Compound microbial agent as well as preparation method and application thereof

Abstract

The invention relates to a compound microbial agent. The compound microbial agent is prepared from bacillus subtilis HMF13 and paenibacillus mucilaginosus HM-7, the preservation number of the bacillus subtilis HMF13 is CGMCC (China General Microbiological Culture Collection Center) No.30479; the preservation number of the paenibacillus mucilaginosus HM-7 is CGMCC (China General Microbiological Culture Collection Center) No. 29734. In the compound microbial agent, the total viable count is not less than 5 * 10 < 8 > CFU / g; and the ratio of the viable count of the bacillus subtilis HMF13 to the viable count of the paenibacillus mucilaginosus HM-7 is (1-2): 1. The compound microbial agent can improve the saline-alkali soil and improve the cold resistance and yield of the saline-alkali wheat.

Description

Technical Field

[0001] The present invention relates to a composite microbial inoculant, a preparation method thereof, and an application thereof. Background Art

[0002] Saline-alkali wheat is a wheat variety adapted to grow in saline-alkali land, with characteristics such as saline-alkali resistance, cold resistance, drought resistance, and water conservation. It has been more than 2,600 years since saline-alkali wheat was planted in Huanghua, Hebei, and it has the characteristics of drought resistance, saline-alkali tolerance, and strong stress resistance. The successful cultivation of saline-alkali wheat not only improves the land use efficiency and effectively improves the land environment, but also has important significance for ensuring food security.

[0003] Although saline-alkali wheat has characteristics such as saline-alkali resistance, cold resistance, drought resistance, and water conservation, due to the low organic matter content in saline-alkali soil and the generally high soil pH (for example, the average pH of the saline-alkali land in Huanghua is as high as 8.6), saline-alkali stress will also affect the growth and development of saline-alkali wheat seedlings and hinder the growth of its plants. Therefore, after saline-alkali wheat is sown, there are still phenomena such as uneven emergence and missing seedlings. Before winter, most wheat seedlings are weak seedlings, with few tillers and few secondary roots.

[0004] The number of tillers of winter wheat before winter refers to the number of new buds formed on a single wheat plant after winter wheat enters winter and before it starts to grow in spring. Generally, tillers before winter are more likely to form ears, and they can form larger ears. The ear formation rate of tillers in spring is relatively low, and the ear heads are slightly smaller. Therefore, if you want high wheat yields, you should try to increase the tillers before winter and promote the tillers before winter.

[0005] In addition, the root system of winter wheat is the basis for its growth. The main root of winter wheat is the root that grows first when the seed germinates, usually relatively thick and has strong absorption ability. As winter wheat grows, secondary roots will gradually grow, and these secondary roots form a network structure around the main root, which helps to expand the absorption area of the root system. Research shows that an increase in the number of secondary roots before winter is beneficial to increasing the photosynthetic area of the plant, promoting the tiller growth and dry matter accumulation of the above-ground part.

[0006] The cold resistance of winter wheat refers to the ability of winter wheat to resist low temperatures. In recent years, winter wheat frost damage has occurred to varying degrees almost every year, having an adverse impact on the yield of winter wheat. Due to its growth in saline-alkali soil, saline-alkali wheat is more vulnerable to the harm of winter low temperatures than non-saline-alkali land wheat in the plain area.

[0007] The biological improvement method in the improvement methods of saline-alkali land has unique advantages. By applying a microbial inoculant to saline-alkali soil, the microorganisms in the inoculant can reproduce in the soil, thereby improving various performance indicators of the soil. Therefore, in view of the current situation of soil salinization, it is of extremely important significance to research and develop a microbial composite inoculant for saline-alkali land improvement. Summary of the Invention

[0008] The purpose of the present invention is to provide a compound microbial inoculant and a preparation method thereof, which can improve saline-alkali soil, enhance the cold resistance and yield of dry alkali wheat.

[0009] The present invention adopts the following technical solutions: A compound microbial inoculant, which includes Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7. Both of the above strains are preserved in the China General Microbiological Culture Collection Center, with the address in Beijing, China; the preservation number of Bacillus oceanisediminis HMF13 is CGMCC No. 30479, and the preservation date is April 30, 2024; the preservation number of Paenibacillus mucilaginosus HM-7 is CGMCC No. 29734, and the preservation date is January 23, 2024.

[0010] In the compound microbial inoculant, the total viable bacteria count is not less than 5×10 8 CFU / g; the ratio of the viable bacteria count of Bacillus oceanisediminis HMF13 to that of Paenibacillus mucilaginosus HM-7 is 1 - 2:1.

[0011] Preferably, the ratio of the viable bacteria count of Bacillus oceanisediminis HMF13 to that of Paenibacillus mucilaginosus HM-7 is 2:1.

[0012] In the compound microbial inoculant, it further includes granular organic fertilizer, binder and anti-caking agent.

[0013] In the compound microbial inoculant, the binder is caramel color; the organic matter content of the granular organic fertilizer is not less than 50%; the anti-caking agent is talc powder and diatomaceous earth with a mass ratio of 1:1.

[0014] A preparation method of the above compound microbial inoculant, which includes the following steps: (1) Prepare Bacillus oceanisediminis HMF13 bacterial powder and Paenibacillus mucilaginosus HM-7 bacterial powder respectively; (2) Mix the Bacillus oceanisediminis HMF13 bacterial powder and Paenibacillus mucilaginosus HM-7 bacterial powder in proportion to obtain a bacterial powder mixture, and then mix the bacterial powder mixture evenly with granular organic fertilizer, binder and anti-caking agent.

[0015] In the preparation method, the Bacillus oceanisediminis HMF13 bacterial powder or Paenibacillus mucilaginosus HM-7 bacterial powder is obtained by mixing the fermentation broth and diatomaceous earth in a mass ratio of 5 - 15:1 and freeze-drying.

[0016] In the preparation method, the effective viable bacteria count of Bacillus oceanisediminis HMF13 bacterial powder is not less than 2×10 10 CFU / g, and the effective viable bacteria count of Paenibacillus mucilaginosus HM-7 bacterial powder is not less than 1×10 10 CFU / g.

[0017] In the preparation method, the mass ratio of the bacterial powder mixture, granular organic fertilizer, binder and anti-caking agent is 5-20:1000:1-10:5-15.

[0018] Application of the above-mentioned compound microbial inoculum in improving saline-alkali soil.

[0019] Application of the above-mentioned compound microbial inoculum in promoting the growth of drought-tolerant and alkali-tolerant wheat.

[0020] Application of the above-mentioned compound microbial inoculum in promoting the growth of drought-tolerant and alkali-tolerant wheat planted in saline-alkali soil.

[0021] Application of the above-mentioned compound microbial inoculum in enhancing the cold resistance of drought-tolerant and alkali-tolerant wheat.

[0022] The beneficial effects of the present invention are as follows: The present invention uses Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 to prepare a compound microbial inoculum, which has a good growth-promoting effect on drought-tolerant and alkali-tolerant wheat. In particular, it can increase the number of tillers before winter and the number of secondary roots of drought-tolerant and alkali-tolerant wheat, reduce the pH of saline-alkali soil, increase the organic matter content of saline-alkali soil, enhance the cold resistance of drought-tolerant and alkali-tolerant wheat, and finally significantly increase the yield per mu of drought-tolerant and alkali-tolerant wheat. Description of the drawings

[0023] Figure 1 Colony morphology of Bacillus oceanisediminis HMF13 cultured on Gibbson modified medium.

[0024] Figure 2 Morphology of Bacillus oceanisediminis HMF13 under an optical microscope after Gram staining.

[0025] Figure 3 Phylogenetic tree of Bacillus oceanisediminis HMF13 constructed based on 16S rDNA.

[0026] Figure 4 Biocompatibility test result A between Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7. Among them, the left side is the front view of the petri dish, and the right side is the reverse view of the petri dish.

[0027] Figure 5 Biocompatibility test result B between Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7. Among them, the left side is the front view of the petri dish, and the right side is the reverse view of the petri dish.

[0028] Figure 6 Cultivation performance of Paenibacillus mucilaginosus HM-7 on Gibbson modified medium under different salt concentrations. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with embodiments and the accompanying drawings. The protection scope of the present invention is not limited to the embodiments. Any modifications made by those skilled in the art within the scope defined by the claims (such as changing the halophytic wheat to wheat, etc.) also fall within the protection scope of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The reagents used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0030] Example 1 Characteristics of Bacillus oceanisediminis HMF13 Bacillus oceanisediminis ( Oceanobacillus sp .) HMF13 was isolated and purified from the root soil of the halophyte Phragmites australis in the saline-alkali land of Gaotuo Village, Liutaizhuang Town, Changli County, Qinhuangdao City, Hebei Province, China. It is deposited in the General Microbiology Center of the China Microbial Culture Collection Center, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 30479, and the deposit date is April 30, 2024.

[0031] (1) Morphological characteristics: After the HMF13 strain was cultured on a Gibbson (Gibson) modified medium plate at 30 °C for 3 days, as Figure 1 shown, the single colony of this strain was milky white, with a rough surface and irregular edges. Under an optical microscope, the cells were rod-shaped, with a size of 1-2 μm × (0.5-1 μm), aggregated together in short chains or beaded arrangements. The spores were oval, central, and the terminal was enlarged into a sporangium; Gram staining showed positive, as Figure 2 . The phylogenetic tree of Bacillus oceanisediminis HMF13 constructed based on 16S rDNA is as Figure 3 shown.

[0032] Gibbson (Gibson) modified medium: casein 5.0 g, sodium citrate 3.0 g, yeast extract powder 10.0 g, KCl 2.0 g, peptone 5.0 g, MgSO4·7H2O 2.0 g, NaCl 100 g, pH 9.0, agar 2%, 1000 mL of water.

[0033] (2) Physiological and biochemical characteristics An identification experiment was conducted on the physiological and biochemical characteristics of the Bacillus oceanisediminis HMF13 strain, and the results are shown in Table 1.

[0034] Table 1 Physiological and biochemical characteristics of Bacillus oceanisediminis HMF13 strain .

[0035] Example 2 Biocompatibility between Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 Paenibacillus mucilaginosus ( Paenibacillus mucilaginosus ) HM-7, which was isolated and purified from the soil sample of the high-yield demonstration base of dryland saline wheat in Huanghua City, Cangzhou, Hebei Province, is preserved in the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 29734. The address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is January 23, 2024.

[0036] Activated Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 using LB medium. Use an inoculation loop to pick up the colony of Bacillus oceanisediminis HMF13 and place it in 1 mL of sterile water. Use a sterile pipette to repeatedly blow and wash to mix evenly to prepare a bacterial suspension, and then add it to the non-solidified but not hot LB medium. After shaking well, immediately pour the plate; place the bacterial cake of Paenibacillus mucilaginosus HM-7 in the center of the cooled and solidified plate, mark it well, and repeat 3 times. Place it in a constant temperature incubator at 37 °C for 3 days.

[0037] Observation results showed that ( Figure 4 ) Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 grew together without the production of an inhibition zone, indicating that there was no mutual inhibition between Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 strains.

[0038] Similarly, use an inoculation loop to pick up the colony of Paenibacillus mucilaginosus HM-7 and place it in 1 mL of sterile water. Use a sterile pipette to repeatedly blow and wash to mix evenly to prepare a bacterial suspension, and then add it to the non-solidified but not hot LB medium. After shaking well, immediately pour the plate; place the bacterial cake of Bacillus oceanisediminis HMF13 in the center of the cooled and solidified plate, mark it well, and repeat 3 times. Place it in a constant temperature incubator at 37 °C for 3 days. The results showed that ( Figure 5 ) Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 grew together without the production of an inhibition zone, indicating that there was no mutual inhibition between Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7 strains.

[0039] LB medium formula: 5 g of yeast extract, 10 g of peptone, 10 g of sodium chloride. Place them in a 1000 mL beaker, add 900 mL of distilled water and heat to dissolve. Adjust the pH to 7.2 - 7.4, make up the volume to 1 L with distilled water, and sterilize at 121 °C for 30 minutes for standby.

[0040] Example 3 Performance of salt tolerance of Paenibacillus mucilaginosus HM-7 Gibbson Modified Medium: casein 5.0 g, sodium citrate 3.0 g, yeast extract powder 10.0 g, KCl 2.0 g, peptone 5.0 g, MgSO4·7H2O 2.0 g, NaCl 100 g, pH 9.0, agar 2%, 1000 mL of water.

[0041] Under the condition of pH = 9 in the Gibbson modified medium, the NaCl concentrations of the medium were respectively adjusted to 100 g·L -1 , 120 g·L -1 , 140 g·L -1 , 160 g·L -1 , 180 g·L -1 and 200 g·L -1 to prepare multiple media with different salinities. Bacillus mucilaginosus HM-7 strains were picked and inoculated onto the plates of Gibbson modified media with altered salinities respectively, and then placed in an incubator at 37°C for 5 days. Whether the strains grew or not was used as the criterion to judge whether the strains were salt-tolerant to the corresponding salinities.

[0042] As Figure 6 shown, Bacillus mucilaginosus HM-7 can grow on the media with NaCl concentrations of 100 g·L -1 and 120 g·L -1 respectively.

[0043] Example 4 Preparation of Bacillus oceanisediminis HMF13 Bacterial Powder Using Diatomite as Carrier (1) Preparation of LB liquid medium: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, placed in a 1000 mL beaker, added 900 mL of distilled water and heated to dissolve, adjusted the pH to 7.2 - 7.4, made up the volume to 1 L with distilled water, and sterilized at 121°C for 30 minutes for standby.

[0044] (2) Activation of strains: One loop of Bacillus oceanisediminis HMF-13 colonies was picked and inoculated into a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium, and cultured at a constant temperature of 37°C with shaking at 160 rpm for 24 h for activation.

[0045] (3) Preparation of seed liquid: 4 mL of the activated bacterial liquid was taken and inoculated into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium, and cultured at a constant temperature of 37°C with shaking at 160 rpm for 24 h to obtain the seed liquid.

[0046] (4) Preparation of fermentation broth: 180 mL of the prepared seed liquid was taken and inoculated into a 6 L small fermenter containing 3.5 L of LB liquid medium, and cultured at a constant temperature of 37°C with shaking at 160 rpm for 48 h to obtain the fermentation broth.

[0047] (5) Mix the fermentation broth and diatomaceous earth evenly at a mass ratio of 10:1, and spray them with a freeze dryer to obtain the Bacillus oceanisediminis HMF-13 bacterial powder. After detection, the viable count of effective bacteria is 4.12×10 10 CFU / g.

[0048] Example 5 Preparation of Paenibacillus mucilaginosus HM-7 bacterial powder using diatomaceous earth as a carrier (1) Preparation of LB liquid medium: Weigh 3 g of beef extract, 10 g of peptone, and 5 g of sodium chloride, place them in a 1000 mL beaker, add 900 mL of distilled water, heat and dissolve. Adjust the pH to 7.2 - 7.4, make up the volume to 1 L with distilled water, and sterilize at 121 °C for 30 minutes for later use.

[0049] (2) Activation of the strain: Pick up a loop of Paenibacillus mucilaginosus HM-7 colony and inoculate it into a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium. Incubate it at 160 rpm and 37 °C with constant shaking for 24 h for activation.

[0050] (3) Preparation of the seed liquid: Take 4 mL of the activated bacterial liquid and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium. Incubate it at 160 rpm and 37 °C with constant shaking for 24 h to obtain the seed liquid.

[0051] (4) Preparation of the fermentation broth: Take 180 mL of the prepared seed liquid and inoculate it into a 6 L small fermenter containing 3.5 L of LB liquid medium. Incubate it at 160 rpm and 37 °C with constant shaking for 48 h to obtain the fermentation broth.

[0052] (5) Mix the fermentation broth and diatomaceous earth evenly at a mass ratio of 10:1, and spray them with a freeze dryer to obtain the Paenibacillus mucilaginosus HM-7 bacterial powder. After detection, the viable count of effective bacteria is 3.03×10 10 CFU / g.

[0053] Example 6 Preparation of compound microbial inoculum (1) Materials Binder: Caramel color, purchased from the market, and dissolved in warm water at a weight ratio of 1:1 before use.

[0054] Granular organic fertilizer: Purchased from the market, with the following content: organic matter 50.0%, N+P+K = 9.2%.

[0055] Anti-caking agent: Prepared from talc powder and diatomaceous earth at a mass ratio of 1:1. Both talc powder and diatomaceous earth are purchased from the market.

[0056] (2) Preparation method Mix the Bacillus oceanisediminis HMF13 bacterial powder (the viable count of effective bacteria is 4.12 ×10 prepared in Examples 4 - 510 CFU / g), Paenibacillus mucilaginosus HM-7 bacterial powder (effective viable count is 3.03×10 10 CFU / g), and respectively mixed to obtain bacterial powder mixture 1, bacterial powder mixture 2, and bacterial powder mixture 3 according to the viable count ratios of Bacillus oceanisediminis HMF13 to Paenibacillus mucilaginosus HM-7 of 1∶1, 1.5∶1, and 2∶1

[0057] Mix to prepare 18 g of bacterial powder mixture 1. 7.6 g of Bacillus oceanisediminis HMF13 bacterial powder and 10.4 g of Paenibacillus mucilaginosus HM-7 bacterial powder are required.

[0058] Mix to prepare 18 g of bacterial powder mixture 2. 9.4 g of Bacillus oceanisediminis HMF13 bacterial powder and 8.6 g of Paenibacillus mucilaginosus HM-7 bacterial powder are required.

[0059] Mix to prepare 18 g of bacterial powder mixture 3. 10.7 g of Bacillus oceanisediminis HMF13 bacterial powder and 7.3 g of Paenibacillus mucilaginosus HM-7 bacterial powder are required.

[0060] Take 18 g of each bacterial powder mixture, mix it with 1 kg of granular organic fertilizer, stir for 5 minutes, then add 5 g of caramel color and stir for 5 minutes, and then add 10 g of anti-caking agent and stir for 5 - 8 minutes. (The mass ratio of the bacterial powder mixture, granular organic fertilizer, binder, and anti-caking agent is 18∶1000∶5∶10). The prepared product particles do not stick, cannot be formed into a ball by hand, have a black and bright appearance, and no powder, thus obtaining compound microbial inoculant 1, compound microbial inoculant 2, and compound microbial inoculant 3. After testing, the total viable count of the prepared compound microbial inoculant 1 is 6.05×10 8 CFU / g, the total viable count of compound microbial inoculant 2 is 6.25×10 8 CFU / g, and the total viable count of compound microbial inoculant 3 is 6.36×10 8 CFU / g. The total viable counts of the above compound microbial inoculants all meet the requirement of not less than 5×10 8 cfu / g.

[0061] Example 7 Pot experiment on the application of compound microbial inoculant (1) Test materials Wheat variety: Cangmai 6002.

[0062] Flower pot: with a diameter of 10 cm.

[0063] (2) Test method (a) Preparation of test soil: Soil was collected from the applicant's experimental field, with the following indicators: organic matter 8.76 g / kg, available nitrogen 30.96 mg / kg, available phosphorus 15.98 mg / kg, available potassium 34.28 mg / kg, salt content 0.22 g / kg, and pH 6.58. This soil has a low organic matter content and is prone to hardening after watering. After thoroughly watering it once with 1.2% NaCl and 1% Na2CO3 solutions and drying it to a constant weight, the soil was screened through a 2-mm sieve and used as the test soil. The pH of the test soil was detected by the potentiometric method to be 7.82, and the organic matter content was determined by the external heating method with potassium dichromate to be 8.74 g / kg.

[0064] (b) Pretreatment: The prepared test soil was transferred to flower pots with a diameter of 10 cm, 350 g per pot; plump wheat seeds of the same size were selected and soaked and disinfected in warm water at 50 °C for 30 minutes for later use.

[0065] (c) Experimental design: The experiment was set with 6 treatments, 8 pots for each treatment, and 10 wheat seeds were sown in each pot. Among them, in treatment 1, the seeds were dressed with compound microbial inoculant 1, in treatment 2, the seeds were dressed with compound microbial inoculant 2, in treatment 3, the seeds were dressed with compound microbial inoculant 3, in treatment 4, instead of using a microbial inoculant, the granular organic fertilizer described in Example 6 was used for seed dressing as a blank control (CK), in treatment 5, the seeds were dressed with the single strain Bacillus oceanisediminis HMF13 inoculant, and in treatment 6, the seeds were dressed with the single strain Paenibacillus mucilaginosus HM-7. The dosage of the inoculant for seed dressing in each treatment was 10 g / kg of seeds, and the dosage of the granular organic fertilizer for seed dressing in the blank control (CK) was 10 g of granular organic fertilizer per 1 kg of seed weight. After sowing the wheat in all treatments, they were jointly transferred to a light incubator for continued cultivation. Among them, the Bacillus oceanisediminis HMF13 inoculant and the Paenibacillus mucilaginosus HM-7 inoculant were prepared from the bacterial powders prepared in Examples 4 and 5, and diatomaceous earth was added to adjust the total viable count to 6.3×10 8 CFU / g.

[0066] (4) Detection indicators: After culturing for 30 d, the growth index (plant height) of wheat seedlings in the blank control group, the single-inoculant treatment group, and the compound microbial treatment group under saline-alkali stress was detected. For each treatment, 4 soil samples were randomly collected from the pots, mixed evenly, and 200 g was taken by the quartering method and dried to detect the pH of the dried soil and the soil organic matter content. The method for determining soil organic matter was the external heating method with potassium dichromate.

[0067] (3) Results and analysis The experimental results are shown in Table 2.

[0068] Table 2 Statistical results of the investigated indicators for each treatment .

[0069] As can be seen from the results in Table 2, under the conditions of the pot experiment, applying single bacterial agents and compound bacterial agents both showed a significant trend of reducing the pH of saline-alkali soil. Compared with applying single Bacillus oceanisediminis HMF13 or Paenibacillus polymyxa HM-7, the effect of applying the compound microbial bacterial agent on reducing soil pH was more significant.

[0070] The experiment also showed that applying single bacterial agents and compound bacterial agents could increase the organic matter content of saline-alkali soil and promote wheat growth. The effects of compound microbial bacterial agents 1-3 were significantly better than those of single bacterial agents Bacillus oceanisediminis HMF13 and Paenibacillus polymyxa HM-7. After applying compound microbial bacterial agent 3, the organic matter content increased by 4.85 g / kg compared with the control, and the wheat plant height increased by an average of 7.1 cm compared with the control, which could effectively promote the growth of wheat in saline-alkali land, and Bacillus oceanisediminis HMF13 and Paenibacillus polymyxa HM-7 played a synergistic effect.

[0071] Example 8 Field Application Experiment of Compound Microbial Bacterial Agent (1) Planting Location and Time The experiment was set up in a dryland saline-alkali wheat experimental field in Huanghua. The soil texture of this plot was loam. Before sowing, soil samples were taken for detection, and the data of the soil samples were: pH 8.62, organic matter 9.97 g / kg, salt content 3.93 g / kg, belonging to moderately saline-alkali land. Wheat was sown on October 17, 2023, with a seeding rate of 15 kg / mu. Before sowing, along with the deep plowing of the soil operation, 28 kg / mu of diammonium phosphate was applied. Wheat variety: Jiemai 19.

[0072] (2) Experimental Treatment Design The bacterial agent treatments included single bacterial agents and compound microbial bacterial agent 3 prepared in Example 6 (total viable count was 6.36×10 8 CFU / g). Control treatment 1 was the local fertilizer application method. Control treatment 2 was the use of granular organic fertilizer (organic matter 50.0%, N+P+K = 9.2%, purchased from the market, the same as in Example 5). The fertilizers used in each treatment were applied into the soil by ditch while sowing at the same time as the dryland saline-alkali wheat was sown.

[0073] The soil samples were collected on October 12, 2023 before planting, and on December 12, 2023 after planting. Each treatment is shown in Table 3.

[0074] Table 3 Experimental Design 。

[0075] Same as Example 7, the Bacillus oceanisediminis HMF13 bacterial agent and Paenibacillus polymyxa HM-7 bacterial agent in Table 3 were prepared by applying the bacterial powders prepared in Examples 4 and 5 respectively, and then adding diatomaceous earth to adjust the total viable count to 6.3×10 8 CFU / g.

[0076] Each treatment in Table 3 was set with 3 replicates, and the field plots were arranged in a randomized block design with a plot area of 0.5 mu. On December 12, 2023, three points were randomly determined along the diagonal direction in each plot, and 20 plants were continuously sampled at each point to investigate the tiller number and the number of secondary roots. Soil samples were collected and analyzed for soil pH and organic matter content.

[0077] On March 4, 2024, after the dryland saline wheat turned green and before new tillers appeared, 3 rows (excluding the edge rows) were selected in each plot according to the 3-point sampling method. Then, 1 meter was selected in each row, and all the wheat seedlings within the 1-meter sample section were dug out to investigate the total stem number and the number of dead stems, and the average value was taken to calculate the overwintering dead stem rate.

[0078] Overwintering dead stem rate (%) = Number of dead stems investigated / Total number of stems investigated × 100.

[0079] On June 9, 2024, at the full maturity stage of wheat, three points were randomly sampled along the diagonal in each plot, and 20 spikes were randomly selected at each point (a total of 60 spikes). The average number of effective spikes per square meter was investigated, and the number of effective spikes per mu for each treatment was calculated. Finally, the theoretical yield of each treatment was calculated based on the number of effective spikes, 1000-grain weight, and average number of grains per spike. The survey results are shown in Table 4.

[0080] Table 4 Effects of applying compound microbial inoculant for saline-alkali soil improvement on wheat in Huanghua saline-alkali soil 。

[0081] As can be seen from the above table, after applying the compound microbial inoculant 3 prepared in Example 6 of the present invention, the number of secondary roots and tiller number of wheat both exceeded the control, and were also significantly better than those of the single inoculant Bacillus oceanisediminis HMF13 and the single inoculant Paenibacillus mucilaginosus HM-7. HMF13 and HM-7 can form a synergistic effect. Compared with the control treatment 1, the soil pH of the compound inoculant treatment decreased from 8.55 to 7.67, and the organic matter content increased from 9.96 g / kg to 14.20 g / kg; compared with the control treatment 2, the soil pH of the compound inoculant treatment decreased from 8.47 to 7.67, and the organic matter content increased from 10.16 g / kg to 14.20 g / kg. Similarly, compared with the control, the number of secondary roots and tiller number of wheat in the plots treated with the compound inoculant increased significantly.

[0082] After the dryland saline wheat turned green in spring, the overwintering dead stem rate of the wheat seedlings was investigated. The investigation results showed that for the treatment with the compound inoculant, the overwintering dead stem rate was only 4.30%, far lower than the overwintering dead stem rate values of 12.70% and 10.45% of the control treatment 1 and the control treatment 2, indicating that applying the compound inoculant to plant dryland saline wheat can enhance the cold resistance of dryland saline wheat, thus significantly reducing the overwintering dead stem rate of wheat seedlings.

[0083] The yield measurement results show that the compound microbial inoculant 3 prepared in Example 6 can relieve the inhibition of saline-alkali stress on wheat growth and can significantly increase the wheat yield. From the analysis in Table 4, the number of secondary roots treated with the compound microbial inoculant is almost twice that of the control treatment 1, indicating that the main function of the compound microbial inoculant is to promote root growth. Farmers near this test area reported to the applicant that the wheat roots in the fields treated with the compound microbial inoculant were well-developed before winter, which also indirectly verified the results of this experiment.

[0084] As mentioned above, it is only the optimal specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions within the technical scope disclosed in this application should be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claims.

Claims

1. A compound microbial inoculum, characterized in that, It includes Bacillus oceanisediminis HMF13 and Paenibacillus mucilaginosus HM-7; the preservation number of Bacillus oceanisediminis HMF13 is CGMCC No. 30479; the preservation number of Paenibacillus mucilaginosus HM-7 is CGMCC No. 29734.

2. The compound microbial inoculum according to claim 1, wherein The total viable count is not less than 5×10 8 CFU / g; the ratio of the viable count of Bacillus oceanisediminis HMF13 to Paenibacillus mucilaginosus HM-7 is 1-2:

1.

3. The composite microbial inoculant according to claim 2, wherein It further includes granular organic fertilizer, binder and anti-caking agent.

4. The composite microbial inoculum according to claim 3, wherein The binder is caramel color; the organic matter content of the granular organic fertilizer is not less than 50%; the anti-caking agent is talcum powder and diatomaceous earth with a mass ratio of 1:

1.

5. A preparation method of the composite microbial inoculum according to any one of claims 1 to 4, characterized in that, It includes the following steps: (1) Prepare Bacillus oceanisediminis HMF13 powder and Paenibacillus mucilaginosus HM-7 powder respectively; (2) Mix the Bacillus oceanisediminis HMF13 powder and Paenibacillus mucilaginosus HM-7 powder in proportion to obtain a powder mixture, and then mix the powder mixture evenly with the granular organic fertilizer, binder and anti-caking agent.

6. The preparation method according to claim 5, characterized in that, The Bacillus oceanisediminis HMF13 bacterial powder or Paenibacillus mucilaginosus HM-7 bacterial powder is obtained by mixing the fermentation broth and diatomite in a mass ratio of 5-15:1 and freeze-drying; the effective viable count of the Bacillus oceanisediminis HMF13 bacterial powder is not less than 2×10 10 CFU / g, and the effective viable count of the Paenibacillus mucilaginosus HM-7 bacterial powder is not less than 1×10 10 CFU / g.

7. The preparation method according to claim 5, characterized in that, The mass ratio of the powder mixture, granular organic fertilizer, binder and anti-caking agent is 5-20:1000:1-10:5-15.

8. Application of a composite microbial inoculant according to any one of claims 1-4 in improving saline-alkali soil.

9. Application of a composite microbial inoculant according to any one of claims 1-4 in promoting the growth of drought-tolerant and alkali-tolerant wheat.

10. Application of a composite microbial inoculant according to any one of claims 1-4 in enhancing the cold resistance of drought-tolerant and alkali-tolerant wheat.

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