of ddH2O. The LAB2 PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 52℃ annealing for 60 s, 72℃ extension for 90 s, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0069] LAB3.2 Utilization gyrBPCR amplification was performed using primers (F: 5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′; R: 5′-AGCAGGGTACGGATGTGCGAGCCTCNACRTCNGCRTCNGTCAT-3′). The LAB3.2 PCR reaction system (25 μL) consisted of 1 μL each of forward and reverse primers, 1 μL of DNA template, 12.5 μL of Taq mix, and 9.5 μL of ddH2O. The LAB3.2 PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min. After the PCR products were verified by 1% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Finally, the sequencing results were cleaned and assembled, and BLAST homology comparison was performed in NCBI. Similar sequences and exogenous gene sequences were selected, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 11.0 software. The reliability of the tree was verified by Bootstrap 1000.

[0070] like Figure 11 As shown in A and C, LAB2 has a genetic distance of less than 0.1 from *Staphylococcus sciuri* DSM20345 (AY688097) and NCTC12103 (LS483305), with a self-expansion support of 100%. Submitted to GenBank, it received accession numbers PX468769 and PX962284. Therefore, the taxonomic position of LAB2 is confirmed as *Staphylococcus sciuri*. Figure 11 As shown in -B, LAB3.2 has a genetic distance of 0 from both *Exiguobacterium acetylicum* DSM20416 (MN587893) and *Exiguobacterium acetylicum* DSM20416 (DQ019163), with a self-expansion support of 100. Submitted to GenBank, it received accession number PX468770, confirming LAB3.2's taxonomic position as *Exiguobacterium acetylicum*.

[0071] Step 5: Optimize the proportions of the superior compound microbial strain formula selected in Step 4.

[0072] 5.1 Optimization of bacterial strain ratio Set L16 (4) 5Orthogonal experiments (Table 2) were conducted by activating each strain on LB plates and then inoculating them into liquid LB medium (50 mL / 150 mL Erlenmeyer flasks). The cultures were incubated at 30 °C and 180 r / min for 36 h to achieve a concentration of 10⁻⁶. 8 After reaching CFU / mL, the culture medium was inoculated again according to the treatment ratios in Table 1, and the mixture was fermented for 36 h before its concentration was measured. OD 600 .

[0073] Table 2. Experimental Design for Optimal Volume Ratio of Each Strain ; like Figure 12 As shown, the OD600 value of treatment T16 was significantly higher (P < 0.05), reaching 2.54. Comparing with Table 1, it was found that the inoculation amounts of strains M3, LAB3.2, LAB2, MP6, and SYP202 involved in T16 were 9%, 9%, 3%, 7%, and 5%, respectively. Therefore, the optimal ratio of the optimal compound bacterial system A3 is M3:LAB3.2:LAB2:MP6:SYP202 = 9:9:3:7:5.

[0074] Example 3: Application of compound microbial agents This embodiment mainly provides the disease prevention and growth promotion effects of the compound microbial agent in Example 1 on onion basal rot, the changes in the nutrient composition of crop leaves and rhizosphere soil, and its impact on onion quality.

[0075] 1. Materials and Methods 1.1 Materials Test pathogen: Fusarium oxysporum ( F. oxysporum ).

[0076] The tested plant variety was onion (Hongxi, Henan Zhongxi Agricultural Technology Co., Ltd.).

[0077] The tested compound microbial inoculant was Tuweiwei No. 9.

[0078] 1.2 Determination of the efficacy of compound microbial inoculants against crop root rot diseases Onion seedlings approximately 5 cm in length and with uniform bulb size were selected and transplanted into flowerpots, one seedling per pot. Different treatments were set up: a control group (CK) treated with pathogen drenching; and four treatment groups: D1 (pathogen drenching + stock solution of compound bacterial culture), D2 (pathogen drenching + 100-fold dilution of compound bacterial culture), and D3 (pathogen drenching + 1000-fold dilution of compound bacterial culture), with each treatment replicated five times. Specifically, on day 20 after transplanting, CK, D1, D2, and D3 were each inoculated with 30 mL of pathogen spore suspension (spore concentration 2×10⁻⁶). 6CFU / mL). After 7 days, the control (CK) was drenched in LB broth (30 mL / plant), and on days 1, 2, and 3, 4 × 10⁻⁶ CFU / mL was used. 8 The root system was irrigated with a CFU / mL compound bacterial fermentation broth (30 mL / plant). After 15 days, the disease incidence was recorded, and the disease index was calculated to determine the control efficacy.

[0079] Disease index = ∑(Number of diseased plants at each level × Disease level value) / Total number of plants surveyed × Highest level value × 100 Prevention and control efficacy (%) = (Incidence rate in control group - Incidence rate in treatment group) / Incidence rate in control group × 100 1.3 Growth-promoting effect of compound microbial inoculants and their impact on soil properties Onion seedlings approximately 5 cm in length and with similar bulb size were selected and transplanted into flowerpots, one seedling per pot. Two treatments were established: a control group (CK) and a treatment group (F1) (using A3 compound bacterial culture stock solution), with each treatment replicated three times. Specifically, the control group was drenched in the fermentation culture solution (30 mL / plant), while the treatment group was drenched in the A3 compound bacterial culture solution (30 mL / plant, concentration 4 × 10⁻⁶). 8(CFU / mL). After 15 days, plant characteristics such as plant height, stem diameter, root length, corm, IAA, POD, SOD, and MDA were measured in both the control and treatment groups. Rhizosphere soil characteristics such as available nitrogen, available phosphorus, available potassium, microbial biomass carbon, microbial biomass nitrogen, microbial biomass phosphorus, soil pH, soluble salt concentration, and soil microbial quantity were also measured. Quality characteristics such as soluble sugar content and flavonoid content were also measured. IAA content was determined using the Plant IAA ELISA Kit; POD content was determined using the Peroxidase (POD) Activity Assay Kit (boxbio); SOD content was determined using the Superoxide Dismutase (SOD) Activity Assay Kit (boxbio); MDA content was determined using the Malnodialdehyde (MDA) Content Assay Kit (boxbio); SPAD content was determined using the Plant Chlorophy II Content Assay Kit (boxbio); available nitrogen was determined using the alkaline hydrolysis diffusion method; available phosphorus was determined using the sodium bicarbonate extraction molybdenum antimony colorimetric method; available potassium was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES); soluble sugar content was determined using the Plant Soluble Sugar Content Assay Kit (boxbio); flavonoid content was determined using the Plant Flavonoids Content Assay Kit (boxbio); protein content was determined using the Bradford Protein Content Assay Kit (boxbio); and soil microbial abundance was determined using the plate count method.

[0080] 1.4 Data Analysis The raw data were processed using Excel 2024, and the variance analysis was performed using SPSS 27.0.

[0081] 2 Results and Analysis 2.1 The efficacy of compound microbial agents in preventing onion basal rot like Figure 13As shown in Tables A and B, the optimal compound microbial agent "Tuweiwei No. 9" showed good control efficacy against onion basal rot. As shown in Table 5-1, the disease index of the control group CK (pathogen root irrigation) was 86.67, the disease index of the treatment groups D1 (pathogen root irrigation + undiluted compound microbial agent "Tuweiwei No. 9") was 46.66, the disease index of D2 (pathogen root irrigation + compound microbial agent "Tuweiwei No. 9" diluted 100 times) was 68.87, and the disease index of D3 (pathogen root irrigation + compound microbial agent "Tuweiwei No. 9" diluted 1000 times) was 73.33, with control efficiencies of 73.3%, 53.3%, and 40%, respectively.

[0082] Table 5-1 Efficacy of compound microbial inoculant "Tuweiwei No. 9" against onion basal rot disease ; 2.2 Growth-promoting effect of compound microbial agents 2.2.1 Effects on nutrient composition of crop leaves like Figure 14 As shown in A, B, C, and D, the compound microbial inoculant "Tuweiwei No. 9" significantly improved onion plant height, stem diameter, root length, and bulb size. P <0.05), among which the plant height increased by 31.88%, the stem diameter increased by 50.68%, and the root length and bulb increased by 47.86% and 17.04%, respectively. Figure 14 According to E, F, G, H, and I, after applying Tuweiwei No. 9, the content of nutrients such as IAA, POD, MDA, and chlorophyll in onion leaves increased significantly. P <0.05), of which IAA content was 120.27 nmol / g, an increase of 23.54 nmol / g compared to the control group; POD content was 9484 μg / g, an increase of 1923.33 μg / g; chlorophyll content was 1.19 mg / g, an increase of 0.67 mg / g; SOD content was 30.42 μg / g, a significant decrease of 12.6 μg / g; and MDA content decreased by 1.78 μg / g.

[0083] 2.2.2 Impact on Soil Nutrients like Figure 15 As shown in A, B, and C, after applying the compound microbial agent "Tuweiwei No. 9", the available nitrogen, available phosphorus, and available potassium in the soil all increased significantly. P <0.05). The available nitrogen content was 909.17 mg / kg, an increase of 123.34 mg / kg compared to the control group; the available phosphorus content was 577.46 mg / kg, an increase of 131.35 mg / kg; while the available potassium content was 42.01 mg / kg, an increase of only 10.04 mg / kg compared to the control group. Figure 15As shown in -D, E, and F, after applying Tuweiwei No. 9, the microbial biomass nitrogen, microbial biomass phosphorus, and microbial biomass carbon all increased significantly. P <0.05). The microbial biomass nitrogen content was 96.62 mg / kg, an increase of 68.93 mg / kg compared to the control group; the microbial biomass phosphorus content was 62.29 mg / kg, an increase of 18.25 mg / kg; and the microbial biomass carbon content was 512.45 mg / kg, an increase of 314.28 mg / kg compared to the control group. For example... Figure 15 According to -G and H, the soil pH decreased by 0.5 and the soluble salt concentration decreased by 44.46 ms / cm.

[0084] 2.2.3 Impact on soil microbial abundance like Figure 16 -As shown in A, B, and C, after applying the optimal compound microbial agent "Tuweiwei No. 9", the number of bacteria, actinomycetes, and fungi all increased significantly. P <0.05), the number of fungi in the rhizosphere soil of onions was 9.5×10. 7 The CFU / g level was significantly higher than the control, with an increase of 30.52%. The bacterial count was 1.12 × 10⁻⁶. 14 The CFU / g level increased by 0.12 × 10⁻⁶ compared to the control CK. 14 CFU / g. The number of actinomycetes in the treatment group was 3.57 × 10⁻⁶. 11 The CFU / g count increased by 0.73 × 10⁻⁶ compared to the treatment group. 11 CFU / g.

[0085] 2.2.4 Impact on Onion Quality like Figure 17 As shown in Figures A, B, and C, after applying the compound microbial agent "Tuweiwei No. 9," the content of soluble protein, soluble sugar, and flavonoids in onions all significantly increased. P <0.05), the soluble sugar content increased from 31.09 mg / g to 29.88 mg / g, an increase of 1.21 mg / g, the soluble protein content increased by 0.048 mg / g, and the flavonoid content was 5.42 mg / g, an increase of 1.81 mg / g compared with the control group.

[0086] In summary, this compound microbial agent can rapidly restore the number of beneficial soil bacteria, inhibit the proliferation of pathogenic microorganisms, improve soil nutrients, and effectively enhance the stress resistance and growth and development capacity of plants, thereby significantly improving crop yield and quality.

[0087] The above-described embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, The inoculum consists of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonum LAB3-2; Among them, the preservation number of Trichoderma harzianum M3 is CCTCC NO: M 2026146; The preservation number of Bacillus belyssus MP6 is CCTCC NO: M 20242192; The accession number for Bacillus halophilus SYP202 is CCTCC NO: M 2022358; The accession number for Staphylococcus aureus LAB2 is CCTCC NO: M 2026144; The preservation number of Microbacterium acetylcholine LAB3-2 is CCTCC NO: M 2026145.

2. The compound microbial agent according to claim 1, characterized in that, The microbial agent is composed of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in a volume ratio of a:b:c:d:e; wherein a, b, c, d, and e are odd numbers from 3 to 9. The concentrations of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in the inoculum were all greater than 10. 8 CFU / mL.

3. The compound microbial agent according to claim 2, characterized in that, The bacterial agent is composed of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2 in a volume ratio of 9:9:3:7:

5.

4. A method for preparing a composite microbial agent according to any one of claims 1-3, characterized in that, The method includes: Step 1: Take rhizosphere soil from onions and isolate and screen Trichoderma harzianum M3; Step 2: Take rhizosphere soil from onions and isolate and screen salt- and alkali-tolerant bacterial strains; Step 3: The salt-tolerant and alkali-tolerant strains screened in Step 2, as well as the excellent disease-preventing and growth-promoting strains in the strain resource bank, are subjected to salt-lowering and alkali-lowering identification to screen out salt-lowering and alkali-lowering strains. Step 4: Construct a compound bacterial system by combining the salt-lowering and alkali-lowering strains screened in Step 3 with Trichoderma harzianum M3 screened in Step 1 and Bacillus belyss MP6, a superior disease-preventing and growth-promoting strain from the bacterial strain resource bank, and screen out the superior compound bacterial system formula. Step 5: Optimize the proportions of the superior compound microbial strain formulas selected in Step 4 to determine the optimal volume ratio of the superior compound microbial strain formulas.

5. The method for preparing a composite microbial agent according to claim 4, characterized in that, Step 2 involves isolating and screening salt- and alkali-tolerant strains, including: Step 201: Take onion rhizosphere soil and prepare solutions with a concentration of 10... -2 10 -3 10 -4 10 -5 CFU / mL soil suspension, each concentration gradient was repeated 3 times, and 100 μL was spread on each MRS solid medium and BCP medium plate and incubated at 37 ℃ for 2-3 days; Step 202: Select colonies that show a calcium dissolution zone or turn the culture medium yellow, streak them on a plate, and incubate at 30 °C; repeat the streaking process, subculture 3 times, and obtain single colonies; Step 203: Inoculate the activated bacterial strain at a rate of 1% into LB liquid medium with a NaCl concentration of 10% and a pH of 10, and incubate at 30 ℃ for 36 h. Measure the OD value. 600 Value, based on OD 600 The strains with salt and alkali tolerance were selected through screening.

6. The method for preparing a composite microbial agent according to claim 5, characterized in that, The salt- and alkali-tolerant strains screened in step 203 include Staphylococcus aureus LAB2 and Microbacterium acetylacetonate LAB3-2.

7. The method for preparing a composite microbial agent according to claim 6, characterized in that, Step 3, screening for salt-lowering and alkali-lowering strains, includes: Step 301: In LB liquid medium with a NaCl concentration of 1.5% and a pH of 9, inoculate the salt-tolerant and alkali-tolerant strains selected in Step 2 and the superior disease-preventing and growth-promoting strains from the strain resource bank at a 1% inoculation rate, and culture with shaking at 30 ℃ and 180 r / min; among which, the superior disease-preventing and growth-promoting strains from the strain resource bank include MX38, MLB36, MN47, MFL379, MFL22, MX66, MX50, Bacillus halophilus SYP202, MX69, and MX27; Step 302: After 48 h and 72 h, take appropriate amounts of fermentation samples and determine the salt concentration of the culture medium before and after fermentation by nitrate titration. Screen strains with significant salt-reducing effects. The salt-reducing strains screened are: Staphylococcus aureus LAB2, Bacillus halophilus SYP202, MX50, and MFL22. Step 303: In LB liquid medium with NaCl concentration of 1.5% and pH value of 9, inoculate the salt-tolerant and alkali-tolerant strains obtained in step 2 and the excellent disease-preventing and growth-promoting strains in the strain resource bank at an inoculation rate of 1%, and culture them with shaking at 30 ℃ and 180 r / min. Step 304: After 48 h and 72 h, take appropriate amounts of fermentation samples and use a pH meter to measure the pH value of the culture medium before and after fermentation. Screen strains with significant alkali-reducing effects. The screened alkali-reducing strains are: Acetylmicrobacterium LAB3-2, Staphylococcus aureus LAB2, and MX38.

8. The method for preparing a composite microbial agent according to claim 7, characterized in that, The superior compound bacterial strain formula selected in step 4 consists of Trichoderma harzianum M3, Bacillus belye MP6, Bacillus halophilus SYP202, Staphylococcus aureus LAB2, and Microbacterium acetylacetonate LAB3-2.

9. The method for preparing a composite microbial agent according to claim 8, characterized in that, The optimal volume ratio of the superior compound microbial strain formula determined in step 5 is 9:7:5:9:

3.

10. The application of the compound microbial agent according to any one of claims 1-3, characterized in that, Includes any one of the following A1-A4: A1: Application in the control of onion basal rot; A2: Application in improving onion growth morphology indicators; A3: Application in improving the nutritional content of onion leaves; A4: Application in improving onion quality indicators.

11. The application of the compound microbial agent according to any one of claims 1-3, characterized in that, Includes any one of the following B1-B3: B1: Application in increasing microbial biomass nitrogen, microbial biomass phosphorus, and microbial biomass carbon in soil; B2: Application in reducing soluble salts and soil pH in soil; B3: Applications in increasing the number of microorganisms in soil.

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