Bacillus velezensis LB17, K87 and MP6 as well as complex microbial inoculant, preparation method and application of bacillus velezensis LB17, K87 and MP6

Abstract

The invention provides bacillus velezensis LB17, K87 and MP6 as well as a complex microbial inoculant, a preparation method and application thereof, and belongs to the technical field of biocontrol bacteria. The method comprises the following steps: separating rhizosphere and rhizosphere soil of characteristic crops such as highland barley, lily, tomato and pepper collected in the Ganqing region to obtain 305 pure cultures, carrying out multiple screening to obtain three strains of bacillus velezensis LB17, K87 and MP6, and optimizing the inoculum size, the components and proportion of a culture medium and fermentation conditions to obtain the bacillus velezensis LB17, K87 and MP6 strains of bacillus velezensis LB17, K87 and MP6 strains of bacillus velezensis LB17, K87 and MP6 strains of bacillus velezensis. And finally, a compound fermentation inoculant, namely the Taiji No.7, which is jointly fermented by three strains of bacillus velezensis LB17, K87 and MP6 is obtained. The Taipian 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 the plants. The complex microbial inoculant provided by the invention provides a powerful guarantee for prevention and control of root rot diseases of special habitat crops in the Ganqing region.

Description

Technical Field

[0001] The invention relates to the technical field of biocontrol bacteria, and in particular to Bacillus velez LB17, K87, MP6 and composite bacterial agents thereof, as well as a preparation method and application thereof. Background Art

[0002] Gansu and Qinghai (Gansu and Qinghai) are located at a high altitude, with sufficient sunshine, low annual average temperature and large temperature difference, which are suitable for the planting and growth of highland barley, lily and various plateau summer vegetables. Among them, highland barley likes cool and cold-resistant weather, and is distributed in Gannan Tibetan Autonomous Prefecture of Gansu Province, the surrounding areas of Xining City of Qinghai Province, Haibei Prefecture, Hainan Prefecture, Haixi Prefecture, Huangnan Prefecture, Yushu Prefecture and Guoluo Prefecture. It is rich in nutritional value, has the functions of improving human immunity and preventing cardiovascular and cerebrovascular diseases, and is the main food crop for the local Tibetan people. Lanzhou lily likes cool and humid weather, and is mainly distributed in Lanzhou City and surrounding areas. It has a sweet and crisp taste, reduces visual fatigue, has anti-oxidation, regulates the human immune system, and stabilizes blood sugar. It is a famous health food. Peppers and tomatoes are the main categories of plateau summer vegetables in Gansu Province, and their contribution rate to the increase in income of vegetable farmers exceeds 60%, which is the pillar industry of local rural areas. However, due to the many mountains in Gansu and Qinghai, the agricultural area accounts for a small proportion of the province's area, and the cultivated area accounts for 12.62% and 2.84% of the province's area respectively. Continuous cropping is serious, and soil-borne diseases such as root rot are prone to occur.

[0003] Root rot diseases are common on highland barley. Studies have found that four diseases, including Fusarium root rot, common root rot, Microdochium root rot, and Clonostachys rosea, are common types of root rot of highland barley in Gansu and Qinghai. Fusarium root rot is the most widely distributed and has a variety of pathogens, with an average incidence of about 20%. Its dominant pathogens are Fusarium avenaceum and Fusarium equiseti. The average incidence of common root rot is 5% to 15%, and the dominant pathogen is Bipolarissorokiniana. The pathogens of Microdochium root rot and Clonostachys rosea are Microdochium bolleyi and Clonostachys rosea, respectively. They only occur sporadically in Qinghai, but they are serious and have a mortality rate of 100%. Lily root rot diseases are serious and hidden, including two common disease types: wilt and root rot. The wilt disease is widely distributed and has a high incidence rate, reaching more than 70% in some areas. Fusarium oxysporum is often the dominant pathogen group and has strong pathogenicity. The incidence rate of lily root rot is about 25%, among which Fusarium oxysporum lily-specific type is the dominant pathogen of lily root rot. The incidence rate of root rot of vegetables such as peppers and tomatoes is generally between 20% and 30%. When peppers are infected with root rot, the yield is reduced by more than 60% in severe cases. When tomatoes are infected with root rot, the yield is generally reduced by 60% to 90%, and even no yield is produced. The dominant pathogens are Fusarium oxysporum and Fusarium oxysporum.

[0004] Biocontrol bacteria refer to a type of beneficial microorganisms that can kill or reduce the number of pathogens to control the occurrence and development of plant diseases and can be used to prevent and control plant diseases. Its essence is to use the antibiotic, competitive, parasitic, and bacteriolytic effects between or within microbial species, or through the active metabolites produced by beneficial microorganisms to inhibit the survival and activity of certain pathogenic microorganisms. The main types of biocontrol bacteria are bacteria, fungi, and actinomycetes. Bacteria have the characteristics of many species, high reproductive capacity, complex metabolic activities and many products, diverse modes of action on pathogens, short life cycle, and easy artificial cultivation. Biocontrol bacteria and their metabolites play an important role in both naturally occurring biological control and human application of biological control activities.

[0005] Bacillus sp. has the advantages of fast reproduction and metabolism, strong vitality, large bacterial cells, ability to produce spores with strong stress resistance, long storage period, and convenient use. It is an ideal biocontrol microorganism and has been widely used in agricultural production. Among them, Bacillus velezensis is a new species of Bacillus, which has the effect of promoting plant growth and inhibiting plant pathogens in agriculture, and has broad application prospects in the field of biological control. However, microbial agents are mostly live bacteria products, with strong regionality, and it is difficult to adapt to the introduction of agents, and the stability is poor. In addition, Gansu and Qinghai are in special environments such as cold drought and high altitude. Existing biocontrol agents are difficult to achieve the prevention and control effect on the root rot and root rot of crops such as highland barley, lily, and vegetables in the special habitats of Gansu and Qinghai. Therefore, the development of effective biocontrol microorganisms and microbial agents in the special habitats of Gansu and Qinghai is of great significance for ensuring the safety of agricultural production and the quality of agricultural products. Summary of the invention

[0006] The purpose of the present invention is to provide Bacillus Velez LB17, K87, MP6 and composite bacterial agent thereof, so as to solve the problem that root rot is difficult to prevent and control in special habitats in Gansu and Qinghai regions.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a composite bacterial agent, which is prepared by mixed fermentation of Bacillus Velez LB17, Bacillus Velez K87 and Bacillus Velez MP6.

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

[0010] S1. Inoculate Bacillus Velezii LB17, K87, and MP6 into slant culture medium respectively, and culture the slant culture at 28-32° C. for 23-25 ​​h to obtain slant culture;

[0011] S2. The slant cultures of Bacillus Velez LB17, K87, and MP6 were inoculated into the seed liquid medium respectively; the culture was carried out at 28 to 32° C. and 130 to 150 rpm for 22 to 26 hours to obtain the seed culture liquid of Bacillus Velez LB17, K87, and MP6;

[0012] S3. The seed culture solution of Bacillus Velez LB17, K87 and MP6 was mixed to obtain a mixed bacterial solution;

[0013] S4. Inoculate the mixed bacterial liquid into the fermentation medium at an inoculation rate of 7-9%, and culture at 32-36° C. and 180-220 rpm for 32-36 hours.

[0014] Preferably, the volume ratio of the seed culture solution of Bacillus velez LB17, K87, and MP6 is: 3.5-4.5:2.5-3.5:5.5-6.5.

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

[0016] Preferably, the fermentation medium comprises components in the following concentrations: mannitol 9-11 g / L, corn syrup powder 14-16 g / L, MnSO4·H2O 14-16 g / L, and the remainder is water.

[0017] The present invention also provides the use of the composite bacterial agent in at least one of the following:

[0018] (1) Application in the prevention and treatment 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 solani, Fusarium equisetum, Fusarium oxysporum, Fusarium solani, Fusarium ovale or Microsporum spp.; and the plant diseases include root rot and wilt.

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

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

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

[0027] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention isolates 305 pure cultures from the rhizosphere and rhizosphere soil of characteristic crops such as highland barley, lily, tomato and pepper collected in Gansu and Qinghai, obtains three strains of Bacillus Velez LB17, K87 and MP6 through multiple screening, and finally obtains a composite fermentation agent, Tuweiwei No. 7, which is fermented by three strains of Bacillus Velez LB17, K87 and MP6 by optimizing the inoculation amount, components, ratio and fermentation conditions of the culture medium. Tuweiwei No. 7 can effectively prevent pathogenic microorganisms and plant diseases such as root rot and wilt, and can promote plant growth and improve its stress resistance. The composite bacterial agent provided by the present invention provides a strong guarantee for the prevention and control of root rot diseases of crops in special habitats in Gansu and Qinghai. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing the antibacterial effect of re-screening of some antagonistic strains.

[0029] Figure 2 The growth of strains in different salt concentration gradients.

[0030] Figure 3 This is a comparative diagram of safety testing of some strains.

[0031] Figure 4 A graph showing the acid resistance of each strain.

[0032] Figure 5 This is a graph showing the alkali resistance of each strain.

[0033] Figure 6 The graph shows the salt tolerance of each strain.

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

[0035] Figure 8 It is the inhibition rate of excellent composite bacterial system against pathogenic fungi.

[0036] Fig. 9 This is a phylogenetic tree based on 16S rDNA universal primer sequences.

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

[0038] Fig.11 This is a graph showing the experimental results for optimizing the best ratio of composite bacteria.

[0039] Fig.12 Figure 2 shows the results of the seed culture medium composition optimization experiment.

[0040] Fig.13 This is a graph showing the experimental results for optimizing the ratio of seed culture medium components.

[0041] Fig.14 This is a graph showing the experimental results for optimizing the fermentation conditions of the seed culture medium.

[0042] Fig.15 This is a graph showing the experimental results for optimizing the fermentation medium composition.

[0043] Fig.16 This is a graph showing the experimental results for optimizing the ratio of fermentation medium components.

[0044] Fig.17 This is a diagram of the experimental results for optimizing fermentation culture conditions.

[0045] Fig.18 These are some illustrations of the disease-resistant effects of Tuweiwei No. 7 on highland barley.

[0046] Fig.19 These are some illustrations of the growth-promoting effect of Tuweiwei No. 7 on highland barley.

[0047] Fig. 20 These are some illustrations of the disease-resistant effects of Tuweiwei No. 7 on lily.

[0048] Fig.21 These are some illustrations of the growth-promoting effects of Tuweiwei No. 7 on lily.

[0049] Fig. 22 These are some illustrations of the disease-resistant effects of Tuweiwei No. 7 on tomatoes.

[0050] Fig.23 These are some illustrations of the growth-promoting effects of Tuweiwei No. 7 on tomatoes.

[0051] Fig.24 These are some illustrations of the disease-resistant effects of Tuweiwei No. 7 on peppers.

[0052] Fig.25 These are some illustrations of the growth-promoting effect of Tuweiwei No. 7 on pepper.

[0053] Microorganisms Deposit Instructions

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

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

[0056] The Bacillus velezensis MP6 provided by the present invention is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242192, a deposit date of October 14, 2024, and a deposit address of Wuhan University. DETAILED DESCRIPTION

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

[0058] Example 1. Strain screening

[0059] 1. Sample collection and initial screening

[0060] The rhizosphere and rhizosphere soil of highland barley, lily, tomato and pepper, characteristic crops in Gansu and Qinghai, were collected and transported to the laboratory at low temperature. The rhizosphere soil bacteria were initially isolated by the plate spreading method, and then the colonies with different morphologies were purified by the plate streak method. The culture medium used was LB medium (Luria-Bertani), and a total of 305 pure cultures were obtained.

[0061] 2. Initial screening of functional strains

[0062] 1. Initial screening of antagonistic bacteria

[0063] After 305 test strains were activated by the plate streak method at 30°C for 48 h, the plate confrontation method was used to screen strains with antagonistic function, that is, the activated pathogenic fungus cake (d = 0.6 cm) was inoculated in the center of the PDA culture medium (the test pathogenic fungi included: Fusarium avenaceum, Fusarium equiseti, Fusarium Oxysporum, Fusarium solani, Bipolaris sorokiniana, Microdochium bolleyi, all from the Economic Crop Disease Research Laboratory, Institute of Plant Protection, Gansu Academy of Agricultural Sciences), and activated bacteria were inoculated at equal distances around the culture medium. After culturing in a constant temperature incubator at 25°C for 7 days, the diameter of the inhibition zone was determined.

[0064] The results are shown in Table 1, and a total of 167 antagonistic bacteria were screened. Among them, 15 strains had good antagonistic effects on Fusarium avenae, and the diameter of K87 inhibition zone was the largest, which was 15.28 mm, and the diameters of the inhibition zones of the other strains were between 8.78 mm and 15.11 mm. There were 54 strains with good antagonistic effects on Fusarium equisetifolia, of which 21 had an inhibition zone diameter greater than 30 mm, and more than 59% of the antagonistic strains had varying degrees of antagonistic effects on the other pathogens. There were 27 strains with antagonistic effects on Fusarium oxysporum, and the diameter of the inhibition zone ranged from 22 mm to 33 mm. There were 35 strains with antagonistic effects on Fusarium solani, and the maximum inhibition zone diameter could reach 38.52 mm, and the minimum inhibition zone diameter was only 10.8 mm, with a large difference in antagonistic ability. Only 6 strains had antagonistic effects on Helminthosporium radicans, with the inhibition zone ranging from 12.69 mm to 21.71 mm. At the same time, these 6 strains had antagonistic effects on more than 3 other pathogens in this study. There were 32 strains with antagonistic effects on Microsporum radicans, with the largest inhibition zone diameter of 30.99 mm and the smallest diameter of 16.94 mm.

[0065] Table 1 Inhibitory diameters against 6 pathogenic antagonists

[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), the same below.

[0070] 2. The activated bacteria were inoculated onto the Montgena organophosphate plate, and after 5 days, the formation of a phosphate-dissolving zone was observed, and the diameter of the phosphate-dissolving zone was measured. The results are shown in Table 2. A total of 109 organophosphate-dissolving bacteria were screened, among which MP6, MP41, and K87 had the largest phosphate-dissolving zones, which were 37.79 mm, 35.67 mm, and 32.89 mm, respectively. The diameters of the phosphate-dissolving zones of the remaining strains ranged from 5.16 mm to 27.49 mm.

[0071] Table 2 Diameter of dissolved phosphorus in primary screening

[0072]

[0073]

[0074] 3. Rescreening of Antagonistic Bacteria

[0075] The strain selected from 2 with both antagonistic and phosphate-dissolving effects was inoculated into LB culture medium. After incubation in a shaking incubator at 30°C and 180 r / min for 48 h, the bacterial solution was placed in a 10 mL centrifuge tube and centrifuged at 4°C and 12000 r / min for 10 min. 1 mL of the supernatant was aspirated with a sterile syringe (10 mL) and filtered through a 0.22 μm microporous filter membrane. The filtrate was spread on a PDA plate to make a poisonous plate. The PDA plate without bacterial solution was used as a control and inoculated with the test pathogenic fungi (bacterial cake diameter = 0.6 cm), respectively. Each treatment was repeated 3 times. After constant temperature incubation at 25°C for 7 days, the diameter of the pathogen was measured and the growth inhibition rate was calculated.

[0076] Growth inhibition rate (%) = (control plate pathogen colony diameter - infected plate pathogen colony diameter) / (control plate colony diameter - inoculated bacterial cake diameter) × 100

[0077] The results are shown in Table 3 and Figure 1 As shown in the figure, after rescreening, some disease-preventing strains had significant effects. Among them, K87 had inhibitory effects on 5 pathogens (F. avenaceum, F. Oxysporum, F. solani, B. sorokiniana, M. bolleyi), and had good antibacterial ability, with an inhibition rate of more than 75%; LB17 had inhibitory effects on 4 pathogens (F. oxysporum, F. solani, B. sorokiniana, M. bolleyi), with an inhibition rate of more than 61%. There were 11 strains with good antibacterial effects on F. avenaceum, with an inhibition rate ranging from 44% to 87%; there were 14 strains with good antibacterial effects on F. equisetum, accounting for 25% of the antagonistic bacteria in the initial screening, with an inhibition rate between 74.9% and 92.88%. Compared with the initial screening results, 59% of the strains still had an antibacterial effect on Fusarium oxysporum, with a total of 16 strains, of which the highest antibacterial rate was 81%; the 6 strains that had an antibacterial effect on Fusarium solani had an antibacterial rate of more than 69%; only 6 strains had a good antibacterial effect on Helicoverpa spp., with the highest inhibition rate of 87.23% and the lowest of 58%. There were 11 strains with a good inhibitory effect on Microsporum, accounting for 34% of the strains with antibacterial ability in the initial screening, and the highest inhibition rate could reach 92%.

[0078] Table 3 Antibacterial rate of antagonistic strains

[0079]

[0080]

[0081] IV. Strain Safety Determination

[0082] 1. Growth of strains under different salt concentration gradients

[0083] Considering that LB medium containing NaCl may affect the growth of highland barley, and the strain grows poorly in WA medium (20 g agar, 1000 mL distilled water), bacteria were randomly selected and inoculated into salt-free LB medium with a NaCl content of 0, WA medium, and standard LB medium with a NaCl content of 1% as controls, and the comparison was made to observe whether there was a significant difference in bacterial growth after 24 hours.

[0084] The results are as follows Figure 2 As shown, it was found that the strain could grow normally after 24 h in LB and salt-free LB medium, but did not grow in WA medium. Therefore, salt-free LB medium was selected for subsequent experiments.

[0085] 2. Determination of pathogenicity of strains by salt-free LB culture method

[0086] The top 50 strains with the highest antibacterial rate were activated by LB culture solution, cultured in a shaking table at 30°C and 180r / min for 48h, and then 1mL of the culture solution was added to a beaker containing a sterile salt-free LB medium (40mL / 50mL) that had been cooled but not solidified to prepare a culture medium containing bacteria. At the same time, the highland barley seeds were germinated. Finally, the seeds with consistent germination were spread flat in the culture medium containing bacteria, 10 seeds per cup, and the salt-free LB culture medium without bacterial solution was used as a control. The culture was cultured at 25°C, 1h of light, and 75% humidity. A small amount of sterile water was sprayed every 8h, and its growth was observed after 10d.

[0087] The results are as follows Figure 3 As shown in Table 4, highland barley grows differently in LB medium connected with different strains. Subsequent studies select strains that can make highland barley grow luxuriantly to carry out subsequent experiments, and avoid using strains that make highland barley sparse or not growing in the seedling stage. As shown in Table 4, a total of 48 strains were detected for the safety of highland barley growth. The good growth in the table is represented by "+", and the poor growth is represented by "-". Among them, highland barley grows poorly on the medium containing 8 strains of bacteria such as K102 and LB44, and all grows well on the medium of the remaining 40 strains of bacteria. It is believed that these 40 strains are safe for highland barley.

[0088] Table 4 Safety test of strains

[0089]

[0090] Note: “+” indicates that the barley plants grow well; “-” indicates that the barley plants grow poorly.

[0091] 5. Determination of growth-promoting properties of strains

[0092] The non-pathogenic strains selected in 4 were activated and inoculated into LB culture medium. They were cultured in a shaking incubator at 30°C and 180 rpm for 48 h. The OD 660When the value of ≥0.5, the following characteristic determination test is carried out:

[0093] Phosphorus solubilization characteristics: Take 1mL of bacterial liquid and inoculate it into PKO inorganic phosphorus and Meng Jinna organic phosphorus culture medium (liquid volume: 50mL / 150mL triangle bottle, the same below), respectively. Repeat 3 times for each strain, and use the culture medium without bacteria as the control. After culturing at 30℃ and 180r / min in a shaking incubator for 7 days, the phosphorus content was determined by the molybdenum antimony colorimetric method.

[0094] Nitrogen fixation characteristics: Take 1 mL of bacterial solution and inoculate it into NFM culture medium, with 3 replicates for each strain. The culture medium without bacteria was used as the control. After culturing at 30℃ and 180r / min for 7 days, the nitrogen content was determined by Kjeldahl method.

[0095] Potassium-solubilizing properties: 1 mL of bacterial solution was inoculated into potassium feldspar culture medium, with 3 replicates for each strain. The culture medium without bacteria was used as the control. After culturing at 30°C and 180 r / min for 7 days, the soluble potassium content in each bacterial solution was determined by flame spectrophotometry.

[0096] IAA secretion characteristics: 1 mL of bacterial solution was inoculated into King's B culture medium, with 3 replicates for each strain, and the uninoculated culture medium was used as the control. After culturing at 30°C and 180 r / min for 5 days, 10 mL of bacterial solution was taken, centrifuged at 10,000 r / min for 5 min according to the method in the reference "Isolation of IAA-producing strains in rhizosphere soil of Salt Claw Claw and analysis of growth-promoting characteristics" (Cheng Xinyu et al. 2024), 5 mL of supernatant was taken, an equal amount 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 liquid at a wavelength of 530 nm was determined by ultraviolet spectrophotometry, and the IAA content in the fermentation liquid per unit volume was calculated against the standard curve.

[0097] Secretion of siderophore characteristics: refer to the method in the literature "Functional characteristics and culture conditions of a white clover rhizosphere siderophore-producing bacteria" (Wei Xin et al. 2024), take 10mL of bacterial solution, centrifuge at 10000r / min for 10min, then take 5mL of supernatant and 5mL of CAS detection solution and mix them thoroughly, let it stand for 1h, and use spectrophotometer to measure the absorbance value (As) at OD630nm. After reacting 5mL of LB liquid culture medium and CAS detection solution, the absorbance value was measured as the reference value (Ar), and the siderophore activity was calculated. The formula is as follows:

[0098] Siderophore activity unit (su) = [(Ar-As) / Ar] × 100%

[0099] Acid, alkali and salt resistance: 1 mL of bacterial solution was inoculated into culture medium with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and into LB culture medium with salt (NaCl) concentrations of 1%, 5%, 10%, 15%, and 20%. The culture was shaken at 30°C and 180 r / min for 48 h, and the OD of each bacterial solution was determined by spectrophotometry. 630 The results are shown in Table 5 and Figure 4 , Figure 5 , Figure 6 Shown

[0100] Table 5 Functional characteristics of excellent strains

[0101]

[0102] As shown in Table 5, LB17 has the strongest ability to secrete siderophores, with a su value of 32%. At the same time, its ability to dissolve inorganic phosphorus is relatively strong, at 1102.95 μg / mL. K113 has the strongest nitrogen fixation ability, at 0.08 g / L, and the nitrogen fixation of the other strains is between 0.04 and 0.06 g / L. K87 has the strongest ability to secrete IAA, which can reach 9.87 mg / L, while the other strains have a weaker ability to secrete IAA, between 5.69 mg / L and 6.78 mg / L, but its potassium-solubilizing ability is weak, only 25.75 mg / L. K85 has the strongest ability to dissolve organic phosphorus, at 1321.23 μg / mL, and the su value of secreted siderophores is above 20%. MP6 has the strongest ability to dissolve inorganic phosphorus, at 1470.69 μg / mL, and also has good ability to dissolve organic phosphorus (1141.86 μg / mL) and dissolve potassium (40.9 mg / L). MP41 has the strongest potassium-dissolving ability, which is 140.33 mg / L.

[0103] Figure 4 To determine the acid resistance of the excellent strains, when the OD of the strains at pH = 7 630 The values ​​were the highest, all greater than 2.0. When pH = 6 and pH = 5, the OD of strain K56 630 The value decreased to 0.96, indicating that the acid resistance of K56 strain was poor. The OD values ​​of the other strains decreased to between 1.5 and 1.7. When the strains were in the culture medium at pH 4, K56 hardly grew, the growth of MP6 was inhibited, and the OD values ​​of MP41, K87, and K33 were 630 The value was higher than 1.6, indicating that MP41, K87, and K33 had good acid resistance. When the strains were at pH = 3, OD 630 The values ​​were close to CK and all below 0.02, indicating that the strains did not grow under pH 3. Therefore, MP41, K87, and K33 were determined to be acid-resistant strains.

[0104] Figure 5The results showed that the OD values ​​of the strains were all greater than 2.0 at pH = 7, and the growth was good. When the pH value of the culture solution was 8, the OD values ​​decreased, but were all greater than 1.5. When the pH reached 9, the OD value decreased significantly (P < 0.05), but the OD values ​​of the LB17 and K113 culture solutions were still greater than 0.5. When the culture solution reached pH = 10, the OD value was extremely low, close to the blank control, indicating that the screened strains were intolerant to alkali concentrations of pH 10 and above. Therefore, strains LB17 and K113 were determined to be alkali-resistant strains.

[0105] Figure 6 The results showed that the OD values ​​of the strains were all greater than 2.0 when the salt concentration was 1%, and the growth was good. When the salt concentration reached 5%, the OD value decreased significantly (P<0.05), and the OD values ​​of strains K85, K113, K33, and K56 were all less than 1.2. When the salt concentration reached 10%, the OD values ​​of K113, K33, and K56 were close to the blank control, and the OD value was close to 0.2, indicating that the 10% salt concentration completely inhibited the growth of K113, K33, and K56, but the OD values ​​of LB17, MP41, K85, and K87 were still greater than 1.1. When the salt concentration reached 15% and 20%, the OD values ​​of each strain were extremely low, close to the blank control, and the difference was not significant (P<0.05), indicating that each strain was intolerant to salt concentrations of 15% and above. Therefore, strains LB17, MP41, K85, and K87 were determined to be salt-tolerant strains.

[0106] VI. Construction of a composite microbial strain for disease prevention and growth promotion

[0107] The pairwise cross-marking method was used to determine the antagonism between superior strains. Figure 7 It was shown that there was antagonism between K87 and MP41, but no antagonism between other strains, so it was avoided to use K87 and MP41 at the same time in the composite bacterial system.

[0108] Then, strains with no antagonistic effects on each other were selected and combined with different pathogens and crops to form composite bacterial strains. Each bacterial strain was repeated 3 times. The phosphorus solubility, nitrogen fixation, potassium solubilization, IAA secretion and iron carrier secretion capacity of each composite bacterial strain were determined according to the method in the fifth method. A comprehensive analysis was performed and the results are shown in Table 6. The combinations of the composite bacterial strains are as follows:

[0109] T1: K87+LB17; T2: K87+LB17+MP6; T3: K87+LB17+MP6+K85

[0110] T4: K56+LB17; T5: K56+LB17+K113; T6: K56+LB17+MP6+K113;

[0111] T7: K33+LB17; T8: K33+LB17+MP6; T9: K33+LB17+MP6+K113;

[0112] T10: MP6+K85+MP41+K113; T12: K113+MP41; T13: LB17+K113+MP41.

[0113] Table 6 Functional characteristics of excellent composite bacterial system

[0114]

[0115]

[0116] Table 6 shows that among the 12 groups of composite bacterial systems, T2 has the highest nitrogen fixation and potassium solubilization, which are 0.212 g / L and 86.28 mg / L, respectively. Composite bacterial system T13 has the highest organic phosphorus solubility, inorganic phosphorus solubility and iron carrier secretion, which are 482 μg / mL, 425.35 μg / mL, and the su value is 47%, respectively. Composite bacterial system T4 has the strongest ability to secrete IAA, which is 51.63 mg / L. Topsis comprehensive evaluation was performed, and the statistics (CI) of T2 and T13 were 0.611 and 0.579, respectively, which were higher than other groups, and were determined to be excellent composite bacterial system formulas.

[0117] Finally, the excellent composite strains T2 and T13 were selected to test their antibacterial ability against six pathogenic fungi, including Microsporum, Fusarium equisetum, Helminthosporium oleraceum, Fusarium solani, Fusarium oxysporum and Fusarium avenae (the method is the same as that of "three"). The results are as follows Figure 8 shown.

[0118] Figure 8 The results showed that the composite bacterial strain T2 had stronger antibacterial activity than T13, with the highest antibacterial rate against Fusarium equisetifolia, reaching 87.69%, followed by Fusarium solani and Microspore, with antibacterial rates of 77.84% and 76.71%, respectively, and the antibacterial rates against Fusarium avenae, Fusarium oxysporum, and Echinops umbilical cordyceps were 60.26%, 65.71%, and 64.46%, respectively. The composite bacterial strain T13 also had stronger antibacterial activity against the tested fungi, with the strongest inhibitory effect against Echinops umbilical cordyceps, reaching 64.45%, followed by Fusarium equisetifolia, with an antibacterial rate of 63.68%, and the antibacterial rates against Microspore and Fusarium avenae, reaching 47.45% and 53.99%, respectively, while the antibacterial rates against Fusarium solani and Fusarium oxysporum were lower, reaching 19.47% and 19.56%, respectively. In summary, the antibacterial activity of T2 was generally higher than that of T13, so T2 was the optimal composite bacterial strain.

[0119] Example 2. Identification of LB17, K87, and MP6 strains

[0120] A DNA extraction kit (TIANGEN centrifugal column type) was used to extract the DNA of the strains involved in the optimal composite bacterial system screened in Example 1 according to its instructions, and 16S rDNA universal primers (SEQ ID NO.1, SEQ ID NO.2) and gyrB primers (SEQ ID NO.3, SEQ ID NO.4) were used for amplification.

[0121] Universal Primer:

[0122] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1),

[0123] 1492R:5'-TACGGCTACCTTGTTACGACTT-3'(SEQ ID NO.2)

[0124] gyrB Primers:

[0125] F: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' (SEQ ID NO.3),

[0126] R: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3' (SEQ ID NO.4)

[0127] The total volume of the reaction system was 25 μL, 1 μL each of the forward and reverse primers, 1 μL of DNA template, 12.5 μL of 2×PCRMix, and 9.5 μL of ddH2O; the reaction procedure was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and 72°C final extension for 10 min. After passing the 1% agarose gel electrophoresis test, the sequencing was entrusted to Sangon Biotech (Shanghai) Co., Ltd. Finally, the sequencing results were compared with BLAST homology in NCBI, and similar sequences and exogenous gene sequences were selected. The phylogenetic tree was constructed using the neighbor-joining method in MEGA11.0, and its credibility was tested by Bootstrap 1000 repetitions. The results are as follows: Fig. 9 , Fig.10 shown.

[0128] Fig. 9The results showed that based on the 16S rDNA sequence analysis, the genetic distance between LB17 and Bacillus velezensis (ON597433.1) was less than 0.01, and the bootstrap support rate was 99 after 1000 repetitions; the genetic distance between MP6 and K87 and Bacillus velezensis (MW578391.1) was less than 0.01, and the bootstrap support rate was 97. The accession numbers obtained by submitting their sequences to GenBank were PQ211017, PQ211018, and PQ211019, respectively. Therefore, it was preliminarily determined that LB17, K87, and MP6 were Bacillus velezensis. Further identification of their gyrB sequences revealed that ( Fig.10 ), the genetic distance between LB17, K87 and the standard strain of Bacillus velezensis BCRC 17467T (DQ903176.1) was 0, and the support rate of 1000 repetitions was 98; the genetic distance between MP6 and the standard strain of Bacillus velezensis NRRL 41580T (EU138622.1) was also 0, and the support rate of 1000 repetitions was 99. The sequences were submitted to GenBank and the accession numbers obtained were PQ144581, PQ144582, and PQ144583, respectively. Therefore, LB17, K87, and MP6 strains were determined to be Bacillus velezensis.

[0129] Example 3. Optimization of the best ratio of composite bacterial system T2

[0130] The test strains LB17, K87, and MP6 were activated and inoculated into LB liquid medium at the same time. The shaking speed was 180 r / min and the temperature was kept at 30°C for 10 8 CFU / mL, four levels (3%, 4%, 5%, 6%) of orthogonal design were performed to compare the amount of bacteria, and L9 (4 3 ) Orthogonal experiment, the orthogonal design scheme is shown in Table 7, and the optimal inoculation amount is determined according to the OD value of the composite bacterial solution. The results are shown in Fig.11 shown.

[0131] Table 7L9(4 3 )Orthogonal design table

[0132] Test 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％

[0133] Fig.11 It shows that in the fourth test group (A4), OD 600The highest value was 1.45 and was significantly higher than other treatments (P<0.05). The OD values ​​of the other experimental groups were between 1.153 and 1.27. This indicates that the fermentation effect is best when the inoculation amount of LB17, K87, and MP6 is 4%, 3%, and 6%, respectively, that is, when the inoculation volume ratio is 4:3:6. The optimized composite bacterial strain was named "Tuweiwei No. 7", hereinafter referred to as "Tuweiwei No. 7".

[0134] Example 4. Seed culture medium optimization

[0135] 1. Optimization of seed culture medium composition

[0136] A single-factor experiment was used to replace the components in 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 3 replicates per treatment. After culturing under the optimized fermentation conditions, the OD of the composite bacterial solution was measured using an ELISA reader. 600 value.

[0137] The results are as follows Fig.12 As shown in the figure, when the seed culture medium composition was optimized and soluble starch was used as the sole carbon source in the initial LB medium, the OD 600 The value was the highest, which was 1.55 and significantly higher than other treatments (P<0.05). When sucrose, sodium citrate, and lactose were the only carbon source, OD 600 The values ​​of soluble starch and NH4Cl were all less than 1.3, so soluble starch was the optimal carbon source for Tuweiwei No. 7 when it was the only carbon source. When the nitrogen source trypsin in the initial LB medium was replaced by NH4Cl as the only nitrogen source, the OD of the composite bacterial system 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 600 The value is between 1.114 and 1.308. Therefore, when NH4Cl is the only nitrogen source, it is the optimal nitrogen source for Tuweiwei No. 7. When the inorganic salt NaCl in the initial LB medium is replaced by MgSO4·7H2O as the inorganic salt, the OD of the composite bacterial system 600 The highest value was 1.243 and the difference was significant (P<0.05). K2HPO4·3H2O, CaCl2·2H2O, MnSO4·7H2O OD 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 No. 7.

[0138] 2. Optimization of the proportion of seed culture medium components

[0139] According to Table 8, design L9(4 3 ) Orthogonal experiment, the optimal ratio between the above-mentioned optimized optimal culture components soluble starch (1%, 1.5%, 2%), NH4Cl (NH4Cl: 1%, 1.5%, 2%), MgSO4·7H2O (1%, 1.5%, 2%) was optimized. Specifically: the seed solution of Tuweiwei No. 7 was added to the culture solution of each treatment group, the shaking speed was 180r / min, and the OD was measured after constant temperature culture at 30℃ for 48h. 600 value.

[0140] Table 8L9(43) Orthogonal design table

[0141] Test 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％

[0142] The results are as follows Fig.13 As shown, Fig.13 The results showed that the OD value of group B1 was the highest, which was 1.63, and the OD values ​​of the other experimental groups ranged from 1.11 to 1.57, with significant differences (P<0.05). That is, the optimal ratio of soluble starch: NH4Cl: MgSO4·7H2O was 2:1:1.

[0143] Example 5. Optimization of seed liquid fermentation conditions

[0144] The composite bacterial strain Tuweiwei No. 7 was subjected to an orthogonal design with five factors and four levels, including different initial pH values ​​(6.0, 6.5, 7, 7.5), inoculation amounts (2%, 4%, 6%, 8%), oscillation rates (140, 160, 180, 200 r / min), culture temperatures (22, 26, 30, 34°C) and culture times (18, 24, 30, 36 h). 5 ) Orthogonal experiment, the orthogonal design scheme is shown in Table 9, and the OD of the composite bacterial solution is determined. 600 value.

[0145] Table 9L16(45) orthogonal design table

[0146]

[0147]

[0148] The results are as follows Fig.14 As shown, the OD of Tuweiwei No. 7 under different fermentation conditions 600 There were significant differences in the values ​​(P<0.05). The OD value of group Z16 was the highest, which was 1.88. Therefore, the optimal fermentation conditions were pH 7.5, inoculation volume 8%, rotation speed 140r / min, temperature 30℃, and fermentation time 24h.

[0149] Example 6. Optimization of fermentation medium in fermenter

[0150] 1. Optimization of fermentation medium composition

[0151] LB medium: 10 g tryptone, 5 g yeast powder, 10 g NaCl, 20 g agar, 1000 mL distilled water, pH 7.0-7.2.

[0152] A single-factor experiment was used to replace the original LB medium with different carbon sources (1% corn starch, 1% soluble starch, 1% mannitol, 1% maltodextrin); nitrogen sources (1% corn syrup powder, 1% yeast powder, 1% urea, 1% soybean powder, 1% soybean meal powder) and inorganic salts (1% MnSO4·H2O, 1% CaCL2, 1% MgSO4, 1% CuSO4, 1% CaCO3), with 3 replicates per treatment. After culturing under the optimized fermentation conditions, the OD of the composite bacterial solution was measured using an ELISA reader. 600 value.

[0153] The results are as follows Fig.15 As shown, when mannitol was the only carbon source, the OD 600 The value was the highest, which was 1.4 and significantly higher than that of other treatments (P<0.05). When corn starch, soluble starch, and maltodextrin were the only carbon source, OD 600 Between 1.16 and 1.3; when corn syrup powder is the only nitrogen source, the OD of the composite bacterial system 600 The highest composite bacterial strain OD 600 The highest value was 1.35 and the difference was significant (P<0.05). The OD 600 The value is between 1.22 and 1.28; when MnSO4·H2O is the only inorganic salt, the OD of the composite bacterial system 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 Tuweiwei No. 7.

[0154] 2. Optimization of the optimal ratio of fermentation medium components

[0155] According to Table 10, design L9(4 3 ) Orthogonal experiment, the optimal ratio between the above-mentioned optimized optimal culture components mannitol (1%, 1.5%, 2%), corn syrup powder (NH4Cl: 1%, 1.5%, 2%), and MnSO4·H2O (1%, 1.5%, 2%) was optimized. Specifically: the seed liquid of Tuweiwei No. 7 was added to the fermentation culture medium of each treatment group, the shaking speed was 180r / min, and the OD was measured after constant temperature cultivation at 30℃ for 48h.600 Value, the result is Fig.16 shown.

[0156] Table 10L9(43) orthogonal design table

[0157] Test 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％

[0158] Fig.16 The results showed that the OD value of the C6 group, the optimal fermentation medium of the T2 composite bacteria system, was the highest, which was 1.75, and the OD value of the other experimental groups ranged from 1.33 to 1.63, that is, the optimal ratio of mannitol: corn syrup powder: MnSO4 H2O was 1:1.5:1.5.

[0159] Example 7. Optimization of fermentation conditions in fermenter

[0160] The composite bacterial strain Tuweiwei No. 7 was subjected to an orthogonal design with five factors and four levels, including different initial pH values ​​(6.0, 6.5, 7, 7.5), inoculation amounts (2%, 4%, 6%, 8%), oscillation rates (140, 160, 180, 200 r / min), culture temperatures (22, 26, 30, 34°C) and culture times (18, 24, 30, 36 h). 5 ) Orthogonal experiment, the orthogonal design scheme is shown in Table 11, and the OD of the composite bacterial solution is determined. 600 Value, the result is Fig.17 shown.

[0161] Table 11L16(45) orthogonal design table

[0162] Test No. PH Inoculation amount 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

[0163] Fig.17 It shows that the OD of the composite bacterial strain under different fermentation conditions of the fermentation medium 600 There were significant differences in the values ​​(P<0.05). Among them, the F4 composite strain had the highest OD value of 1.87. Therefore, the optimal fermentation conditions were pH 6, inoculation volume 8%, rotation speed 200r / min, temperature 34℃, and fermentation time 36h.

[0164] Example 8. Effect of "Tuweiwei No. 7" on the prevention and treatment of barley root rot and its growth-promoting effect on barley

[0165] 1. Preparation of Tuweiwei No. 7 fermentation broth:

[0166] Seed liquid culture medium: soluble starch 20g, NH4Cl 10g, MgSO4·7H2O 10g, water 1L; adjust pH to 7.5 and sterilize.

[0167] Fermentation medium: mannitol 10g, corn syrup powder 15g, MnSO4·H2O 15g, water 1L; adjust pH to 6 for sterilization.

[0168] Preparation of compound bacterial agent:

[0169] 1. Inoculate Bacillus Velezii LB17, K87, and MP6 into slant culture medium respectively, and culture the slant culture at 30°C for 24 hours to obtain slant culture;

[0170] 2. Inoculate the slant cultures of Bacillus Velez LB17, K87, and MP6 into the seed liquid culture medium respectively; culture at 30°C and 140 rpm for 24 hours to obtain the seed culture liquid of Bacillus Velez LB17, K87, and MP6;

[0171] 3. Mix the seed culture solutions of Bacillus Velezii LB17, K87 and MP6 in a volume ratio of 4:3:6 to obtain a mixed bacterial solution;

[0172] 4. Inoculate the mixed bacterial solution into the fermentation medium at an inoculation rate of 8%, and culture at 34°C, 200 rpm for 34 hours to obtain the composite bacterial agent.

[0173] 2. The effect of Tuweiwei No. 7 on preventing barley root rot and promoting its growth

[0174] The disease prevention experiment was divided into 6 treatments, CK1, CK2, CK3 and M1, M2, M3, with 3 replicates and 10 plants per replicate. The growth promotion experiment was divided into 2 treatments, CK4 and M4, with 3 replicates and 10 plants per replicate. Highland barley seedlings with similar growth potential were selected and marked, and spores with a concentration of 2×10 6 CFU / mL of mixed spore suspension of Fusarium avenaceum and Fusarium equiseti, 30 mL of Microdochium bolleyi, and 30 mL of Bipolaris sorokiniana were used. After 4 days, the roots of CK1, CK2, CK3, and CK4 were irrigated with modified LB medium (liquid fermentation medium) (30 mL / plant). At the same time, M1, M2, M3, and M4 were irrigated with 4×10 8 CFU / mL of Tuweiwei No. 7 fermentation liquid was irrigated with roots (30mL / plant), and after 10 days, the disease index of highland barley plants, the number of soil microorganisms, the nutrient composition of highland barley rhizosphere soil and the physiological indicators of highland barley growth were measured.

[0175] Disease index = ∑ (number of diseased plants × representative value) / total number of plants × representative value of the most severe disease × 100

[0176] Prevention effect (%) = (incidence rate of control group - incidence rate of treatment group) / incidence rate of control group × 100

[0177] The results of the disease prevention experiment group Tuweiwei 7 on the prevention of barley root rot are as follows Fig.18 As shown in Table 12.

[0178] Table 12 The efficacy of Tuweiwei No. 7 on barley root rot

[0179] Treatment group number Disease index Effectiveness% 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

[0180] Depend on Fig.18 As shown in Table 12, the plant height and stem diameter of highland 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 heights were 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, with an increase range of 13.28% to 34%; the increase range of stem diameter was 55% to 65.8%; the protective effects of Tuweiwei No. 7 against Fusarium root rot, Microsporum root rot and common root rot were 72.3%, 61.2% and 75.1%, respectively.

[0181] Determination of soil microorganism quantity: Weigh 1g of soil and dissolve it in 10mL of sterile water. Shake it on a shaker at 180r / min for 10min to form a uniform soil suspension. Pipette 1mL of soil suspension into a 9mL sterile water test tube to obtain 10 -1 The soil suspension was subjected to the same operation steps to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 of soil suspension.

[0182] (1) Determination of soil bacteria count: 10 -4 , 10 -5 , 10 -6 200 μL of the soil suspension was aspirated and inoculated into the beef peptone medium. The medium was evenly spread on the surface of the beef peptone medium with a spreader, and the number of bacteria was determined by plate counting.

[0183] (2) Determination of soil fungi quantity: 10 -1 , 10 -2 , 10 -3 200 μL of soil suspension was aspirated and inoculated into Martin's medium, which was evenly spread on the surface of Martin's medium with a spreader, and the number of fungi was determined by the plate count method.

[0184] (3) Determination of soil actinomycete count: 10 -3 , 10-4 , 10 -5 200 μL of the soil suspension was aspirated and inoculated into the modified Gao's medium No. 1. It was evenly spread on the surface of the modified Gao's medium No. 1 using a spreader, and the number of actinomycetes was determined by the plate counting method.

[0185] The number of strains per gram of sample = (C / V) × M (C: represents the average number of colonies grown on the plate at a certain dilution; V: represents the volume of diluent used when coating the plate; M: represents the dilution multiple)

[0186] The results of soil microbial quantity determination are shown in Table 13.

[0187] Table 13 Effect of Tuweiwei 7 on the number of rhizosphere microorganisms in highland barley soil

[0188]

[0189]

[0190] Table 13 shows that the bacteria and actinomycetes in the rhizosphere soil of highland barley in the treatment groups M1, M2, and M3 (Tuweiwei 7+pathogen root irrigation) were significantly higher than those in the control groups CK1, CK2, and CK3 (pathogen root irrigation) (P<0.05). The number of bacteria in M1, M2, and M3 was 3.203×10 7 CFU / g, 2.93×10 7 CFU / g, 3.224×10 7 CFU / g, which was significantly higher than that of M4, M5, and M6. The bacteria in the rhizosphere soil increased greatly, ranging from 70.18% to 76.7%, and the number of actinomycetes was 2.138×10 6 CFU / g, 1.996×10 6 CFU / g, 1.918×10 6 CFU / g, respectively, increased by 1.691×10 6 CFU / g, 1.492×10 6 CFU / g, 1.21×10 6 CFU / g. The number of soil fungi in treatment groups M1, M2, and M3 was significantly lower than that in control groups M4, M5, and M6 (P<0.05). The number of fungi in M1, M2, and M3 was 1.827×10 4 CFU / g, 1.622×10 4 CFU / g, 1.539×10 4 CFU / g, decreased by 5.417×10 4 CFU / g, 6.736×10 4 CFU / g, 7.129×10 4CFU / g.

[0191] Determination of the growth-promoting effect of the growth-promoting experimental group on highland barley:

[0192] The growth of highland barley was observed, and the growth-promoting experimental group was selected to determine the growth-promoting function of Tuweiwei No. 7 on highland barley. The available nitrogen in the rhizosphere soil of highland barley was determined by the alkaline diffusion method; the available phosphorus was determined by the molybdenum antimony colorimetric method extracted with sodium bicarbonate; the available potassium was determined by inductively coupled plasma emission spectrometry; the microbial biomass nitrogen, microbial biomass carbon and microbial biomass phosphorus were determined by chloroform fumigation extraction; the superoxide dismutase SOD in highland barley leaves was determined by the nitroblue tetrazolium photoreduction method; the malondialdehyde content was determined by the TBA colorimetric method; the peroxidase POD content was determined by the guaiacol colorimetric method; the total chlorophyll content was determined by the dual-wavelength colorimetric method; the auxin IAA content was determined by GA1 liquid chromatography-mass spectrometry, and the results were as follows: Fig.19 As shown in Table 14 and Table 15.

[0193] Table 14 Effects of Tuweiwei 7 on physiological parameters of highland barley leaves

[0194]

[0195] Table 15 Effects of Tuweiwei 7 on nutrients in rhizosphere soil of highland barley

[0196]

[0197] Fig.19 It shows that the highland barley inoculated with the composite microbial agent grows more densely and luxuriantly in the seedling stage; Table 14 shows that the highland barley treatment group (M4) treated with the composite microbial agent Tuweiwei No. 7 has significantly increased IAA, SOD, MDA, and chlorophyll contents compared with the control group (CK4) (P < 0.05), and the POD content has decreased. Among them, the IAA content increased by 90.825ng / g, the SOD content increased from 686.86μg / g to 843.79μg / g, the MDA and chlorophyll contents increased by 19.83μg / g and 67.68mg / g, respectively, and the POD content decreased from 336.98μg / g to 257.47μg / g, indicating that Tuweiwei No. 7 has a growth-promoting effect on highland barley plants.

[0198] Table 15 shows that the nutrients in the rhizosphere soil of highland barley in the treatment group (M4) with the application of the composite microbial agent increased significantly (P < 0.05) compared with the control group (CK4), such as the contents of available nitrogen, available phosphorus, and available potassium. The content of available phosphorus increased most significantly, with the content of the control group (CK4) being 98.35 mg / kg and the content of the treatment group (M4) being 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 shows that the use of microbial agents helps to improve the nutrients in the rhizosphere soil of highland barley. The fertility of the rhizosphere soil of highland barley in the treatment group (M4) increased significantly (P < 0.05) compared with the control group (CK4). Among them, the biological phosphorus content of M4 increased by 2.5 times compared with CK4, the biological nitrogen content increased from 18.95 mg / kg to 38.79 mg / kg, and the biological carbon content increased from 110.64 mg / kg to 178.31 mg / kg, indicating that the use of Tuweiwei No. 7 compound bacterial agent helps to improve the fertility of the rhizosphere soil of highland barley.

[0199] Example 9. The effect of "Tuweiwei No. 7" on the prevention and treatment of lily wilt disease and its growth-promoting effect on lilies

[0200] Disease prevention experimental group: Lily bulbs were planted, and lilies with the same growth were set as CK1 and J1 treatments, with 3 replicates and 10 plants in each replicate.

[0201] Growth promotion experimental group: lily bulbs were planted, and lilies with the same growth were set as CK2 and J2 treatments, with 3 replicates and 10 plants in each replicate.

[0202] Lily seedlings with the same growth after 20 days of planting were selected, and J1 and CK1 were inoculated with spores at a concentration of 2×10 6 CFU / mL of mixed spore suspension of Fusarium oxysporum and Fusarium solani was inoculated with pathogens for 13 days and 19 days, and then the roots of CK1 and CK2 were irrigated with modified LB culture medium (liquid fermentation medium) (30 mL / plant). 8 CFU / mL of Tuweiwei No. 7 was irrigated roots (30 mL / plant) on J1 and J2, and 20 days later, the disease index of lily plants, the number of soil microorganisms, the nutrient composition of lily rhizosphere soil and the growth physiological indicators of lily were measured, and the measurement methods were the same as those in Example 8.

[0203] The disease index test results and growth conditions of lily plants are shown in Fig. 20 and as shown in Table 16.

[0204] Table 16 The efficacy of Tuweiwei No. 7 on lily root rot

[0205] Treatment group number Disease index Effectiveness% 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

[0206] The results are shown in Table 5. The plant height and stem diameter of lilies in the treatment group J1 (Tuweiwei No. 7 + pathogen root irrigation) were significantly higher than those in the 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. Compared with CK1, the plant height increased by 65.1% and the stem diameter increased by 19.2%. The prevention effect of Tuweiwei No. 7 composite bacterial agent on lily root rot was 68.6%.

[0207] The number of rhizosphere microorganisms of lily 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 control group CK1 (pathogen root irrigation) (P<0.05). The number of bacteria in J1 was 2.82×10 7 CFU / g, increased by 2.131×10 compared with CK1 7 CFU / g; the number of actinomycetes is 1.074×10 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 control group CK1 (P<0.05). The number of fungi in J1 was 1.156×10 4 CFU / g, decreased by 5.321×10 4 CFU / g.

[0208] The effect of Tuweiwei No. 7 on the growth promotion of lily Figure 2 As shown, the nutritional components in lily leaves were determined according to the method in Example 8. The soluble protein content in lily bulbs was determined by Coomassie Brilliant Blue colorimetry; the reducing sugar content was determined by DNS colorimetry; and the total dietary fiber content in lily bulbs was determined by enzymatic hydrolysis. The results are shown in FIG. Fig.21 As shown in Table 17 and Table 18.

[0209] Table 17 Effects of Tuweiwei 7 on physiological parameters of lily leaves

[0210] 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

[0211] Fig.21 It shows that the lily seedlings inoculated with the composite bacteria system grow rapidly and robustly. Table 17 shows that the lily treatment group (J2) treated with the Tuweiwei No. 7 composite bacterial agent has significantly increased IAA, SOD, MDA, and chlorophyll contents compared with the control group (CK2) (P < 0.05), and the POD content has decreased. Among them, the IAA content increased significantly by 5 times, from 0.287ng / g to 1.578ng / g, and the SOD, MDA and chlorophyll contents increased by 9.036μg / g, 19.83μg / g, and 67.68mg / g, respectively. The POD content decreased by 0.735μg / g.

[0212] Table 18 Effect of Tuweiwei 7 on lily quality

[0213] 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

[0214] Table 18 shows that the lily treatment group (J2) treated with Tuweiwei No. 7 composite bacterial agent had significantly increased soluble protein, reducing sugar and total dietary fiber contents compared with the control group (CK2) (P < 0.05), with soluble protein content increasing from 2.97 mg / g to 3.94 mg / g, reducing sugar content increasing from 4.45 to 5.83, and total dietary fiber increasing by 5.61%, indicating that the composite bacterial agent is beneficial to improving the quality of lilies.

[0215] The number of microorganisms in the lily rhizosphere is shown in Table 19, and the nutrient components of the lily rhizosphere soil are shown in Table 20.

[0216] Table 19 Effect of Tuweiwei 7 on the number of rhizosphere microorganisms of lily

[0217] deal with Bacteria count Number of actinomycetes 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

[0218] Table 19 shows that the number of soil bacteria and actinomycetes in the 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 number of bacteria in J1 was 2.82×10 7 CFU / g, increased by 2.131×10 compared with CK1 7 CFU / g; the number of actinomycetes is 1.074×10 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 control group CK1 (P<0.05). The number of fungi in J1 was 1.156×10 4 CFU / g, decreased by 5.321×10 4 CFU / g.

[0219] Table 20 Effect of Tuweiwei 7 on the nutrient composition of lily rhizosphere soil

[0220]

[0221] Compared with the control group (CK2), the nutrients in the rhizosphere soil of lily in the treatment group (J2) with composite microbial agent increased significantly (P < 0.05), such as the contents of available nitrogen, available phosphorus and available potassium. The content of available phosphorus increased by 4.87 mg / kg, the content of available potassium increased from 1973.81 mg / kg to 2083.73 mg / kg, and the content of available nitrogen only increased by 0.73 mg / kg. This shows that the use of microbial agent can improve the nutrients in the rhizosphere soil of lily. The fertility of the rhizosphere soil of highland barley in the treatment group (J2) increased significantly (P < 0.05) compared with the control group (CK2). Among them, the content of biological phosphorus and biological nitrogen in J2 increased from 37.50 mg / kg to 47.38 mg / kg, the content of biological carbon increased by 57.48 mg / kg, and the content of biological phosphorus increased from 22.65 mg / kg to 2901 mg / kg, indicating that the use of microbial agent is helpful to improve the fertility of rhizosphere soil of lily.

[0222] Example 10. Effect of "Tuweiwei No. 7" on the prevention and treatment of tomato root rot and the growth promotion of tomato

[0223] Tomato seedlings with similar growth were selected. The disease prevention experimental group was set up with CK1 and X12 treatments, 3 replications, 10 plants per replication; CK2 and X2, 2 treatments, 3 replications, 10 plants per replication. 15 days after tomato planting, X1 and CK1 were inoculated with spores at a concentration of 2×10 6 CFU / mL of mixed spore suspension of Fusarium oxysporum and Fusarium solani, 30 mL, and then irrigate the roots of CK1 and CK2 with modified LB culture medium (liquid fermentation medium) (30 mL / plant) 10 and 21 days after inoculation with pathogens, respectively, and 5×10 8 CFU / mL of Tuweiwei No. 7 was irrigated to the roots (30mL / plant) X2, and after 20 days, the disease index of tomato plants, the number of soil microorganisms, the nutrient composition of tomato rhizosphere soil and the tomato growth physiological indicators were measured. The measuring method was the same as that in Example 8, and the soil samples were stored at low temperature and transported back to the laboratory.

[0224] The growth status of tomatoes in the disease prevention experimental group is as follows Fig. 22 The incidence of tomato root rot and the growth of tomato plants are shown in Table 21.

[0225] Table 21 The efficacy of Tuweiwei No. 7 on tomato root rot

[0226] Treatment group number Disease index Effectiveness% 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

[0227] Fig. 22The results showed that the tomato plant height and stem diameter of the treatment group X1 (Tuweiwei No. 7 + root irrigation with pathogens) were significantly higher than those of the control group CK1 (root irrigation with pathogens) (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% and 38.5% higher than those of CK1. The prevention effect of Tuweiwei No. 7 on tomato root rot was 79.87%.

[0228] The effects of Tuweiwei No. 7 on tomato rhizosphere soil microorganisms are shown in Table 22.

[0229] Table 22 Effect of Tuweiwei 7 on the number of rhizosphere microorganisms in tomatoes

[0230]

[0231] As shown in Table 22, the number of soil bacteria and actinomycetes in treatment group X1 (Tuweiwei 7+pathogen root irrigation) was significantly higher than that in control group CK1 (pathogen root irrigation) (P<0.05). The number of bacteria in X1 was 2.623×10 7 CFU / g, increased by 1.995×10 7 CF U / g; the number of actinomycetes is 1.531×10 6 CFU / g, increased by 1.004×10 6 CFU / g. The number of soil fungi in treatment group X1 was significantly lower than that in control group CK1 (P<0.05). The number of fungi in X1 was 1.251×10 4 CFU / g, decreased by 6.426×10 4 CFU / g.

[0232] The growth status of tomatoes in the growth promotion experimental group is as follows Fig.23 The effects of Tuweiwei 7 on the nutritional components of tomato leaves are shown in Table 23.

[0233] Table 23 Effects of Tuweiwei 7 on physiological parameters of tomato leaves

[0234] 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

[0235] Fig.23It shows that the application of the composite fungicide Tuweiwei No. 7 tomato seedlings grew robustly, and the plant height was significantly higher than that of the control group. Table 23 shows that the tomato treatment group (X2) treated with the composite fungicide increased significantly (P < 0.05) in comparison with the control group (CK2), and the POD content decreased. Among them, the IAA content increased significantly by 1.8 times, from 60.354ng / g to 113.711ng / g, the MDA increased from 0.851μg / g to 1.183μg / g, the SOD increased by only 4.012μg / g, the chlorophyll content increased from 0.362mg / g to 0.555mg / g, and the POD content decreased by 113.672μg / g.

[0236] The effects of the Tuweiwei No. 7 compound bacterial agent on the nutrient components of tomato rhizosphere soil are shown in Table 24.

[0237] Table 24 Effect of Tuweiwei 7 on nutrients in tomato rhizosphere soil

[0238]

[0239] Table 24 shows that the nutrients in the rhizosphere soil of tomatoes in the treatment group (X2) with the composite microbial agent increased significantly (P < 0.05) compared with the control group (CK2). 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 shows that the use of microbial agents can improve the nutrients in the rhizosphere soil of tomatoes. The fertility of the rhizosphere soil of tomatoes in the treatment group (X2) increased significantly (P < 0.05) compared with the control group (CK2). Among them, the biological phosphorus content and biological nitrogen content of X2 increased from 30.095 mg / kg to 38.091 mg / kg, the biological carbon content increased by 57.879 mg / kg, and the biological phosphorus content increased from 5.167 mg / kg to 25.759 mg / kg, indicating that the use of bacterial agents helps to improve the fertility of tomato rhizosphere soil.

[0240] Example 11. Effect of "Tuweiwei No. 7" on the prevention and treatment of pepper root rot and its growth-promoting effect on pepper

[0241] Pepper seedlings with similar growth were selected, and two treatments were set up, CK1 and L1, with three replicates and 10 plants per replicate. Two treatments were set up, CK2 and L2, with three replicates and 10 plants per replicate. 15 days after tomato planting, L1 and CK1 were inoculated with spores at a concentration of 2×10 6CFU / mL of mixed spore suspension of Fusarium oxysporum and Fusarium solani were inoculated with pathogens for 10 and 21 days respectively, and then the roots of CK1 and CK2 were irrigated with modified LB culture medium (liquid fermentation medium) (30 mL / plant). 8 CFU / mL of Tuweiwei No. 7 was irrigated roots (30mL / plant) of L1 and L2. After 20 days, the plant disease index, soil microbial quantity, nutrient composition of tomato rhizosphere soil and tomato growth physiological index were determined. The determination methods were the same as those in Example 10. The soil samples were stored at low temperature and transported back to the laboratory.

[0242] The growth of tomato plants in the disease prevention experimental group is as follows Fig.24 The protective effect of Tuweiwei No. 7 against pepper root rot is shown in Table 25.

[0243] Table 25 The efficacy of Tuweiwei No. 7 on pepper root rot

[0244] Treatment group number Disease index Effectiveness% 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

[0245] Fig.24 As shown in Table 25, the plant height and stem diameter of pepper 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% and 23.6% higher than those of CK1. The prevention effect of Tuweiwei No. 7 on pepper root rot was 79.3%.

[0246] The effects of Tuweiwei No. 7 on rhizosphere soil microorganisms of pepper are shown in Table 26.

[0247] Table 26 Effect of Tuweiwei 7 on the number of microorganisms in the rhizosphere soil of pepper

[0248]

[0249] As shown in Table 15, the number of soil bacteria and actinomycetes in the treatment group L1 (Tuweiwei 7+pathogen root irrigation) was significantly higher than that in the control group CK1 (pathogen root irrigation) (P<0.05). The number of bacteria in L1 was 2.404×10 7 CFU / g, increased by 1.484×10 7 CF U / g; the number of actinomycetes is 1.487×10 6 CFU / g, increased by 1.02×10 6 CFU / g. The number of soil fungi in the treatment group L1 was significantly lower than that in the control group CK1 (P<0.05). The number of fungi in L1 was 1.38×10 4 CFU / g, decreased by 5.981×104 CFU / g.

[0250] The growth status of peppers in the growth promotion experimental group is as follows Fig.25 As shown in Table 27, the effects of Tuweiwei 7 on the nutritional components of pepper leaves are shown in Table 27.

[0251] Table 27 Effects of Tuweiwei 7 on physiological parameters of pepper leaves

[0252] 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

[0253] As shown in Table 17, the pepper treatment group (L2) treated with Tuweiwei 7 showed significant increases in IAA, SOD, MDA, and chlorophyll contents (P < 0.05) compared to the control group (CK2), and a decrease in POD content. Among them, the IAA content increased from 55.854ng / g to 72.561ng / g, and the SOD, MDA, and chlorophyll contents increased by 8.429μg / g, 0.499μg / g, and 0.318mg / g, respectively. The POD content decreased from 367.37μg / g to 221.773μg / g, indicating that Tuweiwei 7 has the effect of promoting growth and improving stress resistance of pepper plants.

[0254] The effect of Tuweiwei No. 7 on the nutrient components of pepper rhizosphere soil is shown in Table 28.

[0255] Table 28 Effect of Tuweiwei 7 on rhizosphere soil nutrients of pepper

[0256]

[0257] Table 27 shows that the nutrient content of the pepper rhizosphere soil in the treatment group (L2) with Tuweiwei 7 increased significantly (P < 0.05) compared with the control group (CK2), such as the content of available nitrogen, available phosphorus, and available potassium. The content of available phosphorus increased from 466.292 mg / kg to 935.251 mg / kg, and the content of available potassium increased from 471.756 mg / kg to 903.773 mg / kg, while the content of available nitrogen only increased by 4.254 mg / kg. This shows that the use of microbial agents can improve the nutrient content of pepper rhizosphere soil. The fertility of pepper rhizosphere soil in the treatment group (L2) increased significantly (P < 0.05) compared with the control group (CK2). Among them, the biological phosphorus content and biological nitrogen content of L2 increased from 37.634mg / kg to 41.636mg / kg, the biological carbon content increased from 212.455mg / kg to 318.459mg / kg, and the biological phosphorus content increased from 5.943mg / kg to 31.670mg / kg, indicating that the use of bacterial agents helps to improve the fertility of the rhizosphere soil of peppers.

[0258] It can be seen from the above embodiments that the present invention provides Bacillus Velezii LB17, K87, MP6 and their composite bacterial agent - "Tuweiwei No. 7", which 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.

[0259] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite bacterial agent, characterized in that: It is prepared by mixed fermentation of Bacillus Velez LB17, Bacillus Velez K87 and Bacillus Velez MP6.

2. The method for preparing the composite bacterial agent according to claim 1, characterized in that: The following steps are involved: S1. Inoculate Bacillus Velezii LB17, K87, and MP6 into slant culture medium respectively, and culture the slant culture at 28-32° C. for 23-25 ​​h to obtain slant culture; S2. The slant cultures of Bacillus Velez LB17, K87, and MP6 were inoculated into the seed liquid medium respectively; the culture was carried out at 28 to 32° C. and 130 to 150 rpm for 22 to 26 hours to obtain the seed culture liquid of Bacillus Velez LB17, K87, and MP6; S3. The seed culture solution of Bacillus Velez LB17, K87 and MP6 was mixed to obtain a mixed bacterial solution; S4. Inoculate the mixed bacterial liquid into the fermentation medium at an inoculation rate of 7-9%, and culture at 32-36° C. and 180-220 rpm for 32-36 hours.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the seed culture solution of Bacillus velez LB17, K87, and 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 in concentrations: 19-21 g / L of soluble starch, 9-11 g / L of NH4Cl, MgSO4·7H2O, and the balance is water.

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

6. Use of the composite bacterial agent according to claim 1 in at least one of the following: (1) Application in the prevention and treatment of plant pathogens; (2) Application in the prevention and control of 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 plant rhizosphere soil.

7. The use according to claim 6, characterized in that: The plant pathogens include Fusarium solani, Fusarium equisetum, Fusarium oxysporum, Fusarium oxysporum, Fusarium solani, Helminthosporium salsa or Microsporum salsa; the plant diseases include root rot and wilt.

8. A strain of Bacillus Velezii LB17, characterized in that It is deposited in the China Center for Type Culture Collection, the deposit number is CCTCCNO: M 20242190, the deposit date is October 14, 2024, and the deposit address is Wuhan University.

9. A strain of Bacillus Velezii K87, characterized in that: It is deposited in the China Center for Type Culture Collection, the deposit number is CCTCCNO: M 20242191, the deposit date is October 14, 2024, and the deposit address is Wuhan University.

10. A strain of Bacillus Velezii MP6, characterized in that It is deposited in the China Center for Type Culture Collection, the deposit number is CCTCCNO: M 20242192, the deposit date is October 14, 2024, and the deposit address is Wuhan University.

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