Bacillus velezensis lb17, k87, mp6 and complex microbial inoculant, preparation method and application

Abstract

The application provides bacillus velezensis LB17, K87, MP6 and a compound microbial inoculant thereof, a preparation method and application, and belongs to the technical field of biocontrol bacteria.The application collects rhizosphere and rhizosphere soil of characteristic crops such as highland barley, lily, tomato and pepper in the Gansu-Qinghai region, and obtains 305 pure cultures through separation, obtains three strains of bacillus velezensis LB17, K87 and MP6 through multiple screening, and finally obtains a compound fermentation microbial inoculant, i.e., tuoweiwei No.7, which is fermented by the three strains of bacillus velezensis LB17, K87 and MP6 through optimization of inoculation amount, components of culture medium, ratio and fermentation conditions.Tuoweiwei No.7 can effectively prevent plant diseases such as pathogenic microorganisms, root rot and fusarium wilt, and can promote plant growth and improve the stress resistance of plants.The compound microbial inoculant provided by the application provides a strong guarantee for the prevention and control of root rot diseases of special habitat crops in the Gansu-Qinghai region.

Description

Technical Field

[0001] This invention relates to the field of biocontrol technology, and in particular to Bacillus belye LB17, K87, MP6 and their compound agents, preparation methods and applications. Background Technology

[0002] The Gansu and Qinghai provinces, with their high altitude, abundant sunshine, low average annual temperature, and large temperature differences, are ideal for the cultivation and growth of highland barley, lilies, and various highland summer vegetables. Highland barley, which thrives in cool, cold-hardy conditions, is distributed in Gannan Tibetan Autonomous Prefecture of Gansu Province, the area surrounding Xining City in Qinghai Province, Haibei Prefecture, Hainan Prefecture, Haixi Prefecture, Huangnan Prefecture, Yushu Prefecture, and Guoluo Prefecture. Rich in nutrients, it enhances immunity and helps prevent cardiovascular diseases, making it a staple food crop for the local Tibetan people. Lanzhou lilies prefer cool, humid environments and are mainly distributed in Lanzhou City and surrounding areas. With a sweet and crisp taste, they reduce eye fatigue, provide antioxidants, regulate the immune system, and stabilize blood sugar, making them a well-known health food. Chili peppers and tomatoes are the main highland summer vegetables in Gansu Province, contributing over 60% to farmers' income and serving as pillar industries in the local rural areas. However, due to the mountainous terrain in the Gansu-Qinghai region, the agricultural area accounts for a small proportion, with cultivated areas accounting for only 12.62% and 2.84% of the province's total area, respectively. Continuous cropping is a serious problem, and soil-borne diseases such as root rot are prone to occur.

[0003] Root rot diseases are widespread in highland barley. Studies have found that four common types of root rot in highland barley in the Gansu and Qinghai regions are Fusarium root rot, common root rot, Microdochium root rot, and Clonostachys rosea root rot. Fusarium root rot is the most widespread and has a diverse range of pathogens, with an average incidence rate of around 20%. Its dominant pathogens are *Fusarium avenaceum* and *Fusarium equiseti*. Common root rot has an average incidence rate of 5%–15%, with the dominant pathogen being *Bipolaris sorokiniana*. Microdochium root rot and Clonostachys rosea root rot are caused by *Microdochium bolleyi* and *Clonostachys rosea*, respectively, and occur sporadically only in Qinghai, but are severely damaging with a 100% mortality rate. Root rot diseases in lilies are serious and insidious, including two common types: wilt and root rot. Fusarium wilt is widespread and has a high incidence rate, reaching over 70% in some areas. Fusarium oxysporum is often the dominant pathogenic group and is highly pathogenic. The incidence of lily root rot is around 25%, with Fusarium solanum-specific lily-specific being the dominant pathogen. The incidence of root rot in vegetables such as peppers and tomatoes is generally between 20% and 30%. Severe root rot infection in peppers can reduce yield by more than 60%, while tomato root rot infection generally results in yield reductions of 60% to 90%, or even total crop failure. The dominant pathogens in these cases are Fusarium oxysporum and Fusarium solanum.

[0004] Biocontrol bacteria refer to a class of beneficial microorganisms that can kill or reduce the number of pathogenic organisms to control the occurrence and development of plant diseases. Essentially, they utilize inter- or intra-species antimicrobial, competitive, hyperparasitic, or lytic interactions, or the active metabolites produced by beneficial microorganisms, to inhibit the survival and activity of certain pathogenic microorganisms. The main types of biocontrol bacteria include bacteria, fungi, and actinomycetes. Bacteria are characterized by their diversity, high reproductive capacity, complex metabolic activities and numerous products, diverse modes of action against pathogens, short life cycles, and ease of artificial cultivation. Biocontrol bacteria and their metabolites play a crucial role in both naturally occurring biological control and human-application biological control activities.

[0005] Bacillius sp. possesses advantages such as rapid reproduction and metabolism, strong vitality, large cell size, production of highly resistant spores, long shelf life, and ease of use, making it an ideal biocontrol microorganism widely used in agricultural production. Among them, Bacillus velezensis, a new species in the genus Bacillus, promotes plant growth and inhibits plant pathogens in agriculture, showing broad application prospects in the field of biological control. However, most microbial agents are live bacteria products, highly regional, and difficult to adapt to, exhibiting poor stability when introduced. Furthermore, the Gansu-Qinghai region is characterized by cold, arid, and high-altitude environments, making existing biocontrol agents ineffective against root rot and other diseases in crops such as barley, lilies, and vegetables under these unique habitats. Therefore, developing effective biocontrol microorganisms and agents for the special habitats of Gansu-Qinghai is of great significance for ensuring agricultural safety and the quality of agricultural products. Summary of the Invention

[0006] The purpose of this invention is to provide Bacillus belyssus LB17, K87, MP6 and their compound inoculants to solve the problem of root rot that is difficult to control in the special habitat of Gansu and Qinghai.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a compound microbial agent prepared by mixed fermentation of Bacillus bellis LB17, Bacillus bellis K87 and Bacillus bellis MP6.

[0009] The present invention also provides a method for preparing the compound microbial agent, comprising the following steps:

[0010] S1. Bacillus belye LB17, K87 and MP6 were inoculated into slant culture medium and cultured on slant at 28~32℃ for 23~25h to obtain slant culture;

[0011] S2. The slant cultures of Bacillus belyss LB17, K87 and MP6 were inoculated into seed culture medium; and cultured at 28-32℃ and 130-150rpm for 22-26h to obtain seed culture solutions of Bacillus belyss LB17, K87 and MP6.

[0012] S3. Mix the seed culture solutions of Bacillus belyssus LB17, K87, and MP6 to obtain a mixed bacterial solution;

[0013] S4. Inoculate the mixed bacterial solution into the fermentation medium at an inoculation rate of 7-9%, and incubate at 32-36℃ and 180-220rpm for 32-36 hours to obtain the final product.

[0014] Preferably, the volume ratio of seed culture medium for Bacillus belyssus LB17, K87, and MP6 is 3.5~4.5: 2.5~3.5: 5.5~6.5.

[0015] Preferably, the seed culture medium comprises the following components at the following concentrations: soluble starch 19-21 g / L, NH4Cl 9-11 g / L, MgSO4·7H2O, and the balance being water.

[0016] Preferably, the fermentation medium comprises the following components at the following concentrations: mannitol 9-11 g / L, corn steep liquor powder 14-16 g / L, MnSO4·H2O 14-16 g / L, with the balance being water.

[0017] The present invention also provides the application of the aforementioned compound microbial agent in at least one of the following:

[0018] (1) Application in the prevention and control of plant pathogens;

[0019] (2) Application in the prevention and control of plant diseases;

[0020] (3) Application in promoting plant growth and improving plant stress resistance;

[0021] (4) Application in increasing the number of beneficial microorganisms in soil;

[0022] (5) Application in improving the fertility of plant rhizosphere soil.

[0023] Preferably, the plant pathogens include Fusarium oxysporum, Fusarium equisetifolium, Fusarium oxysporum, Fusarium solanifolium, Helicobacter pylori, or Micrococcus simonii; the plant diseases include root rot and wilt.

[0024] The present invention also provides a strain of Bacillus belye LB17, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242190, deposit date October 14, 2024, and deposit address Wuhan University.

[0025] The present invention also provides a strain of Bacillus belye K87, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242191, deposit date October 14, 2024, and deposit address Wuhan University.

[0026] The present invention also provides a strain of Bacillus belye MP6, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242192, deposit date October 14, 2024, and deposit address Wuhan University.

[0027] By adopting the above technical solution, this invention has the following beneficial effects: This invention isolated 305 pure cultures from the rhizosphere and rhizosphere soil of characteristic crops such as highland barley, lily, tomato, and pepper collected from the Gansu-Qinghai region. After multiple screenings, three strains of *Bacillus belye*—LB17, K87, and MP6—were obtained. Through optimization of the inoculum size, culture medium components, ratios, and fermentation conditions, a compound fermentation agent—Tuweiwei No. 7—was finally obtained, co-fermented by the three strains of *Bacillus belye*—LB17, K87, and MP6. Tuweiwei No. 7 can effectively prevent pathogenic microorganisms and plant diseases such as root rot and wilt, and can also promote plant growth and improve its resistance. The compound fermentation agent provided by this invention provides a strong guarantee for the prevention and control of root rot diseases in crops with special habitats in the Gansu-Qinghai region. Attached Figure Description

[0028] Figure 1 This image shows the antibacterial effect of rescreening some antagonistic strains.

[0029] Figure 2 The growth of strains at different salt concentration gradients is shown.

[0030] Figure 3 This is a comparative illustration of the safety testing results for some strains.

[0031] Figure 4 This is a graph showing the acid resistance of each strain.

[0032] Figure 5 The graph shows the alkali tolerance of each strain.

[0033] Figure 6 This is a graph showing the salt tolerance of each strain.

[0034] Figure 7 This is a diagram showing the antagonistic effects between some strains.

[0035] Figure 8 The inhibition rate of superior compound bacterial strains against pathogenic fungi.

[0036] Figure 9 This is a phylogenetic tree based on the universal primer sequence of 16S rDNA.

[0037] Figure 10 This is a phylogenetic tree based on the gyr B universal primer sequence.

[0038] Figure 11 The figure shows the experimental results of optimizing the ratio of the compound bacterial strain.

[0039] Figure 12 Figure showing the results of the experiment to optimize the composition of the seed culture medium.

[0040] Figure 13 The figure shows the results of the experiment on optimizing the composition ratio of seed culture medium.

[0041] Figure 14 Figure shows the results of the experiment to optimize the fermentation conditions of the seed culture medium.

[0042] Figure 15 Figure showing the results of the experiment to optimize the composition of the fermentation medium.

[0043] Figure 16 The figure shows the results of the experiment to optimize the composition ratio of the fermentation medium.

[0044] Figure 17 Figure showing the experimental results of optimizing fermentation culture conditions.

[0045] Figure 18 Illustrations showing the disease resistance of Tuweiwei No. 7 to highland barley.

[0046] Figure 19 Illustrations showing the effects of Tuweiwei No. 7 on the growth promotion of highland barley.

[0047] Figure 20 Illustrations showing the disease resistance of Tuweiwei No. 7 to lilies.

[0048] Figure 21 Illustrations showing the effects of Tuweiwei No. 7 on promoting lily growth.

[0049] Figure 22 Illustrations showing the disease resistance of Tuweiwei No. 7 to tomatoes.

[0050] Figure 23 Illustrations showing the effects of Tuweiwei No. 7 on promoting tomato growth.

[0051] Figure 24 Illustrations showing the disease resistance of Tuweiwei No. 7 to chili peppers.

[0052] Figure 25 Illustrations showing the effects of Tuweiwei No. 7 on promoting the growth of chili peppers.

[0053] Microbial Preservation Instructions

[0054] The Bacillus velezensis LB17 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242190, deposit date October 14, 2024, and deposit address Wuhan University.

[0055] The Bacillus velezensis K87 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242191, deposit date October 14, 2024, and deposit address Wuhan University.

[0056] The Bacillus velezensis MP6 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242192, deposit date October 14, 2024, and deposit address Wuhan University. Detailed Implementation

[0057] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1. Strain Screening

[0059] I. Sample Collection and Initial Screening

[0060] Rhizosphere and rhizosphere soils of characteristic crops such as highland barley, lily, tomato, and pepper in Gansu and Qinghai regions were collected and transported to the laboratory at low temperature. Rhizosphere soil bacteria were initially isolated using the plate spread method, and then different morphological colonies were purified using the streak plate method. All culture media used were LB medium (Luria-Bertani), and a total of 305 pure cultures were obtained.

[0061] II. Initial Screening of Functional Strains

[0062] 1. Initial screening of antagonistic bacteria

[0063] After activating 305 test strains by streak plating at 30℃ for 48 h, strains with antagonistic function were screened using the plate confrontation method. Activated pathogenic fungal discs (d=0.6 cm) were inoculated in the center of PDA medium (the tested pathogenic fungi included: *Fusarium avenaceum*, *Fusarium equiseti*, *Fusarium oxysporum*, *Fusarium solani*, *Bipolaris sorokiniana*, and *Microdochium bolleyi*, all derived from the Economic Crop Diseases Research Laboratory of the Institute of Plant Protection, Gansu Academy of Agricultural Sciences). Simultaneously, activated bacteria were inoculated at equal intervals around the discs. The medium was then incubated at 25℃ for 7 days, and the diameter of the inhibition zone was measured.

[0064] The results are shown in Table 1. A total of 167 antagonistic bacteria were screened. Among them, 15 strains showed good antagonistic effects against *Fusarium oxysporum*, with K87 having the largest inhibition zone diameter of 15.28 mm. The inhibition zone diameters of the remaining strains ranged from 8.78 mm to 15.11 mm. 54 strains showed good antagonistic effects against *Fusarium equisetifolium*, with 21 strains having inhibition zone diameters greater than 30 mm. More than 59% of the antagonistic strains also showed varying degrees of antagonistic effects against other pathogens. 27 strains showed antagonistic effects against *Fusarium oxysporum*, with inhibition zone diameters ranging from 22 mm to 33 mm. 35 strains showed antagonistic effects against *Fusarium solani*, with inhibition zone diameters ranging from a maximum of 38.52 mm to a minimum of only 10.8 mm, indicating a significant difference in antagonistic ability. Only 6 strains showed antagonistic effects against *Hymenobacter oryzae*, with inhibition zones ranging from 12.69 mm to 21.71 mm. These 6 strains also showed antagonistic effects against at least 3 other pathogenic bacteria identified in this study. 32 strains showed antagonistic effects against *Microcystis aeruginosa*, with inhibition zones ranging in diameter from 16.94 mm to 30.99 mm.

[0065] Table 1. Inhibition diameters of antagonistic bacteria against 6 pathogens

[0066]

[0067]

[0068]

[0069] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences (P < 0.05), and the same applies below.

[0070] 2. The activated bacteria were inoculated onto Monkina organophosphate plates. After 5 days, the formation of phosphate-solubilizing zones was observed, and their diameters were measured. The results are shown in Table 2. A total of 109 organophosphate-solubilizing bacteria were screened. Among them, MP6, MP41, and K87 had the largest phosphate-solubilizing zones, at 37.79 mm, 35.67 mm, and 32.89 mm, respectively. The diameters of the phosphate-solubilizing zones of the remaining strains ranged from 5.16 mm to 27.49 mm.

[0071] Table 2. Diameter of phosphorus dissolved in the initial screening

[0072] Strain number Diameter (mm) of the phosphorus solution zone Strain number Diameter (mm) of the phosphorus solution zone Strain number Diameter (mm) of the phosphorus solution zone Strain number Diameter (mm) of the phosphorus solution zone Strain number Diameter (mm) of the phosphorus solution zone CK 0.00±0.0001 K38 11.21±0.003 K115 8.88±0.005 K17 10.78±0.199 MP8 8.83±0.028 K86 7.33±0.016 K37 10.97±0.039 K116 9.68±0.018 K6 9.76±0.019 MP12 13.64±0.039 K87 32.89±0.009 K79 12.59±0.012 K92 11.81±0.004 K5 7.76±0.019 MP13 13.68±0.01 K89 8.65±0.054 K12 7.01±0.005 K93 8.77±0.026 MP1 10.84±0.033 MP14 9.6±0.001 K91 10.27±0.027 K71 8.58±0.015 K94 11.54±0.021 MP7 11.19±0.051 MP16 6.75±0.071 K100 8.25±0.034 MP41 35.67±0.022 K95 7.41±0.018 MP10 11.23±0.067 MP17 7.82±0.035 K102 17.67±0.201 MP6 37.79±0.015 K72 11.21±0.01 MP11 10.19±0.051 K110 8.5±0.032 K117 9.93±3.336 MP9 16.49±0.018 K73 9.98±0.009 MP29 10.23±0.067 K112 6.9±0.004 K118 8.37±0.037 MP5 9.69±0.009 K74 12.98±0.009 MP28 9.23±0.067 K113 8.7±0.668 K119 14.29±0.004 MP3 8.71±0.006 K75 9.45±0.012 MP26 7.81±0.066 K114 11.28±0.001 LB7 9.59±0.038 K53 7.76±0.047 MP31 7.45±0.012 MP25 8.73±0.068 K34 10.9±0.014 LB9 15.56±0.064 K52 10.48±0.025 MP32 12.66±0.013 LB21 13.23±0.069 LB17 16.69±0.01 LB10 9.48±0.129 K61 8.38±0.009 MP33 9.24±0.015 LB22 8.99±0.058 K16 11.22±0.403 LB11 7.9±0.041 K62 13.26±0.016 K63 8.4±0.009 LB23 10.29±0.047 K45 13.74±0.01 LB16 11.68±0.001 K67 8.47±0.019 K35 7.3±0.022 LB25 8.29±0.047 K57 13.21±0.009 LB18 8.85±0.043 K85 27.49±0.011 K21 8.5±0.009 LB28 9.17±0.027 K11 7.85±0.027 LB24 12.77±0.031 K90 9.53±0.102 K22 9.7±0.008 LB38 9.84±0.017 K42 11.05±0.012 LB27 6.24±0.035 K108 8.58±0.287 K23 10.26±0.028 LB3 10.25±0.019 K58 8.1±0.007 LB30 12.39±0.007 K107 7.89±0.005 K24 8.64±0.003 LB2 7.25±0.019 K65 10.77±0.007 LB31 16.68±0.207 LB36 11.68±0.013 K25 7.9±0.004 LB29 13.28±0.018 K43 10.98±0.014 LB32 8.32±0.035 LB8 13.36±0.024 K26 7.75±0.209 K40 7.43±0.017 K44 11.43±0.013 LB33 11.62±0.035 LB6 7.38±0.082 K27 7.48±0.418 K41 8.56±0.015 K33 16.31±0.02 LB34 5.16±0.921 LB5 8.93±0.001 K28 10.79±0.42 MP36 7.31±0.014 MP2 16.34±0.038 LB35 7.6±0.006 LB1 8.72±0.025 K31 10.94±0.42 MP39 9.23±0.014 K104 9.85±0.03 LB37 7.41±0.018 K103 11.08±0.03 K13 13.26±0.417 MP40 7.23±0.014 K15 11.62±0.393 LB40 10.29±0.004 K105 10.86±0.022 K14 8.69±0.393 K111 10.91±0.002 MP34 12.24±0.015

[0073] III. Antagonistic Bacterial Rescreening

[0074] The strains selected from step 2 that exhibit both antagonistic and phosphate-solubilizing effects were inoculated into LB broth and cultured at 30°C and 180 r / min for 48 h. The bacterial solution was then transferred to 10 mL centrifuge tubes and centrifuged at 12000 r / min for 10 min at 4°C. 1 mL of the supernatant was aspirated using a sterile syringe (10 mL) and filtered through a 0.22 µm microporous membrane. The filtrate was spread onto PDA plates to prepare virus-containing plates, with uncoated PDA plates serving as controls. The tested pathogenic fungi (cup diameter = 0.6 cm) were inoculated into each plate, with three replicates per treatment. After incubation at 25°C for 7 days, the diameter of the pathogenic fungi was measured, and the growth inhibition rate was calculated.

[0075] Growth inhibition rate (%) = (Coronavirus colony diameter on control plate - Coronavirus colony diameter on infected plate) / (Coronavirus colony diameter on control plate - Inoculated mycelium diameter) × 100

[0076] The results are shown in Table 3 and Figure 1 As shown, some strains exhibited significant inhibitory effects after secondary screening. K87 showed inhibitory effects against all five pathogens (F. avenaceum, F. oxysporum, F. solani, B. sorokinina, and M. bolleyi), with good inhibition rates exceeding 75%. LB17 showed inhibitory effects against all four pathogens (F. oxysporum, F. solani, B. sorokinina, and M. bolleyi), with inhibition rates exceeding 61%. Eleven strains showed good inhibitory effects against Fusarium avenaceum, with inhibition rates ranging from 44% to 87%. Fourteen strains showed good inhibitory effects against Fusarium equisetifolium, accounting for 25% of the initial antagonistic strains, with inhibition rates ranging from 74.9% to 92.88%. Compared with the initial screening results, 59% of the strains (16 strains in total) still showed inhibitory effects against *Fusarium oxysporum*, with the highest inhibition rate being 81%. Six strains showed inhibitory effects against *Fusarium solanum*, with inhibition rates all above 69%. Only six strains showed good inhibitory effects against *Hylocereus buergerianum*, with the highest inhibition rate being 87.23% and the lowest being 58%. Eleven strains (34% of the initially screened strains) showed good inhibitory effects against *Micrococcus pluvialis*, with the highest inhibition rate reaching 92%.

[0077] Table 3 Antibacterial rate of antagonistic strains

[0078]

[0079] IV. Safety testing of strains

[0080] 1. Growth of strains under various salt concentration gradients

[0081] Considering that LB medium containing NaCl may affect the growth of barley, and that the strains on WA medium (20 g agar, 1000 mL distilled water) did not grow well, bacteria were randomly selected and inoculated onto salt-free LB medium with 0 NaCl, WA medium, and standard LB medium with 1% NaCl as a control. The bacterial growth was compared and observed after 24 h to see if there were any significant differences.

[0082] The results are as follows Figure 2 As shown, the strain grew normally in both LB and salt-free LB media for 24 h, but did not grow in WA medium. Therefore, salt-free LB medium was chosen for subsequent experiments.

[0083] 2. Determination of pathogenicity of strains using the salt-free LB culture method

[0084] The top 50 strains with the highest antibacterial rates were activated by LB broth culture at 30℃ and 180 r / min for 48 h. 1 mL of the bacterial culture was then added to a beaker containing cooled but not solidified sterile, salt-free LB medium (40 mL / 50 mL) to prepare the bacterial culture medium. Simultaneously, barley seeds were treated to promote germination. Finally, 10 seeds with uniform germination were evenly spread in the bacterial culture medium, with a control of salt-free LB medium without bacterial inoculation. The cultures were incubated at 25℃, 1 h of light, and 75% humidity, with a small amount of sterile water sprayed every 8 h. Growth was observed after 10 days.

[0085] The results are as follows Figure 3 As shown, the growth of barley varied in LB medium inoculated with different strains. Subsequent studies selected strains that promoted vigorous barley growth for further experiments, avoiding strains that resulted in sparse or non-existent seedling growth. Table 4 shows the safety of 48 strains for barley growth. In the table, "+" indicates good growth and "-" indicates poor growth. Barley grew poorly on 8 strains including K102 and LB44, but grew well on the remaining 40 strains; therefore, these 40 strains were considered safe for barley.

[0086] Table 4. Safety testing of strains

[0087] strain number Security strain number Security strain number Security strain number Security K102 - N10 + MP41 + MP29 + K87 + K113 + LB33 + LB17 + N10 + LB15 + LB11 + MP6 + K115 + LB28 + LB44 - K33 + N27 + LB64 + MP31 + K56 + MP20 + N20 + LB14 - K37 + K89 + LB24 + LB7 - LB15 + MP12 + MP14 + K79 + LB20 + K91 + LB9 + K34 + LB21 - K85 + MP18 - MP24 + LB26 + MP17 + MP28 + K113 + K71 + MP28 - MP16 + MP35 - N1 +

[0088] Note: "+" indicates that the barley plants are growing well; "-" indicates that the barley plants are growing poorly.

[0089] V. Determination of the growth-promoting characteristics of the strain

[0090] The non-pathogenic strains selected from the four screenings were activated and inoculated into LB broth. They were then cultured at 30°C and 180 r / min in a shaker for 48 h, and the OD was measured. 660When the value is ≥0.5, the following characteristic test shall be performed:

[0091] Phosphorus solubility characteristics: 1 mL of bacterial culture was inoculated into PKO inorganic phosphorus and Mengjina organic phosphorus culture media (volume: 50 mL / 150 mL Erlenmeyer flask, the same below), with 3 replicates for each strain. The culture media without inoculation was used as a control. After culturing in a shaker at 30℃ and 180 r / min for 7 days, the phosphorus content was determined by the molybdenum antimony colorimetric method.

[0092] Nitrogen fixation characteristics: 1 mL of bacterial culture was inoculated into NFM culture medium, with 3 replicates per strain. The culture medium without inoculation was used as a control. After incubation at 30 ℃ and 180 r / min for 7 days, the nitrogen content was determined by the Kjeldahl method.

[0093] Potassium solubility characteristics: 1 mL of bacterial culture was inoculated into potassium feldspar culture medium, with 3 replicates for each strain. The culture medium without inoculation was used as a control. After culturing at 30 ℃ and 180 r / min for 7 days, the soluble potassium content in each bacterial culture was determined by flame spectrophotometry.

[0094] IAA secretion characteristics: 1 mL of bacterial culture was inoculated into King's B culture medium, with 3 replicates per strain. The culture medium without inoculation was used as a control. After culturing at 30 ℃ and 180 r / min for 5 days, referring to the method in the literature "Isolation and Growth-promoting Characteristics Analysis of IAA-producing Strains in Rhizosphere Soil of *Salix babylonica*" (Cheng Xinyu et al., 2024), 10 mL of bacterial culture was taken, centrifuged at 10000 r / min for 5 min, and 5 mL of supernatant was taken. An equal volume of Salkowski colorimetric reagent was added and the volume was adjusted to 10 mL. After reacting at room temperature in the dark for 30 min, the OD value of the fermentation broth at a wavelength of 530 nm was measured by ultraviolet spectrophotometer. The IAA content per unit volume was calculated by referring to the standard curve.

[0095] Siderophore secretion characteristics: Following the method described in the literature "Functional characteristics and culture conditions of a white clover rhizosphere siderophore-producing bacterium" (Wei et al., 2024), 10 mL of bacterial culture was centrifuged at 10000 r / min for 10 min. Then, 5 mL of the supernatant was thoroughly mixed with 5 mL of CAS detection solution. After standing for 1 h, the absorbance (As) at OD630 nm was measured using a spectrophotometer. The absorbance (Ar) of 5 mL each of LB liquid culture medium and CAS detection solution was used as a reference value. Siderophore activity was calculated using the following formula:

[0096] Siderophore activity unit (su) = [(Ar-As) / Ar] × 100%

[0097] Acid, alkali, and salt tolerance characteristics: 1 mL of bacterial suspension was inoculated into culture media with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and into LB culture media with NaCl concentrations of 1%, 5%, 10%, 15%, and 20%. The cultures were incubated at 30 ℃ and 180 r / min for 48 h on a shaker. The OD values ​​of each bacterial suspension were measured spectrophotometrically. 630 The values ​​and measurement results are shown in Table 5 and Figure 4 , Figure 5 , Figure 6 As shown

[0098] Table 5 Functional characteristics of superior strains

[0099] strain number Nitrogen fixation capacity (g / L) Dissolved inorganic phosphorus (μg / mL) Dissolved organophosphorus compounds (μg / mL) Potassium solubility (mg / L) Secretion of IAA (mg / L) Secretion of siderophores (su) CK 0.00±0.0001 0.00±0.0001 0.00±0.0001 0.00±0.0001 0.00±0.0001 0.00±0.000 1 LB17 0.05±0.0002 d 1102.95±0.02 b 586.45±0.03 e 43.17±0.02 b 5.69±0.001 g 0.32±0.043 a K113 0.08±0.0004 a 909.84±0.04 d 744.68±0.07 d 33.7±0.02 g 6.78±0.005 c 0.31±0.0003 a K87 0.06±0.0016 c 367.63±0.13 h 432.79±0.1 g 25.75±0.02 h 9.87±0.003 a 0.26±0.0003 b K85 0.06±0.002 c 949.69±0.09 c 1321.23±0.09 a 35.52±0.03 f 5.79±0.006 f 0.26±0.0012 b MP6 0.06±0.002 c 1470.69±0.2 a 1141.86±0.04 c 40.9±0.02 c 6.67±0.023 d 0.17±0.0003 c K33 0.04±0.0003 e 569.64±0.01 f 436.47±0.03 f 36.23±0.03 d 5.95±0 e 0.17±0.0003 c K56 0.04±0.0003 f 458.87±0.02 g 336.92±0.04 h 35.86±0.03 e 6.65±0.012 d 0.13±0.0007 c MP41 0.05±0.0004 d 742.27±0.02 e 1272.8±0.04 b 140.33±0.03 a 6.85±0.008 b 0.13±0.0006 c

[0100] As shown in Table 5, LB17 exhibited the strongest siderophore secretion capacity (su value 32%) and also demonstrated strong inorganic phosphorus solubility (1102.95 μg / mL). K113 showed the strongest nitrogen fixation capacity (0.08 g / L), while the nitrogen fixation capacity of the other strains ranged from 0.04 to 0.06 g / L. K87 showed the strongest IAA secretion capacity (9.87 mg / L), while the other strains showed weaker IAA secretion capacity (5.69 mg / L to 6.78 mg / L), but also exhibited weak potassium solubilization capacity (25.75 mg / L). K85 showed the strongest organic phosphorus solubilization capacity (1321.23 μg / mL) and a siderophore su value exceeding 20%. MP6 showed the strongest inorganic phosphorus solubilization capacity (1470.69 μg / mL) and also demonstrated good organic phosphorus solubilization (1141.86 μg / mL) and potassium solubilization (40.9 mg / L) capabilities. MP41 exhibits the strongest potassium solubilizing ability, at 140.33 mg / L.

[0101] Figure 4 To determine the acid tolerance of superior strains, the OD values ​​of the strains at pH 7 were measured. 630 The values ​​were the highest, all greater than 2.0. When pH=6 and pH=5, the OD values ​​of strain K56 were... 630 The OD value decreased to 0.96, indicating that strain K56 has poor acid tolerance. The OD values ​​of the other strains also decreased, ranging from 1.5 to 1.7. When the cultures were in a pH 4 medium, K56 showed almost no growth, MP6 growth was inhibited, and the OD values ​​of MP41, K87, and K33 were [not specified]. 630 A value higher than 1.6 indicates that MP41, K87, and K33 have good acid resistance. When each strain is at pH 3, the OD... 630 The values ​​were close to the CK and all below 0.02, indicating that none of the strains grew under pH 3 conditions. Therefore, MP41, K87, and K33 were identified as acid-resistant strains.

[0102] Figure 5The results showed that the OD values ​​of the strains were all greater than 2.0 at pH 7, indicating good growth. When the pH of the culture medium was 8, the OD values ​​decreased, but remained greater than 1.5. At pH 9, the OD values ​​decreased significantly (P < 0.05), but the OD values ​​of LB17 and K113 cultures remained greater than 0.5. At pH 10, the OD values ​​were extremely low, close to the blank control, indicating that the screened strains were intolerant to alkaline concentrations at pH 10 and above. Therefore, strains LB17 and K113 were identified as alkali-tolerant strains.

[0103] Figure 6 The results showed that at a salt concentration of 1%, the OD values ​​of all strains were greater than 2.0, indicating good growth. When the salt concentration reached 5%, the OD values ​​decreased significantly (P<0.05), with strains K85, K113, K33, and K56 all having OD values ​​less than 1.2. At a salt concentration of 10%, the OD values ​​of K113, K33, and K56 were close to the blank control, with OD values ​​close to 0.2, indicating that a 10% salt concentration completely inhibited the growth of K113, K33, and K56. However, the OD values ​​of LB17, MP41, K85, and K87 remained greater than 1.1. At salt concentrations of 15% and 20%, the OD values ​​of all strains were extremely low, close to the blank control, and the differences were not significant (P<0.05), indicating that all strains were intolerant to salt concentrations of 15% and above. Therefore, strains LB17, MP41, K85, and K87 were identified as salt-tolerant strains.

[0104] VI. Construction of a disease-preventing and growth-promoting compound microbial system

[0105] The antagonistic effect between superior strains was determined by the pairwise streak method. Figure 7 The results showed that K87 and MP41 had an antagonistic effect, while the other strains did not have an antagonistic effect. Therefore, it is advisable to avoid using K87 and MP41 simultaneously in a compound bacterial strain.

[0106] Then, strains without antagonistic interactions were selected and combined with different pathogens and crops to form complex bacterial systems. Each system was replicated three times. The phosphorus solubility, nitrogen fixation, potassium solubility, IAA secretion, and siderophore secretion capacity of each complex bacterial system were measured according to the method described in section five. A comprehensive analysis was then performed, and the results are shown in Table 6. The combinations of each complex bacterial system are as follows:

[0107] T1: K87+LB17; T2: K87+LB17+MP6; T3: K87+LB17+MP6+K85

[0108] T4: K56+LB17; T5: K56+LB17+K113; T6: K56+LB17+MP6+K113;

[0109] T7: K33+LB17; T8: K33+LB17+MP6; T9: K33+LB17+MP6+K113;

[0110] T10: MP6+K85+MP41+K113; T12: K113+MP41; T13: LB17+K113+MP41.

[0111] Table 6 Functional characteristics of superior compound bacterial strains

[0112] Complex bacterial strain Nitrogen fixation capacity (g / L) Potassium solubility (mg / L) Dissolved organophosphorus content (μg / mL) Dissolved inorganic phosphorus content (μg / mL) IAA content (mg / L) Ferric load (su) Comprehensive analysis T1 0.107±0.0002 e 84±0.0947 b 300.56±2.03g 351.86±2.3 d 9.94±0.015m 0.28±0.0012 f 0.302 T2 0.212±0 a 86.28±0.0945 a 346.5±0.77 e 413. 11±2.03c 16.91±0.0462 h 0.33±0.0003 e 0.611 T3 0.019±0.0001 k 71.78±0.095f 437.6±2.03 b 419.23±1.53b 18.56±0.03e 0.18±0.0013 j 0.376 T4 0.012±0.0001 l 43.01±0 h 255.4±0 i 299.8±0.77 f 51.63±0.0901 a 0.21±0.0003 i 0.464 T5 0.105±0.0002 f 39.72±0.0317 l 322.77±2.03f 328.13±3.05e 19.39±0.03d 0.37±0.0003 c 0.434 T6 0.011±0 l 43.97±0 g 263.25±3.63i 273±0.77 g 20.28±0 c 0.18±0.0015 k 0.249 T7 0.045±0.0001 i 41.3±0.0003k 386.31±1.33d 409.43±0. 15c 15.59±0 j 0.26±0.0009 g 0.32 T8 0.018±0 k 42. 12±0.0313 j 403.91±0.77c 413.25±2.66c 15±0.0347kl 0.34±0.0009 d 0.335 T9 0. 149±0 b 80.4±0 e 259.22±1.53i 275.5±2.63 g 18.28±0.0173 f 0. 11±0 m 0.471 T10 0.037±0.0003 j 42. 19±0.0317 j 281.43±1.53h 298.27±2.03f 23.54±0.0173 b 0.23±0.0006 h 0.306 T12 0.074±0.0025 h 30.37±0.0313 m 212.98±8.68k 233. 19±1.53i 14.98±0.0177 l 0.16±0.0009 l 0.234 T13 0. 141±0 c 81.26±0 c 482±0.77 a 425.35±0 a 15. 1±0.0459 k 0.47±0.0009 a 0.579

[0113] Table 6 shows that among the 12 compound bacterial strains, T2 had the highest nitrogen fixation and potassium solubilization capacity, at 0.212 g / L and 86.28 mg / L, respectively. Compound bacterial strain T13 had the highest organic phosphorus solubilization, inorganic phosphorus solubilization, and siderophore secretion capacity, at 482 μg / mL, 425.35 μg / mL, and a su value of 47%, respectively. Compound bacterial strain T4 had the strongest IAA secretion capacity, at 51.63 mg / L. In the Topsis comprehensive evaluation, the CIs for T2 and T13 were 0.611 and 0.579, respectively, higher than the other groups, thus identifying them as superior compound bacterial strain formulations.

[0114] Finally, the superior compound bacterial strains T2 and T13 were selected to determine their inhibitory effects against six tested pathogenic fungi: *Microcystis aeruginosa*, *Fusarium oxysporum*, *Helicobacter pylori*, *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium oxysporum* (method as described in "III"). The results are as follows: Figure 8 As shown.

[0115] Figure 8 The results showed that the T2 compound strain exhibited stronger antibacterial activity than the T13 strain, with the highest inhibition rate against *Fusarium equisetifolium* (87.69%), followed by *Fusarium solanum* and *Microcystis aeruginosa* (77.84% and 76.71%, respectively). The inhibition rates against *Fusarium oxysporum*, *Fusarium oxysporum*, and *Hypericum spp.* were 60.26%, 65.71%, and 64.46%, respectively. The T13 compound strain also showed strong antibacterial activity against the tested fungi, with the strongest inhibition against *Hypericum spp.* (64.45%), followed by *Fusarium equisetifolium* (63.68%). The inhibition rates against *Microcystis aeruginosa* and *Fusarium oxysporum* were 47.45% and 53.99%, respectively, while the inhibition rates against *Fusarium oxysporum* and *Fusarium oxysporum* were lower, at 19.47% and 19.56%, respectively. In summary, T2 generally exhibits higher antibacterial activity than T13, therefore T2 is the optimal mixed bacterial strain.

[0116] Example 2. Identification of LB17, K87, and MP6 strains

[0117] DNA was extracted from the strains involved in the optimal composite bacterial strain screened in Example 1 using a DNA extraction kit (TIANGEN centrifuge column type) according to its instructions. The DNA was then amplified using 16S rDNA universal primers (SEQ ID NO.1, SEQ ID NO.2) and gyrB primers (SEQ ID NO.3, SEQ ID NO.4).

[0118] Universal primers:

[0119] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1),

[0120] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO.2)

[0121] gyrB primers:

[0122] F: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' (SEQ ID NO.3),

[0123] R: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3' (SEQ ID NO.4)

[0124] The total reaction volume was 25 μL, containing 1 μL each of forward and reverse primers, 1 μL of DNA template, 12.5 μL of 2×PCR Mix, and 9.5 μL of ddH2O. The reaction program was: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 10 min. After passing 1% agarose gel electrophoresis, sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. Finally, the sequencing results were compared using BLAST homology 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. The reliability was verified using Bootstrap 1000. The results are shown below. Figure 9 , Figure 10 As shown.

[0125] Figure 9The analysis based on 16S rDNA sequences revealed that LB17 has a genetic distance of less than 0.01 from Bacillus velezensis (ON597433.1), with a support rate of 99% after 1000 replicates; MP6 and K87 also have a genetic distance of less than 0.01 from Bacillus velezensis (MW578391.1), with a support rate of 97%. Their sequences were submitted to GenBank and obtained accession numbers PQ211017, PQ211018, and PQ211019, respectively. Therefore, LB17, K87, and MP6 were preliminarily identified as Bacillus velezensis. Further identification of their gyrB sequences revealed (… Figure 10 The genetic distance between LB17 and K87 and the Bacillus velezensis standard strain BCRC 17467T (DQ903176.1) is 0, with a support rate of 98% after 1000 replicates. The genetic distance between MP6 and the Bacillus velezensis standard strain NRRL 41580T (EU138622.1) is also 0, with a support rate of 99% after 1000 replicates. Their sequences were submitted to GenBank and obtained accession numbers PQ144581, PQ144582, and PQ144583, respectively. Therefore, strains LB17, K87, and MP6 are identified as Bacillus velezensis.

[0126] Example 3. Optimal formulation optimization of compound bacterial strain T2

[0127] After activation, the tested strains LB17, K87, and MP6 were inoculated together into LB liquid medium and incubated at 30°C with a shaker speed of 180 rpm for 10 weeks. 8 CFU / mL, and an orthogonal design with four levels (3%, 4%, 5%, 6%) was used to conduct L9 (4 3 The orthogonal experiment was conducted, and the orthogonal design scheme is shown in Table 7. The optimal inoculum quantity was determined based on the measured OD value of the composite bacterial solution. The results are as follows: Figure 11 As shown.

[0128] Table 7 L9(4) 3 Orthogonal design table

[0129] Experimental group number LB17 K87 MP6 A1 3% 3% 3% A2 4% 4% 4% A3 3% 4% 5% A4 4% 3% 6% A5 5% 5% 3% A6 6% 4% 4% A5 5% 5% 3% A8 4% 5% 5% A9 6% 3% 4%

[0130] Figure 11 The results showed that in test group 4 (A4), OD 600The highest OD value was 1.45, significantly higher than other treatments (P<0.05). The OD values ​​of the remaining experimental groups ranged from 1.153 to 1.27. This indicates that the optimal fermentation effect was achieved when the inoculum amounts of LB17, K87, and MP6 were 4%, 3%, and 6%, respectively, i.e., the inoculum volume ratio was 4:3:6. The optimized composite strain was named "Tuweiwei No. 7" and will be referred to as "Tuweiwei No. 7" from now on.

[0131] Example 4. Seed Culture Medium Optimization

[0132] 1. Optimization of seed culture medium composition

[0133] Single-factor experiments were conducted, replacing the components of the original LB medium with different carbon sources (1% sucrose, 1% sodium citrate, 1% lactose, 1% soluble starch); nitrogen sources (1% NH4Cl, 1% (NH4)2SO4, 1% NaNO3, 1% yeast extract); and inorganic salts (1% MgSO4·7H2O, 1% K2HPO4·3H2O, 1% CaCl2·2H2O, 1% MnSO4·7H2O), with three replicates per treatment. After culturing under optimized fermentation conditions, the OD of the composite bacterial culture was measured using a microplate reader. 600 value.

[0134] The results are as follows Figure 12 As shown, when the seed culture medium composition was optimized by using soluble starch as the sole carbon source in the initial LB medium, the OD of Tuweiwei 7 was... 600 The highest value was 1.55, significantly higher than other treatments (P<0.05). OD was highest when sucrose, sodium citrate, or lactose were the sole carbon sources. 600 All values ​​were less than 1.3, therefore soluble starch was the optimal carbon source for Tuweiwei 7 when used as the sole carbon source; when the nitrogen source in the initial LB medium, tryptone, was replaced with NH4Cl as the sole nitrogen source, the OD of the complex bacterial strain was... 600 The highest value was 1.405, and the difference was significant (P<0.05). The OD values ​​of (NH4)2SO4, yeast extract, and NaNO3 were also significant. 600 The value is between 1.114 and 1.308, therefore, NH4Cl is the optimal nitrogen source for Tuweiwei No. 7 when used as the sole nitrogen source; when the inorganic salt NaCl in the initial LB medium is replaced with MgSO4·7H2O, the OD of the composite strain is... 600 The highest value was 1.243, and the difference was significant (P<0.05). The OD values ​​for K2HPO4·3H2O, CaCl2·2H2O, and MnSO4·7H2O were [missing values]. 600 The values ​​are 1.209, 1.217, and 1.164, respectively. Therefore, when MgSO4·7H2O is the only inorganic salt, it is the optimal inorganic salt for Tuweiwei 7.

[0135] 2. Optimization of seed culture medium component ratio

[0136] Design L9 (4) according to Table 8 3 An orthogonal experiment was conducted to optimize the optimal ratios of the above-mentioned optimal culture components: soluble starch (1%, 1.5%, 2%), NH4Cl (NH4Cl: 1%, 1.5%, 2%), and MgSO4·7H2O (1%, 1.5%, 2%). Specifically, the seed culture of *Tuweiwei No. 7* was inoculated into the culture medium of each treatment group, and cultured at 30℃ for 48 hours on a shaker at 180 r / min. The OD values ​​were then measured. 600 value.

[0137] Table 8 L9(43) Orthogonal Design Table

[0138] Experimental group number Soluble starch <![CDATA[NH4Cl]]> <![CDATA[MgSO4·7H2O]]> B1 2% 1% 1% B2 1.5% 1% 1.5% B3 1% 1% 2% B4 1% 1.5% 1% B5 1.5% 1.5% 1.5% B6 2% 1.5% 2% B7 1.5% 2% 1% B8 2% 2% 1.5% B9 1% 2% 2%

[0139] The results are as follows Figure 13 As shown, Figure 13 The results showed that group B1 had the highest OD value of 1.63, while the OD values ​​of the other experimental groups ranged from 1.11 to 1.57, with significant differences (P<0.05). The optimal ratio of soluble starch:NH4Cl:MgSO4·7H2O was 2:1:1.

[0140] Example 5. Optimization of Seed Liquid Fermentation Conditions

[0141] A five-factor, four-level orthogonal design was used to study the compound microbial strain Tuwei No. 7 with different initial pH (6.0, 6.5, 7, 7.5), inoculum amount (2%, 4%, 6%, 8%), shaking rate (140, 160, 180, 200 r / min), culture temperature (22, 26, 30, 34 ℃), and culture time (18, 24, 30, 36 h). The L16(4)4(t ... 5 Orthogonal experiment, the orthogonal design scheme is shown in Table 9, and the OD of the compound bacterial solution was determined. 600 value.

[0142] Table 9 L16(45) Orthogonal Design Table

[0143] Test No. PH Inoculation volume rotational speed temperature time Z1 6 2% 140 22 18 Z2 6 4% 160 26 24 Z3 6 6% 180 30 30 Z4 6 8% 200 34 36 Z5 6.5 2% 160 30 36 Z6 6.5 4% 140 34 30 Z7 6.5 6% 200 22 24 Z8 6.5 8% 180 26 18 Z9 7 2% 180 34 24 Z10 7 4% 140 30 18 Z11 7 6% 200 26 36 Z12 7 8% 160 22 30 Z13 7.5 2% 200 26 30 Z14 7.5 4% 180 22 36 Z15 7.5 6% 160 34 18 Z16 7.5 8% 140 30 24

[0144] The results are as follows Figure 14 As shown, the OD of Tuweiwei No. 7 under different fermentation conditions 600 The values ​​showed significant differences (P<0.05). Among them, group Z16 had the highest OD value of 1.88. Therefore, the optimal fermentation conditions were pH 7.5, inoculum size of 8%, rotation speed of 140 r / min, temperature of 30℃, and fermentation time of 24h.

[0145] Example 6. Optimization of fermentation medium in fermenter

[0146] 1. Optimization of fermentation medium composition

[0147] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 20 g agar, 1000 mL distilled water, pH 7.0~7.2.

[0148] Single-factor experiments were conducted, replacing the components of the original LB medium with different carbon sources (1% corn starch, 1% soluble starch, 1% mannitol, 1% maltodextrin); nitrogen sources (1% corn steep liquor powder, 1% yeast powder, 1% urea, 1% soybean flour, 1% soybean meal powder); and inorganic salts (1% MnSO4·H2O, 1% CaCl2, 1% MgSO4, 1% CuSO4, 1% CaCO3), with three replicates per treatment. After cultivation under optimized fermentation conditions, the OD of the composite bacterial solution was measured using a microplate reader. 600 value.

[0149] The results are as follows Figure 15 As shown, when mannitol is the sole carbon source, the OD of Tuweiwei 7 is... 600 The highest value was 1.4, significantly higher than other treatments (P<0.05) when corn starch, soluble starch, and maltodextrin were the sole carbon sources, resulting in an OD value that was significantly higher than other treatments. 600 The OD value of the complex bacterial strain was between 1.16 and 1.3; when corn steep liquor was the sole nitrogen source, the OD value of the complex bacterial strain was... 600 The highest value of the compound bacterial strain OD 600 The highest value was 1.35, and the difference was significant (P<0.05). The OD values ​​of yeast powder, urea, soybean flour, and soybean meal were... 600 The value is between 1.22 and 1.28; when MnSO4·H2O is the only inorganic salt, the OD value of the complex bacterial strain is... 600 The highest value was 1.243, and the difference was significant (P<0.05). CaCl2, MgSO4, CuSO4, CaCO3, OD 600 The values ​​are 1.247, 1.275, 1.133, and 1.188, respectively. Therefore, when MnSO4·H2O is the only inorganic salt, it is the optimal inorganic salt for T.V. 7.

[0150] 2. Optimal ratio of fermentation medium components

[0151] Design L9 (4) according to Table 10 3 An orthogonal experiment was conducted to optimize the optimal ratios of mannitol (1%, 1.5%, 2%), corn steep liquor powder (NH4Cl: 1%, 1.5%, 2%), and MnSO4·H2O (1%, 1.5%, 2%), the optimal culture components mentioned above. Specifically, the seed culture of Tuwei No. 7 was inoculated into the fermentation broth of each treatment group, and cultured at 30℃ for 48 hours on a shaker at 180 r / min. OD values ​​were then measured. 600Value, result as Figure 16 As shown.

[0152] Table 10 L9(43) Orthogonal Design Table

[0153] Experimental group number Mannitol Corn syrup powder <![CDATA[MnSO4·H2O]]> C1 2% 1% 1% C2 1.5% 1% 1.5% C3 2% 1% 2% C4 1% 1.5% 1% C5 1.5% 1.5% 1.5% C6 1% 1.5% 1.5% C7 1.5% 2% 1% C8 2% 2% 1.5% C9 1% 2% 2%

[0154] Figure 16 The results showed that the optimal fermentation medium C6 group of the T2 composite strain had the highest OD value of 1.75, while the other experimental groups ranged from 1.33 to 1.63. This indicates that the optimal ratio of mannitol: corn steep liquor powder: MnSO4 H2O was 1:1.5:1.5.

[0155] Example 7. Optimization of fermentation conditions in fermenter

[0156] A five-factor, four-level orthogonal design was used to study the compound microbial strain Tuwei No. 7 with different initial pH (6.0, 6.5, 7, 7.5), inoculum amount (2%, 4%, 6%, 8%), shaking rate (140, 160, 180, 200 r / min), culture temperature (22, 26, 30, 34 ℃), and culture time (18, 24, 30, 36 h). The L16(4)4(t ... 5 Orthogonal experiment, the orthogonal design scheme is shown in Table 11, and the OD of the compound bacterial solution was measured. 600 Value, result as Figure 17 As shown.

[0157] Table 11 L16(45) Orthogonal Design Table

[0158] Test No. PH Inoculation volume rotational speed temperature time F1 6 2% 140 22 18 F2 6 4% 160 26 24 F3 6 6% 180 30 30 F4 6 8% 200 34 36 F5 6.5 2% 160 30 36 F6 6.5 4% 140 34 30 F7 6.5 6% 200 22 24 F8 6.5 8% 180 26 18 F9 7 2% 180 34 24 F10 7 4% 140 30 18 F11 7 6% 200 26 36 F12 7 8% 160 22 30 F13 7.5 2% 200 26 30 F14 7.5 4% 180 22 36 F15 7.5 6% 160 34 18 F16 7.5 8% 140 30 24

[0159] Figure 17 The results showed that the OD of the compound bacterial strain under different fermentation conditions in the fermentation medium... 600 The values ​​showed significant differences (P<0.05). Among them, the F4 complex strain had the highest OD value, at 1.87. Therefore, the optimal fermentation conditions were pH 6, inoculum size 8%, rotation speed 200 r / min, temperature 34℃, and fermentation time 36 h.

[0160] Example 8. The preventive and therapeutic effects of "Tuweiwei No. 7" on barley root rot and its growth-promoting effect on barley.

[0161] 1. Preparation of fermentation broth for Tuweiwei No. 7:

[0162] Seed culture medium: 20g soluble starch, 10g NH4Cl, 10g MgSO4·7H2O, 1L water; adjust pH to 7.5 and sterilize.

[0163] Fermentation medium: 10g mannitol, 15g corn steep liquor powder, 15g MnSO4·H2O, 1L water; adjust pH to 6 and sterilize.

[0164] Preparation of compound microbial agents:

[0165] 1. Bacillus belye LB17, K87 and MP6 were inoculated into slant culture medium and cultured on slant culture medium at 30℃ for 24 h to obtain slant culture;

[0166] 2. Slant cultures of Bacillus belyss LB17, K87, and MP6 were inoculated into seed culture medium and cultured at 30℃ and 140 rpm for 24 h to obtain seed culture solutions of Bacillus belyss LB17, K87, and MP6, respectively.

[0167] 3. Mix the seed culture solutions of Bacillus belyssus LB17, K87 and MP6 in a volume ratio of 4:3:6 to obtain a mixed bacterial solution;

[0168] 4. Inoculate the mixed bacterial solution into the fermentation medium at an inoculation rate of 8%, and incubate at 34℃ and 200rpm for 34 hours to obtain the compound bacterial agent.

[0169] 2. The preventive and growth-promoting effects of Tuwei No. 7 on barley root rot.

[0170] The disease prevention experiment consisted of six treatments: CK1, CK2, CK3, and M1, M2, M3, with three replicates and ten plants per replicate. The growth promotion experiment consisted of two treatments: CK4 and M4, with three replicates and ten plants per replicate. Barley seedlings with similar growth were selected, marked, and spores at a concentration of 2 × 10⁻⁶ were inoculated into CK1, CK2, and CK3, respectively. 6 30 mL of a mixed spore suspension of *Fusarium avenaceum* and *Fusarium equiseti* (CFU / mL), 30 mL of a spore suspension of *Microdochium bolleyi*, and 30 mL of a spore suspension of *Bipolaris sorokiniana* were administered. Four days later, the roots of plants CK1, CK2, CK3, and CK4 were drenched with a modified LB broth (liquid fermentation medium) (30 mL / plant). Simultaneously, plants M1, M2, M3, and M4 were treated with 4 × 10⁻⁶ spores. 8 The roots were irrigated with 30 mL of the fermentation liquid of Tuweiwei No. 7 (CFU / mL) and the disease index of barley plants, soil microbial quantity, rhizosphere soil nutrient composition and growth physiological indicators of barley were measured 10 days later.

[0171] Disease index = ∑(Number of disease-severe plants × Representative value) / Total number of plants × Representative value of the most severe disease level × 100

[0172] Prevention efficacy (%) = (Incidence rate in control group - Incidence rate in treatment group) / Incidence rate in control group × 100

[0173] The efficacy of Tuweiwei No. 7 in controlling barley root rot in the disease control experimental group is as follows: Figure 18 As shown in Table 12.

[0174] Table 12. Control efficacy of Tuweiwei No. 7 against barley root rot

[0175] Processing group number Disease index % of the protective effect Plant height / cm Stem diameter / mm CK1 85±3.908a / 19.75±2.894b 0.058±0.010b M1 23.5±4.743b 72.3% 30.11±1.241a 0.114±0.012a CK2 86.5±3.162a / 19.89±3.378b 0.042±0.013b M2 25.25±5.197b 61.2% 30.22±1.276a 0.123±0.010a CK3 86.25±2.124a / 26.63±1.651b 0.051±0.015b M3 21.5±5.676b 75.1% 30.71±1.198a 0.114±0.011a

[0176] Depend on Figure 18 As shown in Table 12, the plant height and stem diameter of barley in treatment groups M1, M2, and M3 were significantly higher than those in control groups M4, M5, and M6 (P<0.05). The plant height was 30.11, 30.22, and 30.71 cm, respectively, which were 10.36 cm, 10.33 cm, and 4.08 cm higher than those in CK1, CK2, and CK3, respectively, with an increase range of 13.28% to 34%. The stem diameter increased by 55% to 65.8%. The control efficacy of Tuweiwei No. 7 against Fusarium root rot, Micrococcus root rot, and common root rot was 72.3%, 61.2%, and 75.1%, respectively.

[0177] Soil microbial quantity determination: Weigh 1 g of soil and dissolve it in 10 mL of sterile water. Shake on a shaker at 180 r / min for 10 min to form a homogeneous soil suspension. Add 1 mL of the soil suspension to a 9 mL sterile water test tube to obtain 10... -1 Following the same operational steps, 10 soil suspensions were obtained. -2 10 -3 10 -4 10 -5 10 -6 Soil suspension.

[0178] (1) Determination of soil bacterial count: 10 -4 10 -5 10 -6 200 μL of soil suspension was inoculated into beef peptone medium and spread evenly on the surface of the beef peptone medium using a spreader. The bacterial count was determined by plate counting.

[0179] (2) Determination of soil fungal abundance: 10 -1 10 -2 10 -3 200 μL of soil suspension was inoculated into Martin's medium and spread evenly on the surface of Martin's medium using a spreader. The number of fungi was determined by plate counting.

[0180] (3) Determination of soil actinomycete count: 10 -310 -4 10 -5 200 μL of soil suspension was inoculated into modified Gao's No. 1 medium and spread evenly on the surface of the modified Gao's No. 1 medium using a spreader. The number of actinomycetes was determined by plate counting method.

[0181] Number of bacterial strains per gram of sample = (C / V) × M (C: represents the average number of colonies growing on a plate at a certain dilution; V: represents the volume of diluent used when plating; M: represents the dilution factor)

[0182] The results of soil microbial abundance determination are shown in Table 13.

[0183] Table 13 Effects of Tuweiwei No. 7 on the number of rhizosphere microorganisms in barley soil

[0184] deal with <![CDATA[Number of bacteria / (×10 7 CFU / g)]]> <![CDATA[Number of actinomycetes / (×10 6 CFU / g)]]> <![CDATA[Number of fungi / (×10 4 CFU / g)]]> CK1 0.913±0.0349b 0.448±0.0186b 7.244±0.0167a M1 3.203±0.0436a 2.139±0.006a 1.827±0.0356b CK2 0.874±0.012b 0.504±0.017b 8.359±0.1111a M2 2.93±0.0425a 1.996±0.015a 1.623±0.0078b CK3 0.755±0.0228b 0.708±0.0056b 8.668±0.0953a M3 3.224±0.1033a 1.918±0.0425a 1.539±0.0152b

[0185] Table 13 shows that the number of bacteria and actinomycetes in the rhizosphere soil of barley in treatment groups M1, M2, and M3 (Tuweiwei 7 + pathogen root irrigation) was significantly higher than that in the control groups CK1, CK2, and CK3 (pathogen root irrigation) (P<0.05). The bacterial counts in M1, M2, and M3 were 3.203 × 10⁻⁶. 7 CFU / g, 2.93×10 7 CFU / g, 3.224×10 7 The CFU / g concentration was significantly higher than that of M4, M5, and M6, with a large increase in bacteria in the rhizosphere soil, ranging from 70.18% to 76.7%, and the number of actinomycetes was 2.138 × 10⁻⁶. 6 CFU / g, 1.996×10 6 CFU / g, 1.918×10 6 CFU / g increased by 1.691×10 6 CFU / g, 1.492×10 6 CFU / g, 1.21×10 6 CFU / g. The soil fungal counts in treatment groups M1, M2, and M3 were significantly lower than those in control groups M4, M5, and M6 (P<0.05). The fungal counts in M1, M2, and M3 were 1.827 × 10⁻⁶. 4 CFU / g, 1.622×10 4 CFU / g, 1.539×10 4 CFU / g decreased by 5.417×10 compared to CK1, CK2, and CK3. 4 CFU / g, 6.736×10 4 CFU / g, 7.129×10 4 CFU / g.

[0186] Determination of the growth-promoting effect of the growth-promoting experimental group on highland barley:

[0187] The growth of highland barley was observed, and the growth-promoting function of Tuweiwei No. 7 was tested in the selected growth-promoting experimental group. Available nitrogen in the rhizosphere soil 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; microbial biomass nitrogen, microbial biomass carbon, and microbial biomass phosphorus were determined using the chloroform fumigation extraction method; superoxide dismutase (SOD) in highland barley leaves was determined using the nitrogen-blue tetrazolium photochemical reduction method; malondialdehyde (MDA) content was determined using the TBA colorimetric method; peroxidase (POD) content was determined using the guaiacol colorimetric method; total chlorophyll content was determined using the dual-wavelength colorimetric method; and auxin IAA content was determined using GA1 by liquid chromatography-mass spectrometry. The results are as follows: Figure 19 As shown in Tables 14 and 15.

[0188] Table 14 Effects of Tuweiwei No. 7 on physiological parameters of barley leaves

[0189] deal with IAA (ng / g) POD (μg / g) SOD (μg / g) MDA (μg / g) Chlorophyll (mg / g) CK4 62.918±0.0463b 336.98±0.0627a 39.739±0.0357b 0.7487±0.0112b 0.3597±0.0067b M4 153.743±0.0197a 257.47±0.0089b 46.5621±0.0357a 1.1609±0.0012a 0.593±0.006a

[0190] Table 15 Effects of Tuweiwei No. 7 on rhizosphere soil nutrients in highland barley

[0191] deal with Available nitrogen mg / kg Available phosphorus mg / kg Available potassium mg / kg Biological nitrogen mg / kg Biochar mg / kg Biological phosphorus mg / kg CK4 438.508±0.004b 98.3503±0.0086b 686.861±0.1006b 18.953±0.0105b 110.6385±0.0035b 4.226±0.0825b M4 462.2517±0.0484a 466.1257±0.1607a 843.7903±0.0058a 38.7873±0.3346a 178.311±0.0198a 10.357±0.0046a

[0192] Figure 19 The results showed that barley seedlings treated with the compound microbial agent grew more densely and vigorously during the seedling stage. Table 14 showed that compared with the control group (CK4), the barley treatment group (M4) treated with Tuweiwei No. 7 compound microbial agent had significantly increased IAA, SOD, MDA, and chlorophyll contents (P < 0.05), while the POD content decreased. Specifically, the IAA content increased by 90.825 ng / g, the SOD content increased from 686.86 μg / g to 843.79 μg / g, the MDA and chlorophyll contents increased by 19.83 μg / g and 67.68 mg / g, respectively, and the POD content decreased from 336.98 μg / g to 257.47 μg / g, indicating that Tuweiwei No. 7 has a growth-promoting effect on barley plants.

[0193] Table 15 shows that the nutrient content of the rhizosphere soil of barley in the treatment group (M4) treated with compound microbial agents was significantly increased compared with that of the control group (CK4) (P < 0.05). The contents of available nitrogen, available phosphorus, and available potassium were all increased, with the most significant increase in available phosphorus. The content in the control group (CK4) was 98.35 mg / kg, while that in the treatment group (M4) was 466.12 mg / kg. The contents of available nitrogen and available potassium increased by 23.75 mg / kg and 156.93 mg / kg, respectively. This indicates that the use of microbial agents helps to improve the nutrient content of the rhizosphere soil of barley. The fertility of the rhizosphere soil of barley in the treatment group (M4) was significantly increased compared with that of the control group (CK4) (P < 0.05). The biogenic phosphorus content of M4 increased by 2.5 times compared with CK4, the biogenic nitrogen content increased from 18.95 mg / kg to 38.79 mg / kg, and the biogenic carbon content increased from 110.64 mg / kg to 178.31 mg / kg, indicating that the use of Tuweiwei No. 7 compound microbial agent helps to improve the fertility of the barley rhizosphere soil.

[0194] Example 9. The control effect of "Tuweiwei No. 7" on lily blight and wilt disease and its growth-promoting effect on lilies.

[0195] Disease prevention experimental group: Lily bulbs were planted, and lilies with uniform growth were set up as CK1 and J1 treatments, with 3 replicates and 10 plants in each replicate.

[0196] Growth-promoting experimental group: Lily bulbs were planted, and lilies with uniform growth were divided into CK2 and J2 treatments, with 3 replicates and 10 plants in each replicate.

[0197] Select lily seedlings that have grown uniformly 20 days after planting, and inoculate J1 and CK1 seedlings with spore concentrations of 2×10⁻⁶. 6 30 mL of a mixed spore suspension of *F. oxysporum* and *F. solani* (CFU / mL) was inoculated with the pathogens. After 13 and 19 days, the roots of CK1 and CK2 were drenched with a modified LB broth (liquid fermentation medium) (30 mL / plant). The solution was then applied at a concentration of 4 × 10⁻⁶ mg / mL. 8 The roots of lily plants were irrigated with CFU / mL of Tuweiwei No. 7 (30 mL / plant) for 20 days. The disease index of lily plants, the number of soil microorganisms, the nutrient composition of lily rhizosphere soil and the physiological indicators of lily growth were measured. The measurement methods were the same as in Example 8.

[0198] The results of the disease index test and the growth status of the lily plants are as follows: Figure 20 As shown in Table 16.

[0199] Table 16. The efficacy of Tuweiwei No. 7 against lily root rot

[0200] Processing group number Disease index % of the protective effect Plant height / cm Stem diameter / mm CK1 87.5±12.076a / 30.23±1.421a 0.328±0.025a J1 27.5±9.884b 68.6% 10.55±1.319b 0.265±0.013b

[0201] The results are shown in Table 16. The plant height and stem diameter of lily in treatment group J1 (Tuweiwei No. 7 + pathogen root irrigation) were significantly higher than those in control group CK1 (pathogen root irrigation) (P<0.05). The plant height and stem diameter of J1 were 30.23 cm and 0.328 cm, respectively, which were 65.1% higher than those of CK1 and 19.2% higher than those of CK1. The control efficacy of Tuweiwei No. 7 compound microbial agent against lily root rot was 68.6%.

[0202] The number of rhizosphere microorganisms in lilies is shown in Table 17. Table 17 shows that the number of soil bacteria and actinomycetes in treatment group J1 (Tuweiwei 7 + pathogen root irrigation) was significantly higher than that in the control group CK1 (pathogen root irrigation) (P<0.05). The bacterial count in J1 was 2.82 × 10⁻⁶. 7 CFU / g increased by 2.131 × 10 compared to CK1. 7 CFU / g; Actinomycete count was 1.074 × 10⁻⁶. 6 CFU / g increased by 0.563×10 6 CFU / g. The number of soil fungi in treatment group J1 was significantly lower than that in the control group CK1 (P<0.05), with a fungal count of 1.156 × 10⁻⁶. 4 CFU / g decreased by 5.321 × 10 compared to CK1. 4 CFU / g.

[0203] The effect of Tuwei No. 7 on promoting lily growth, such as Figure 21 As shown in Table 17, the physiological indicators of the two groups of lily leaves were measured. The nutritional components in the lily leaves were determined using the Coomassie brilliant blue colorimetric method to determine the soluble protein content in the lily bulbs; the reducing sugar content was determined using the DNS colorimetric method; and the total dietary fiber content in the lily bulbs was determined using an enzymatic method. The results are shown in Table 18.

[0204] Table 17 Effects of Tuwei No. 7 on physiological parameters of lily leaves

[0205] deal with IAA (ng / g) POD (μg / g) SOD (μg / g) MDA (μg / g) Chlorophyll (mg / g) CK2 0.2877±0.0031b 4.05±0.0021a 37.582±0.1204b 0.4913±0.0035b 0.3573±0.0057b J2 1.5783±0.004a 3.315±0.0092b 46.6187±0.0047a 0.9299±0.0035a 0.6713±0.0035a

[0206] Figure 21The results showed that the lily seedlings treated with the compound microbial inoculum grew rapidly and vigorously. Table 17 shows that, compared with the control group (CK2), the lily treatment group (J2) treated with Tuweiwei No. 7 compound microbial agent showed a significant increase in IAA, SOD, MDA, and chlorophyll content (P < 0.05), while the POD content decreased. Specifically, the IAA content increased significantly by 5 times, from 0.287 ng / g to 1.578 ng / g. The SOD, MDA, and chlorophyll contents also increased, by 9.036 μg / g, 19.83 μg / g, and 67.68 mg / g, respectively. The POD content decreased by 0.735 μg / g.

[0207] Table 18. Effects of Tuweiwei No. 7 on Lily Quality

[0208] deal with Soluble protein (mg / g) Reducing sugar (mg / g) Total dietary fiber (%) CK2 2.9751±0.1925b 4.4503±0.203b 16.9995±0.0092b J2 3.9407±0.5078a 5.8316±0.2809a 22.6045±0.4236a

[0209] Table 18 shows that, compared with the control group (CK2), the lily treatment group (J2) treated with Tuweiwei No. 7 compound microbial agent had significantly increased soluble protein, reducing sugar, and total dietary fiber content (P < 0.05). The soluble protein content increased from 2.97 mg / g to 3.94 mg / g, the reducing sugar content increased from 4.45 mg / g to 5.83 mg / g, and the total dietary fiber increased by 5.61%. This indicates that the compound microbial agent is beneficial for improving the quality of lilies.

[0210] The number of microorganisms in the rhizosphere of lilies is shown in Table 19, and the nutrient composition of the rhizosphere soil of lilies is shown in Table 20.

[0211] Table 19. Effects of Tuweiwei No. 7 on the number of rhizosphere microorganisms in lilies.

[0212] deal with Bacterial count Actinomycete count Fungal count CK1 0.689±0.0058b 0.511±0.0383b 6.477±0.0976a J1 2.820±0.0299a 1.074±0.0082a 1.156±0.0128b

[0213] Table 19 shows that the number of soil bacteria and actinomycetes in treatment group J1 (Tuweiwei No. 7 + pathogen root irrigation) was significantly higher than that in the control group CK1 (pathogen root irrigation) (P<0.05), with the bacterial count in J1 being 2.82×10⁻⁶. 7 CFU / g increased by 2.131 × 10 compared to CK1. 7 CFU / g; Actinomycete count was 1.074 × 10⁻⁶. 6 CFU / g increased by 0.563×10 6 CFU / g. The number of soil fungi in treatment group J1 was significantly lower than that in the control group CK1 (P<0.05), with a fungal count of 1.156 × 10⁻⁶. 4 CFU / g decreased by 5.321 × 10 compared to CK1. 4 CFU / g.

[0214] Table 20 Effects of Tuweiwei No. 7 on the nutrient composition of rhizosphere soil of lily

[0215] deal with Available nitrogen (mg / kg) Available phosphorus (mg / kg) Available potassium (mg / kg) Biological nitrogen (mg / kg) Biochar (mg / kg) Biological phosphorus (mg / kg) CK2 460.027±0.0046b 935.1107±0.1138b 1973.8115±0.0399b 37.5065±0.0078b 283.7745±0.0375b 22.6527±0.0045b J2 460.7563±0.1216a 939.9817±0.0552a 2083.7253±0.0122a 47.3835±0.0035a 341.2535±0.0064a 29.0077±0.0045a

[0216] Compared to the control group (CK2), the nutrient content of the rhizosphere soil in the treatment group (J2) treated with compound microbial agents was significantly increased (P < 0.05). For example, the contents of available nitrogen, available phosphorus, and available potassium all increased, with available phosphorus increasing by 4.87 mg / kg and available potassium increasing from 1973.81 mg / kg to 2083.73 mg / kg, while available nitrogen only increased by 0.73 mg / kg. This indicates that the use of microbial agents can improve the nutrient content of the rhizosphere soil in lilies. Compared to the control group (CK2), the fertility of the rhizosphere soil in the treatment group (J2) of highland barley was significantly increased (P < 0.05). Specifically, the contents of biogenic phosphorus and biogenic nitrogen in J2 increased from 37.50 mg / kg to 47.38 mg / kg, biogenic carbon increased by 57.48 mg / kg, and biogenic phosphorus increased from 22.65 mg / kg to 2901 mg / kg, indicating that the use of microbial agents helps improve the fertility of the rhizosphere soil in lilies.

[0217] Example 10. The effect of "Tuweiwei No. 7" on the prevention and control of tomato root rot and its effect on promoting tomato growth.

[0218] Tomato seedlings with similar growth were selected. The disease control experimental group consisted of two treatments, CK1 and X1, with three replicates (10 seedlings per replicate); and two treatments, CK2 and X2, with three replicates (10 seedlings per replicate). Fifteen days after planting, X1 and CK1 were inoculated with spores at a concentration of 2 × 10⁻⁶. 6 A mixed spore suspension of *F. oxysporum* and *F. solani* (CFU / mL) was administered in 30 mL. Ten and 21 days after inoculation with the pathogens, the roots of plants CK1 and CK2 were drenched with a modified LB broth (liquid fermentation medium) (30 mL / plant). 8 Two soil samples were irrigated with CFU / mL Tuweiwei No. 7 (30 mL / plant). After 20 days, the disease index of tomato plants, the number of soil microorganisms, the nutrient composition of tomato rhizosphere soil, and the physiological indicators of tomato growth were measured. The measurement methods were the same as in Example 8. The soil samples were stored at low temperature and transported back to the laboratory.

[0219] The growth status of tomatoes in the disease prevention experimental group is as follows: Figure 22 As shown in Table 21, the incidence of tomato root rot and the growth of tomato plants are as follows.

[0220] Table 21. Efficacy of Tuweiwei No. 7 against Tomato Root Rot

[0221] Processing group number Disease index % of the protective effect Plant height / cm Stem diameter / mm CK1 80±19.720a / 14.28±1.725b 0.128±0.011b X1 10±12.10b 79.87% 22.51±5.296a 0.208±0.018a

[0222] Figure 22The results showed that the tomato plant height and stem diameter of the treatment group X1 (Tuweiwei No. 7 + pathogen root irrigation) were significantly higher than those of the control group CK1 (pathogen root irrigation) (P<0.05). As shown in Table 21, the plant height and stem diameter of X1 were 22.51 cm and 0.208 cm, respectively, which were 36.5% higher than those of CK1 and 38.5% higher than those of CK1. The control efficacy of Tuweiwei No. 7 against tomato root rot was 79.87%.

[0223] The effects of Tuweiwei No. 7 on rhizosphere soil microorganisms of tomatoes are shown in Table 22.

[0224] Table 22 Effects of Tuweiwei No. 7 on the number of rhizosphere microorganisms in tomatoes

[0225] deal with <![CDATA[Number of bacteria / (×10 7 CFU / g)]]> <![CDATA[Number of actinomycetes / (×10 6 CFU / g)]]> <![CDATA[Number of fungi / (×10 4 CFU / g)]]> CK1 0.628±0.0033b 0.527±0.0234b 7.677±0.0046a X1 2.623±0.044a 1.531±0.0291a 1.251±0.045b

[0226] As shown in Table 22, the number of soil bacteria and actinomycetes in treatment group X1 (Tuweiwei No. 7 + pathogen root irrigation) was significantly higher than that in control group CK1 (pathogen root irrigation) (P<0.05). The bacterial count in X1 was 2.623×10⁻⁶. 7 CFU / g increased by 1.995×10 compared to CK1. 7 CFU / g; Actinomycete count was 1.531 × 10⁻⁶. 6 CFU / g increased by 1.004 × 10⁻⁶ 6 CFU / g. The number of soil fungi in treatment group X1 was significantly lower than that in the control group CK1 (P<0.05), with a fungal count of 1.251×10⁻⁶. 4 CFU / g decreased by 6.426 × 10 compared to X2. 4 CFU / g.

[0227] The growth status of tomatoes in the growth-promoting experimental group was as follows: Figure 23 As shown in Table 23, the effects of Tuweiwei No. 7 on the nutritional components of tomato leaves are illustrated in the table.

[0228] Table 23 Effects of Tuweiwei No. 7 on physiological indicators of tomato leaves

[0229] deal with IAA (ng / g) POD (μg / g) SOD (μg / g) MDA (μg / g) Chlorophyll (mg / g) CK2 60.354±0.023b 357.349±0.037a 41.644±0.025b 0.851±0.043b 0.362±0.016b X2 113.711±0.171a 243.677±0.011b 45.656±0.031a 1.183±0.020a 0.555±0.006a

[0230] Figure 23The results showed that tomato seedlings treated with the Tuweiwei No. 7 compound microbial agent grew vigorously, with significantly increased plant height compared to the control group. Table 23 shows that, compared to the control group (CK2), the tomato treatment group (X2) treated with the compound microbial agent showed significant increases in IAA, SOD, MDA, and chlorophyll content (P < 0.05), while POD content decreased. Specifically, IAA content increased significantly by 1.8 times, from 60.354 ng / g to 113.711 ng / g, MDA increased from 0.851 μg / g to 1.183 μg / g, SOD increased only by 4.012 μg / g, chlorophyll content increased from 0.362 mg / g to 0.555 mg / g, and POD content decreased by 113.672 μg / g.

[0231] The effects of Tuweiwei No. 7 compound microbial agent on the nutrient composition of tomato rhizosphere soil are shown in Table 24.

[0232] Table 24 Effects of Tuweiwei No. 7 on rhizosphere soil nutrients in tomatoes

[0233] deal with Available nitrogen mg / kg Available phosphorus mg / kg Available potassium mg / kg Biological nitrogen mg / kg Biochar mg / kg Biological phosphorus mg / kg CK2 450.3797±0.023b 122.359±0.012b 592.656±0.016b 30.095±0.016b 122.46±0.028b 5.167±0.004b X2 460.591±0.0285a 210.562±0.021a 808.687±0.067a 38.091±0.009a 180.339±0.020a 25.759±0.019a

[0234] Table 24 shows that the nutrient content of the tomato rhizosphere soil in the treatment group (X2) treated with compound microbial agents was significantly increased compared with the control group (CK2) (P < 0.05). For example, the contents of available nitrogen, available phosphorus, and available potassium all increased. The available phosphorus content increased from 122.359 mg / kg to 210.562 mg / kg, and the available potassium content increased from 592.656 mg / kg to 808.687 mg / kg, while the available nitrogen content only increased by 10.212 mg / kg. This indicates that the use of microbial agents can improve the nutrients in the tomato rhizosphere soil. The fertility of the tomato rhizosphere soil in the treatment group (X2) was significantly increased compared with the control group (CK2) (P < 0.05). The biogenic phosphorus and biogenic nitrogen content of X2 increased from 30.095 mg / kg to 38.091 mg / kg, the biogenic carbon content increased by 57.879 mg / kg, and the biogenic phosphorus content increased from 5.167 mg / kg to 25.759 mg / kg, indicating that the use of microbial agents helps to improve the fertility of the tomato rhizosphere soil.

[0235] Example 11. The control effect of "Tuweiwei No. 7" on pepper root rot and its effect on promoting pepper growth.

[0236] Pepper seedlings with similar growth were selected and divided into two treatments, CK1 and L1, with three replicates and 10 seedlings per replicate. Two treatments, CK2 and L2, were also selected with three replicates and 10 seedlings per replicate. Fifteen days after planting, L1 and CK1 were inoculated with spores at a concentration of 2 × 10⁻⁶. 630 mL of a mixed spore suspension of *F. oxysporum* and *F. solani* (CFU / mL) was inoculated with the pathogens. After 10 and 21 days, the roots of CK1 and CK2 were drenched with a modified LB broth (liquid fermentation medium) (30 mL / plant). A 5×10⁻⁶ dose was applied. 8 The soil samples were irrigated with CFU / mL of Tuweiwei No. 7 (30 mL / plant) for L1 and L2. After 20 days, the plant disease index, soil microbial quantity, tomato rhizosphere soil nutrient composition and tomato growth physiological indicators were measured. The measurement methods were the same as in Example 10. The soil samples were stored at low temperature and transported back to the laboratory.

[0237] The growth of tomato plants in the disease prevention experimental group is as follows: Figure 24 As shown in Table 25, the efficacy of Tuweiwei No. 7 against root rot of pepper is as follows.

[0238] Table 25. Efficacy of Tuweiwei No. 7 against root rot of chili peppers

[0239] Processing group number Disease index % of the protective effect Plant height t / cm Stem diameter / mm CK1 72.5±24.861a / 26.03±0.893b 0.178±0.012b L1 15±17.480b 79.3% 30.99±1.839a 0.22±0.014a

[0240] Figure 24 Table 25 shows that the plant height and stem diameter of peppers in treatment group L1 (Tuweiwei No. 7 + pathogen root irrigation) were significantly higher than those in control group CK1 (pathogen root irrigation) (P<0.05). The plant height and stem diameter of L1 were 30.99 cm and 0.22 cm, respectively, which were 19% higher than those of CK1 and 23.6% higher than those of CK1. The control efficacy of Tuweiwei No. 7 against pepper root rot was 79.3%.

[0241] The effects of Tuweiwei No. 7 on rhizosphere soil microorganisms of chili peppers are shown in Table 26.

[0242] Table 26 Effects of Tuweiwei No. 7 on the number of microorganisms in the rhizosphere soil of pepper.

[0243] deal with <![CDATA[Number of bacteria / (×10 7 CFU / g)]]> <![CDATA[Number of actinomycetes / (×10 6 CFU / g)]]> <![CDATA[Number of fungi / (×10 4 CFU / g)]]> CK1 0.92±0.0062b 0.467±0.0231b 7.3607±0.0459a L1 2.404±0.0080a 1.487±0.0208a 1.38±0.0486b

[0244] As shown in Table 26, the number of soil bacteria and actinomycetes in treatment group L1 (Tuweiwei No. 7 + pathogen root irrigation) was significantly higher than that in control group CK1 (pathogen root irrigation) (P<0.05). The bacterial count in L1 was 2.404×10⁻⁶. 7 CFU / g increased by 1.484 × 10 compared to CK1. 7 CFU / g; Actinomycete count was 1.487 × 10⁻⁶. 6 CFU / g increased by 1.02 × 10⁻⁶ 6 CFU / g. The number of soil fungi in treatment group L1 was significantly lower than that in the control group CK1 (P<0.05), with a fungal count of 1.38×10⁻⁶. 4CFU / g decreased by 5.981 × 10 compared to CK1. 4 CFU / g.

[0245] The growth status of peppers in the growth-promoting experimental group is as follows: Figure 25 As shown in Table 27, the effects of Tuweiwei No. 7 on the nutritional components of chili pepper leaves are as follows.

[0246] Table 27 Effects of Tuweiwei No. 7 on physiological parameters of pepper leaves

[0247] deal with IAA (ng / g) POD (μg / g) SOD (μg / g) MDA (μg / g) Chlorophyll (mg / g) CK2 55.854±0.029b 367.270±0.016a 36.334±5.774b 0.66±0.006b 0.355±0.004b L2 72.561±0.011a 221.773±0.024b 44.763±0.021a 1.159±0.018a 0.673±0.007a

[0248] As shown in Table 27, compared with the control group (CK2), the pepper treatment group (L2) treated with Tuweiwei No. 7 showed significant increases in IAA, SOD, MDA, and chlorophyll contents (P < 0.05), while the POD content decreased. Specifically, the IAA content increased from 55.854 ng / g to 72.561 ng / g, and the contents of SOD, MDA, and chlorophyll all increased, by 8.429 μg / g, 0.499 μg / g, and 0.318 mg / g, respectively. Meanwhile, the POD content decreased from 367.37 μg / g to 221.773 μg / g, indicating that Tuweiwei No. 7 has a growth-promoting effect and enhances the stress resistance of pepper plants.

[0249] The effects of Tuweiwei No. 7 on the rhizosphere soil nutrients of chili peppers are shown in Table 28.

[0250] Table 28 Effects of Tuweiwei No. 7 on rhizosphere soil nutrients in chili peppers

[0251] deal with Available nitrogen (mg / kg) Available phosphorus (mg / kg) Available potassium (mg / kg) Biological nitrogen (mg / kg) Biochar (mg / kg) Biological phosphorus (mg / kg) CK2 458.649±0.031b 466.292±0.372b 471.756±0.120b 37.634±0.163b 212.455±0.021b 5.943±0.018b L2 462.894±0.064a 935.251±0.036a 903.773±0.008a 41.636±0.018a 318.459±0.014a 31.670±0.041a

[0252] Table 28 shows that the rhizosphere soil nutrients in the treatment group (L2) treated with Tuwei No. 7 were significantly increased compared to the control group (CK2) (P < 0.05). For example, the contents of available nitrogen, available phosphorus, and available potassium all increased. Available phosphorus increased from 466.292 mg / kg to 935.251 mg / kg, available potassium increased from 471.756 mg / kg to 903.773 mg / kg, while available nitrogen only increased by 4.254 mg / kg. This indicates that the application of the microbial agent can improve the rhizosphere soil nutrients of chili peppers. The rhizosphere soil fertility in the treatment group (L2) was significantly increased compared to the control group (CK2) (P < 0.05). The biogenic phosphorus and biogenic nitrogen content of L2 increased from 37.634 mg / kg to 41.636 mg / kg, the biogenic carbon content increased from 212.455 mg / kg to 318.459 mg / kg, and the biogenic phosphorus content increased from 5.943 mg / kg to 31.670 mg / kg, indicating that the use of microbial agents helps to improve the fertility of the rhizosphere soil of chili peppers.

[0253] As can be seen from the above embodiments, the present invention provides Bacillus berleis LB17, K87, MP6 and their compound microbial agent "Tuweiwei No. 7". This compound microbial agent can effectively prevent and control crop root rot and wilt in the special habitat of Gansu and Qinghai, and can promote crop growth and provide crop resistance.

[0254] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex microbial agent, characterized in that, Bacillus belesiensis ( Bacillus velezensis LB17, Bacillus belysinus ( Bacillus velezensis K87 and Bacillus belesii ( Bacillus velezensis MP6 was prepared by mixed fermentation; Bacillus velezensis (Bacillus velezensis) Bacillus velezensis LB17, Bacillus velezensis (Bacillus velezensis) Bacillus velezensis K87, and Bacillus velezensis (Bacillus velezensis) Bacillus velezensis MP6 are preserved in China Center for Type Culture Collection, and the preservation numbers are CCTCC NO: M 20242190, CCTCC NO: M 20242191, and CCTCC NO: M 20242192, respectively.

2. The preparation method of the complex bacterial agent of claim 1, characterized in that, comprising the following steps: S1. respectively inoculating Bacillus velezensis LB17, K87, MP6 into slant culture medium, and culturing the slant culture medium at 28-32°C for 23-25h to obtain a slant culture; S2. respectively inoculating the slant culture of Bacillus velezensis LB17, K87, MP6 into seed liquid culture medium; culturing the seed liquid culture medium at 28-32°C, 130-150rpm for 22-26h to obtain a seed culture of Bacillus velezensis LB17, K87, MP6; S3. mixing the seed culture of Bacillus velezensis LB17, K87, MP6 to obtain a mixed bacterial liquid; S4. inoculating the mixed bacterial liquid into fermentation culture medium at an inoculation amount of 7-9%, and culturing the fermentation culture medium at 32-36°C, 180-220rpm for 32-36h to obtain the product.

3. The preparation method according to claim 2, characterized in that, The volume ratio of the seed culture of Bacillus velezensis LB17, K87, MP6 is 3.5-4.5:2.5-3.5:5.5-6.

5.

4. The preparation method according to claim 2, characterized in that, The seed liquid culture medium comprises the following components at the following concentrations: soluble starch 19-21g / L, NH4Cl 9-11g / L, MgSO4·7H2O 9-11g / L, and the balance being water.

5. The preparation method according to claim 2, characterized in that, The fermentation culture medium comprises the following components at the following concentrations: mannitol 9-11g / L, corn syrup powder 14-16g / L, MnSO4·H2O 14-16g / L, and the balance being water.

6. The use of the complex microbial agent of claim 1 in at least one of the following: (1) application in preventing and treating plant pathogenic fungi; (2) application in preventing and treating plant diseases; (3) application in promoting plant growth and improving plant stress resistance; (4) application in increasing the number of beneficial microorganisms in soil; (5) application in improving the fertility of rhizosphere soil of plants.

7. Use according to claim 6, characterized in that, The plant pathogenic fungi include Fusarium avenaceum, F. equiseti, F. oxysporum, F. solani, P. gramineum, or M. phaseolina; the plant diseases include root rot and fusarium wilt.

8. A strain of Bacillus velezensis (LB17), characterized in that, Bacillus velezensis ) LB17, characterized in that, It is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20242190, the preservation date October 14, 2024, and the preservation address Wuhan University.

9. A strain of Bacillus velezensis (B. velezensis) K87, characterized by, Bacillus velezensis ) K87, characterized by, It is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20242191, the preservation date October 14, 2024, and the preservation address Wuhan University.

10. A Bacillus velezensis (Bacillus velezensis) MP6 strain characterized by, Bacillus velezensis ) MP6, characterized in that, It is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20242192, the preservation date October 14, 2024, and the preservation address Wuhan University.

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