Compound microbial agent for nitrogen fixation, carbon sequestration, synergism and stress resistance and application of compound microbial agent

Abstract

The invention discloses a compound microbial agent for nitrogen fixation, carbon sequestration, synergism and stress resistance and application, and belongs to the technical field of functional microbial preparations, the compound microbial agent provided by the invention is prepared by taking combined nitrogen-fixing bacteria and high-yield urease inhibitor bacteria as cores and compounding salt-resistant, biocontrol, growth-promoting and carbon sequestration bacteria. The compound microbial agent provided by the invention can efficiently wake up nitrogen-fixing flora of a root system, accelerate nitrogen conversion in the atmosphere and play a role in nitrogen fixation and synergism on one hand, and can increase the content of organic carbon in soil and improve the fertility of the soil on the other hand, so that the quality of agricultural products is improved, and the purposes of increasing yield and income are achieved. In addition, the compound microbial agent also has stress resistance and disease prevention functions, and can relieve abiotic stress caused by a saline-alkali soil environment, prevent plant diseases caused by bacteria or fungi, improve the survival rate of crop cutting seedlings and enhance the continuous cropping resistance of crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microbial preparations, and in particular relates to a composite microbial agent capable of fixing nitrogen and carbon, enhancing efficiency and resisting stress, and its application. Background Art

[0002] Nitrogen is the mineral nutrient element with the largest demand by plants. Insufficient nitrogen nutrient in the soil is the main factor limiting plant growth. In agricultural production, the application of nitrogen fertilizer can meet the growth needs of crops, but unreasonable and excessive application of nitrogen fertilizer will lead to nitrogen loss and cause a series of environmental problems such as acid rain, soil compaction, and eutrophication of water bodies, which seriously restrict the growth of crops.

[0003] The decomposition rate of chemical nitrogen fertilizers is much greater than the utilization rate of crops, which is the main reason for the low utilization rate of traditional nitrogen fertilizers. Taking urea, which is widely used in nitrogen fertilizers, as an example, its effective action time in the soil is usually only 7 to 14 days. Urease inhibitors can inhibit urease activity and slow down the rate of urea hydrolysis and ammoniaization, thereby reducing nitrogen losses. However, the potential safety issues of compound urease inhibitors and the process cost issues of plant extract urease inhibitors have forced researchers to seek a new way to obtain urease inhibitors.

[0004] Nitrogen-fixing microorganisms provide a new solution for achieving efficient and pollution-free nitrogen application. In nature, some prokaryotic microorganisms can convert nitrogen in the air into ammonia, a process called biological nitrogen fixation. Using biological nitrogen fixation to replace chemical nitrogen fertilizers has great application potential in agricultural production. According to the relationship between nitrogen-fixing microorganisms and crops, biological nitrogen fixation can be divided into two types: symbiotic nodulation nitrogen fixation and non-leguminous combined nitrogen fixation. These are also the two types of microbial nitrogen fixation that can be used in agricultural production so far. Compared with the former, combined nitrogen-fixing bacteria have the advantages of a wide host range and strong adaptability, so they have broader application prospects.

[0005] In recent years, reports on urease inhibitor-producing microorganisms have appeared one after another, including natural strains such as Bacillus subtilis and Bacillus coagulans, as well as artificially modified engineered strains such as Escherichia coli and yeast. The application areas are mainly concentrated in environmental governance and livestock and poultry breeding. At present, there is still a lack of application cases of urease inhibitor-producing microorganisms in agricultural production, especially in agricultural microbial agents. If "combined nitrogen fixation" and "microbial urease inhibitors" can be effectively combined, and the low utilization rate and environmental pollution problems in the use of traditional chemical nitrogen fertilizers can be solved at the same time, then the significance of this technology in reducing nitrogen fertilizers and replacing them, and reducing agricultural costs and emissions will be self-evident.

[0006] The application of microbial agents has played an important role in achieving sustainable agricultural development. CN 118285403A provides an ARC microbial agent (Bacillus laterosporus + Bacillus amyloliquefaciens + Bacillus mucilaginosus + Enterobacter Ludwigii) with a coupled effect of controlling toxicity and fixing nitrogen, which can promote early nodulation and nitrogen fixation of soybeans, early flowering and pod formation, increase the abundance of rhizobia in the rhizosphere of leguminous crops, and increase the number of nodules of leguminous crops. CN 118266480A provides a microbial agent with acid-alkali-resistant nitrogen-fixing tropical Bacillus as the core, which can not only promote plant growth, but also improve the structure of soil microbial communities, increase the richness, diversity, uniformity and species diversity of microbial communities, and increase the content of total nitrogen, effective phosphorus and fast-acting potassium in the soil. The efficiency of nitrogen fixation to ammonia is improved by high-yield ACC deaminase and nitrogenase activity, and the nitrogenase activity can reach up to 233.75 IU / L. However, due to the differences in nitrogen fixation ability or other functional performances of different nitrogen-fixing bacteria, there may be a variety of complex interaction relationships such as antagonism, synergy, and symbiosis between nitrogen-fixing bacteria and other functional strains. Therefore, the development of composite microbial agents with better nitrogen fixation ability or new functions is still a hot topic and difficulty in current research. Summary of the invention

[0007] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a composite microbial agent for nitrogen fixation, carbon fixation, synergistic resistance and its application. The composite microbial agent prepared by the present invention combines nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, and compound salt-tolerant, biocontrol, growth-promoting and carbon-fixing bacteria to achieve efficient utilization of nitrogen fertilizer, improve the quality and yield of agricultural products, and significantly enhance the ability to resist abiotic stress caused by saline-alkali soil environment, which is of great significance to agricultural production.

[0008] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a composite microbial agent comprising: combined nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, salt-tolerant bacteria, biocontrol bacteria, growth-promoting bacteria and carbon-fixing bacteria.

[0009] The combined nitrogen-fixing bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )NBL-B11002; the high-yield urease inhibitor bacteria is Brevibacillus laterosporus ( Brevibacillus laterosporus )BLCC1-1219; the salt-tolerant bacteria is Daban Halobacillus ( Halobacillus dabanensis strain ) NBL-BS214; the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )NBL-AP73; the growth-promoting bacteria is Bacillus subtilis ( Bacillus subtilis )NBL-B12004; the carbon-fixing bacteria is Priesteria gigantea ( Priestia megaterium )C17.

[0010] Among them, the deposit number of Bacillus amyloliquefaciens NBL-B11002 is CCTCC NO: M 20232184, and the strain has been recorded in the published patent CN 117660243 A; Brevibacillus laterosporus BLCC1-1219 has been deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), with a deposit date of September 29, 2024, and a deposit number of CCTCC NO: M 20242118. The strain was isolated from fresh livestock and poultry feces samples and has significant urease inhibitory activity. The urease activity inhibition rate of the strain at different times was determined by the phenol-hypochlorous acid method. The test results showed that the inhibition rate of the strain on urease could reach 92.52% within 30 minutes, 92.61% within 60 minutes, and 97.07% within 120 minutes.

[0011] The deposit number of Daban Halobacillus sp. NBL-BS214 is CCTCC NO: M 20221370, and the strain has been recorded in the published patent CN 116286461 B; Bacillus amyloliquefaciens NBL-AP73 has been deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), with a deposit date of June 19, 2024, and a deposit number of CCTCC NO: M 20241295. The strain has been recorded in the published patent CN 118931780 A. The strain was isolated from a tomato plant with a recent tomato neck rot and root rot disease. The strain was inoculated on a plate containing the tomato neck rot and root rot specialized type pathogen of Fusarium oxysporum, and the plate without NBL-AP73 was used as the control group. The plate was streaked and the inhibition rate was calculated according to the formula: (control group colony diameter-treatment group colony diameter) / control group colony diameter × 100%. The results of the inhibition rate detection experiment showed that the strain had an antibacterial ability of 93.38% against Fusarium oxysporum tomato neck rot and root rot specialized type (FORL), and had a good antibacterial effect. At the same time, the growth conditions of tomato seedlings irrigated with Bacillus amyloliquefaciens NBL-AP73 solution and those not treated with Bacillus amyloliquefaciens NBL-AP73 solution were compared. The results showed that the application of Bacillus amyloliquefaciens NBL-AP73 solution can effectively promote the increase of plant height and stem thickness of tomato seedlings. The results show that Bacillus amyloliquefaciens NBL-AP73 also has a good growth-promoting effect on tomato seedlings.

[0012] The deposit number of Bacillus subtilis NBL-B12004 is CCTCC NO: M 20231506, and the strain has been recorded in the published patent CN 117487699 B; The deposit number of Priesteria gigantea C17 is CCTCC NO: M 20241724, and the strain has been recorded in the published patent CN118853507A; Improving the growth and health of crops by inoculating microbial agents is essentially a process of artificially constructing microbial communities, that is, synthetic microbial communities. In the ecosystem composed of plants, soil and microorganisms, the species diversity of the synthetic microbial community directly determines the resistance stability of the system. Therefore, the preparation of composite microbial agents based on the niche theory is of great significance for improving the environmental adaptability of products, the stability of effects, and achieving overall regulation of community functions.

[0013] Plant secretions (roots, leaves, phloem, etc.) provide nutrients for symbiotic microorganisms and have a shaping effect on their community structure. When inoculated with a single microbial agent, it is easy to increase the metabolic load of crops. The resource utilization overlap rate of the multiple strains described in this application is low, and the established functional zoning is in line with the micro-regulation of crop growth by microorganisms in the context of the material cycle. On the one hand, through the multiple effects of nitrogen fixation, carbon fixation, growth promotion and high-yield urease inhibitor bacteria, the soil can maintain a reasonable carbon-nitrogen ratio under the premise of outputting sufficient nitrogen nutrition, which is beneficial to maintaining soil fertility and fertility and promoting crop growth; on the other hand, through the activation of biological control and salt-tolerant bacteria, the biological stress / abiotic stress response mechanism significantly enhances the adaptability of crops to adversity, which is beneficial to the healthy growth of crops, thereby improving the quality of agricultural products and achieving the purpose of increasing production and income.

[0014] The ratio of the number of live bacteria of nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, salt-tolerant bacteria, biocontrol bacteria, growth-promoting bacteria and carbon-fixing bacteria in the composite microbial agent is (5-6):(1-2):1:1:1:1.

[0015] Preferably, the total number of viable bacteria in the composite microbial agent is ≥ 10 billion / g.

[0016] Furthermore, during the preparation of the composite microbial agent, a carrier may be added according to the requirements of the dosage form.

[0017] Preferably, the composite microbial agent is in the form of any one of granules, wettable powders, suspensions and water dispersible granules.

[0018] In a second aspect, the present invention provides the use of the composite microbial agent in at least one of the following (1) to (6): (1) Nitrogen fixation; (2) Carbon fixation; (3) Promote crop growth; (4) Enhance the stress resistance of crops; (5) Enhance the disease resistance of crops; (6) Improve the ability of crops to resist repeated cropping.

[0019] The nitrogen fixation is specifically manifested in: increasing the total nitrogen content of crops, inhibiting the urease activity in the soil, increasing the number of nitrogen-fixing bacteria in the soil and / or increasing the number of nodules in leguminous crops.

[0020] The carbon fixation is specifically manifested as increasing the organic carbon content in the soil.

[0021] The promotion of crop growth is specifically manifested in: promoting the growth of above-ground and underground parts of crops, increasing crop dry matter accumulation and / or improving crop yield.

[0022] The stress resistance is specifically manifested as: resisting abiotic stress caused by saline-alkali soil environment. The composite microbial agent can reduce ethylene accumulation caused by moderate salt stress and significantly increase the activity of antioxidant enzymes in crops.

[0023] The disease resistance is specifically manifested as: preventing plant diseases caused by bacteria or fungi.

[0024] Furthermore, the plant disease caused by the bacteria or fungi is bacterial soft rot or potato wilt.

[0025] Beneficial effects of the present invention: (1) The composite microbial agent provided by the present invention is prepared with combined nitrogen-fixing bacteria and high-yield urease inhibitor bacteria as the core, and is compounded with salt-tolerant, biocontrol, growth-promoting and carbon-fixing bacteria. The composite microbial agent is prepared by innovatively adjusting the ratio of the components in the components, has good application effect, and is of great significance to agricultural production.

[0026] (2) The composite microbial agent provided by the present invention can, on the one hand, efficiently awaken the root nitrogen-fixing bacteria, accelerate the conversion of atmospheric nitrogen, alleviate the loss of soil nitrogen fertilizer, establish an efficient combined nitrogen fixation system with crops, achieve efficient utilization of nitrogen fertilizer and reduce the amount and increase the efficiency, significantly improve the nitrogen fixation efficiency, play a role in nitrogen fixation and increase the efficiency, and can also increase the soil organic carbon content and improve soil fertility, thereby improving the quality of agricultural products and achieving the purpose of increasing production and income; on the other hand, it also has the function of resisting stress and preventing diseases, resisting abiotic stress caused by saline-alkali soil environment, reducing ethylene accumulation caused by moderate salt stress, significantly increasing the activity of antioxidant enzymes in crops, preventing plant diseases caused by bacteria or fungi, and playing a role in improving the ability of crops to resist continuous cropping. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Comparison of corn plant growth between formula 4 and the control group.

[0028] Figure 2 Growth of wheat plants under different treatments.

[0029] Figure 3 Comparison of the growth of wheat plants in formula 4 and the control group; Figure 3 A is a comparison of the growth of wheat plant height between Formula 4 and the control group. Figure 3 Figure B is a comparison of the root growth of wheat plants in formula 4 and the control group.

[0030] Figure 4 Comparison of the growth of peanut plants between formula 4 and the control group.

[0031] Figure 5 Comparison of cabbage disease conditions between formula 4 and the control group.

[0032] Figure 6 Comparison of disease conditions of potatoes in formula 4 and the control group ("Lucinda V7").

[0033] Figure 7 Comparison of strawberry survival between formula 4 and the control group.

[0034] Figure 8 Comparison of root systems between soybean formula 4 and the control group in the early stage of maturity. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0036] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0037] The deposit number of Bacillus amyloliquefaciens NBL-B11002 in the embodiment of the present invention is CCTCC NO: M 20232184, and the strain has been recorded in the published patent CN 117660243 A; The Daban Halobacillus sp. NBL-BS214 has a deposit number of CCTCC NO: M 20221370, and the strain has been recorded in the published patent CN 116286461 B; The Bacillus subtilis NBL-B12004 has a deposit number of CCTCC NO: M 20231506, and the strain has been recorded in the published patent CN 117487699 B; The Brevibacillus laterosporus BLCC1-1219 in the embodiment of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), the deposit date is: September 29, 2024, and the deposit number is CCTCC NO: M 20242118.

[0038] The Bacillus amyloliquefaciens NBL-AP73 in the embodiment of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), the deposit date is: June 19, 2024, and the deposit number is CCTCC NO: M 20241295. The strain has been recorded in the published patent CN118931780 A.

[0039] The deposit number of Priesteria gigantea C17 is CCTCC NO: M 20241724, and the strain has been recorded in the published patent CN118853507A; Example 1: Preparation of composite microbial agent Bacillus amyloliquefaciens NBL-B11002, Bacillus laterosporus BLCC1-1219, Bacillus dasakahalophilus NBL-BS214, Bacillus amyloliquefaciens NBL-AP73, Bacillus subtilis NBL-B12004 and Priesteria gigantea C17 bacterial powders were prepared respectively. The preparation method of the bacterial powders was as follows: firstly liquid deep fermentation was carried out, the obtained fermentation liquid was continuously centrifuged, and finally the precipitate obtained by centrifugation was spray-dried. According to this process, the number of live bacteria in the bacterial powders of Bacillus amyloliquefaciens NBL-B11002, Bacillus amyloliquefaciens NBL-AP73, Bacillus subtilis NBL-B12004 and Priesteria gigantea C17 could reach 1.0×10 11 CFU / g, the number of live bacteria in the powder of Halobacillus dabanensis NBL-BS214 can reach 5.0×10 10 CFU / g, the number of live bacteria of Brevibacillus laterosporus BLCC1-1219 can reach 1.0×10 10 CFU / g.

[0040] The Bacillus amyloliquefaciens NBL-B11002 powder, Bacillus laterosporus BLCC1-1219 powder, Bacillus dasakahalophilus NBL-BS214 powder, Bacillus amyloliquefaciens NBL-AP73 powder, Bacillus subtilis NBL-B12004 powder and Priesteria gigantea C17 powder obtained after spray drying are mixed according to the ratio of live bacteria count of 5: 2: 1: 1: 1: 1, and the total live bacteria count in the composite microbial agent after mixing is ≥ 10 billion / g.

[0041] Example 2: Corn potted plant growth promotion test 1. Corn variety: The tested corn variety was “Lainong 14”.

[0042] 2. Experimental method: Flower pots with an inner diameter of 23.5 cm and a depth of 20 cm were selected for the experiment. 3.0 kg of sandy loam was placed in each flower pot. The design of the potted corn experiment is shown in Table 1 (the composition of the inoculants in Formulations 1 to 4 is also applicable to Example 3). All treatments were repeated in 3 pots, and 0.8 g of urea was applied to the control group and each formulation group at a distance of about 8 cm from the soil surface. Corn seeds with the same grain size and fullness were selected for sowing (4 grains per pot). After sowing, each formulation group was flushed with inoculants, and the inoculants of each formulation group were diluted to a total viable count of 2.0×10 6 cfu / mL, 400mL was applied to each pot, and the same amount of water was applied to the clean water group and the control group. The soil at a depth of about 8cm was taken 14 days after sowing to determine the urease activity. The height and stem diameter of corn were measured 45 days after sowing. The total number of soil bacteria and nitrogen-fixing bacteria were also measured, and the average values ​​were calculated. The measurement and detection methods of each index are as follows: (1) Plant height: Use a ruler to measure the height from the soil surface to the top growth point of the plant.

[0043] (2) Stem diameter: measured using a vernier caliper.

[0044] (3) Determination of total nitrogen content in plants: After measuring plant height and stem diameter, cut the aboveground part of the corn plant and divide it into leaves and stems. After the samples are packaged according to treatment, place them in a 105℃ oven for 30 minutes, then lower the temperature to 60-80℃ and continue to dry until constant weight. The dried samples are crushed and sieved, digested using the H2SO4-H2O2 method, and the total nitrogen content of the leaves and stems of the corn plants is determined using the Kjeldahl method.

[0045] (4) Soil urease activity: measured according to the method of the kit (Solabo, BC0120).

[0046] (5) Determination of viable bacterial count in soil: Nutrient agar (NA, Haibo Biotechnology) and Ashuber medium (Haibo Biotechnology) were used to determine the total bacterial count and viable nitrogen-fixing bacteria count in the soil.

[0047] Table 1 Potted corn experiment design 3. Test results Table 2 Determination of corn plant and soil indexes Note: Data are mean ± standard error. Different lowercase letters in the same column indicate significant differences ( P <0.05, the same letters in the same column indicate no significant difference ( P>0.05).

[0048] From Table 2, Figure 1 It can be seen that the growth of corn in each formula group is higher than that in the control group to varying degrees. Among them, the plant height and stem diameter of corn plants in formula group 4 increased by 41.53% and 30.38% respectively compared with the control group. The total nitrogen content of the leaves of corn plants in formula group 1 and formula group 4 increased by 20.73% and 42.38% respectively compared with the control group, and the total nitrogen content of the stems increased by 18.11% and 32.83% respectively compared with the control group. This shows that nitrogen-fixing bacteria can improve the nitrogen supply level, promote nitrogen accumulation in plants, and can play a synergistic role with other functional bacteria. The soil urease activity of formula group 2 and formula group 4 was lower than that of the control group and other formula groups, but higher than that of the water group. This shows that the urease inhibitor produced by Brevibacillus laterosporus BLCC1-1219 can inhibit soil urease activity, reduce the ammoniation rate of chemical nitrogen fertilizers, delay the release and volatilization of urea, and reduce the loss of chemical nitrogen fertilizers. After adding the formula 4 compound microbial agent, the total number of soil bacteria increased significantly, and the number of nitrogen-fixing bacteria increased by 2 orders of magnitude compared with the control group, indicating that each strain can colonize and proliferate in the soil and better play a synergistic role.

[0049] Example 3: Wheat potted growth promotion experiment 1. Experimental materials: The wheat variety used for the experiment was “Jimai 22”.

[0050] 2. Experimental method: A seedling cup with an inner diameter of 8 cm and a depth of 12 cm was selected for the test. 400 g of sand was placed in each seedling cup. 0.1 g of urea was applied to the control group and each formulation group at about 8 cm. Wheat seeds with consistent grain size and fullness were selected, washed several times with distilled water, and then disinfected with 1% sodium hypochlorite for 10 minutes, and then washed several times with distilled water. The sodium hypochlorite attached to the seeds was rinsed clean before sowing. 50 seeds were sown in each pot, and 6 replicates were set for each group. After sowing, each formulation group was flushed with a bacterial agent, and the bacterial agent of each formulation group was diluted to a total viable bacterial count of 2.0×10 6 cfu / mL, 100 mL was applied to each pot, and the control group was flushed with the same amount of water. The specific types of irrigation in each treatment group are shown in Table 3. The wheat plant height, root length, dry weight, leaf total nitrogen content and soil organic carbon content were measured 28 days after sowing, and the average values ​​were calculated. The measurement method is as follows: (1) Plant height: Use a ruler to measure the height from the soil surface to the top growth point of the plant.

[0051] (2) Root length: Carefully dig out the wheat plant with its roots, try to keep the root system intact, gently shake off the soil attached to the root system and wash it. Use a ruler to measure from the base of the root system to the end of the root system.

[0052] (3) Dry weight: After the samples are collected, they are divided into two parts: the aboveground part and the underground part. After the samples are packaged according to the treatment, they are placed in a 105℃ oven for 30 minutes, and then the temperature is lowered to 60-80℃ and continued to be dried until constant weight.

[0053] (4) Determination of total nitrogen content in plants: The dried aboveground samples were crushed and sieved, digested using the H2SO4–H2O2 method, and the total nitrogen content in the aboveground parts of wheat plants was determined using the Kjeldahl method.

[0054] (5) Determination of soil organic carbon content: Determined by K2Cr2O7-H2SO4 volumetric method.

[0055] Table 3 Design of potted wheat experiment 3. Test results Table 4 Determination of wheat growth indexes Note: Data are mean ± standard error. Different lowercase letters in the same column indicate significant differences ( P <0.05, the same letters in the same column indicate no significant difference ( P >0.05).

[0056] Wheat plant growth diagram of different treatment groups ( Figure 2 ), Comparison of wheat plant growth between the four formula groups and the control group ( Figure 3 ) and the statistical data in Table 4 show that the application of formula 4 can significantly promote the growth of the above-ground and underground parts of wheat plants.

[0057] Furthermore, it can be seen from Table 4 that formulas 1, 2, 3, and 4 can promote the growth of the aboveground and underground parts of wheat plants to varying degrees, increase dry matter accumulation, and increase the total nitrogen content of the aboveground parts. Especially for formula 4, its plant height, root length, dry weight, and total nitrogen content of the aboveground parts increased significantly compared with the control group, which were 38.64%, 40.64%, 39.19%, and 27.31% higher than those of the control group, respectively. The soil organic carbon content was 0.55% before the experiment, which was at a relatively low level. Formulas 3 and 4 can promote the conversion of soil inorganic carbon into organic carbon, supplement soil organic matter, and increase the soil organic carbon content by 12.73% and 25.45% respectively compared with before the experiment, which is beneficial to nutrient retention and soil fertility improvement. Because plant growth requires the consumption of organic matter, the soil organic carbon content of the control group, formula 1, and formula 2 groups decreased to varying degrees after the experiment.

[0058] Based on the test results of Examples 2 and 3, Formula 4 (i.e., the composite microbial agent of Example 1 of the present invention) was selected for subsequent tests.

[0059] Example 4: Evaluation of the stress resistance effect of composite microbial agents on peanuts 1. Experimental materials: The peanut variety used for the experiment was “Shanhua No. 9”.

[0060] 2. Test method: Use flower pots with an inner diameter of 23.5 cm and a depth of 20 cm for the test. Select peanut seeds with consistent seed size and fullness for sowing (3 seeds per pot), and put 3.0 kg of sandy, moderately saline-alkali soil (pH = 8.5, salt content 3.5‰) into each flower pot. Peanuts need to be germinated before sowing, and sow when the radicle breaks through the seed coat and grows to 3-5 mm. There are 6 pots in each control group and treatment group. After 7 days of peanut emergence, dilute the composite microbial agent (formula 4 agent) of Example 1 to a total viable count of 2.0×10 6 cfu / mL, 400mL was applied to each pot in the treatment group, and the same amount of water was applied to each pot in the control group. The ethylene release of peanuts was measured 20 days after the application of the four-formula agent, and the growth index and leaf antioxidant enzyme activity of peanuts were measured about 60 days after the application of the four-formula agent, and the average values ​​were calculated.

[0061] 3. Index determination (1) Determination of ethylene release from peanuts: Peanut leaves of equal fresh weight were taken from the control group and the treatment group, and immediately sealed and stored in the dark. The ethylene release from the peanut leaves was determined by gas chromatography, with three replicates for each group. Gas chromatography conditions: GC-2010 gas chromatograph (Shimadzu), hydrogen ion flame (FID) detector, column temperature 45°C, HP-55% Phenyl MethylSiloxane column, detector temperature 250°C, gas H2 flow rate 40 mL / min, combustion air flow rate 450 mL / min, carrier gas N2 flow rate 30 mL / min, non-split injection, injection volume retention time 5 min. The injection volume was 0.1 mL, and the injection was repeated 3 times.

[0062] (2) Determination of antioxidant enzyme activity in peanut leaves: 60 days after the application of the four-microbial formulation, peanut leaves were randomly cut from the 6 pots of the control group and the treatment group. The leaves of 2 pots were thoroughly mixed and used as a replicate. The control group and the treatment group had 3 replicates. The cut leaves were immediately transferred to liquid nitrogen for later use. The activities of SOD superoxide dismutase (Solabo, BC0170), POD peroxidase (Solabo, BC0090) and CAT catalase (Solabo, BC0200) were determined by the kit method.

[0063] (3) Plant height: Use a ruler to measure the straightened height from the soil surface to the top growth point of the peanut plant.

[0064] (4) Length of the first pair of lateral branches: Use a ruler to measure the lengths of the first and second primary branches growing between the axils of the cotyledon nodes of the peanut main stem and take the average value.

[0065] (5) Dry weight of aboveground parts: Collect fresh peanut plants, cut off the roots, and place them in a 105°C oven to dry for 30 minutes. Then reduce the temperature to 80°C and continue drying until constant weight is reached, and then weigh them.

[0066] 4. Test results Table 5 Growth indexes of peanut plants, antioxidant enzyme activities and ethylene release in leaves Note: Data are mean ± standard error. "*" indicates significant difference by T test (P<0.05), and "**" indicates extremely significant difference by T test (P<0.01).

[0067] Depend on Figure 4 It can be seen that compared with the control group, the root system of the peanut plants in the treatment group is thicker and the aboveground growth is more luxuriant.

[0068] As shown in Table 5, the application of the four-microorganism formula can significantly reduce the ethylene release of peanut leaves, reduce the ethylene accumulation caused by moderate salt stress, and relieve the inhibitory effect of high concentration of ethylene on peanut growth. The ethylene release of peanut leaves in the treatment group was only 45.57% of that in the control group, while the plant height, the length of the first pair of lateral branches and the aboveground dry weight increased by 21.92%, 29.48% and 61.41% respectively compared with the control group.

[0069] Moreover, the application of the four-microorganism formula can significantly increase the antioxidant enzyme activity of peanut leaves and activate the plant's stress response mechanism with the removal of reactive oxygen species (ROS) as the core. Compared with the control group, the activities of SOD, POD, and CAT in the peanut leaves of the treatment group increased by 38.39%, 21.98%, and 65.37%, respectively.

[0070] The test results of Example 4 show that the composite microbial agent has a significant effect on improving crop stress resistance, especially abiotic stress caused by saline-alkali soil environment.

[0071] Example 5: Evaluation of the disease prevention effect of composite microbial agents on cabbage 1. Experimental materials: The cabbage variety used for the test was “Tiejia 75” (maye Chinese cabbage).

[0072] 2. Experimental method: The experiment was conducted in Shanggao Manor, Taishan District, Tai'an City, Shandong Province. The experimental plot was 2 mu in total, divided into 2 groups (control group and treatment group), each group was 1 mu. Cabbage seeds with consistent seed size and fullness were selected for sowing. After 2 weeks of cabbage emergence, the treatment group was flushed with 500 times diluted formula four bacterial agent, the dosage of bacterial agent was 1kg / mu, and the control group was flushed with the same amount of water. The basic fertilizer and field management measures of the control group and the treatment group (formula four bacterial agent) were carried out in accordance with the local management methods and remained consistent.

[0073] 3. Determination of protective efficacy (1) Disease classification: 7 days before the cabbage is harvested, the disease area is classified according to the proportion of the leaf area to the disease area in accordance with the "Guidelines for Field Efficacy Tests of Pesticides". 0 is grade 0; 1%-5% is grade 1; 6%-10% is grade 3; 11%-25% is grade 5; 26%-50% is grade 7; >50% is grade 9.

[0074] (2) Disease index: Disease index = ∑ (number of diseased leaves at each level × disease level) / (total number of leaves surveyed × highest disease level) × 100.

[0075] (3) Prevention and treatment effect: Prevention and treatment effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0076] 4. Test results Table 6 Field efficacy test of four-formula microbial agents against Chinese cabbage soft rot As shown in Table 6, the application of formula 4 fungi during the seedling stage can effectively prevent and control the bacterial soft rot of cabbage, with a prevention rate of up to 75.96%. One week before harvest, the old leaves of the cabbage in the control group were widely covered with lesions, and the leaves were seriously soft and cracked, while the cabbage in the group treated with fungi grew well, with new leaves green, and only occasional lesions on old leaves. The disease situation of the cabbage treated with formula 4 fungi and the control group was compared. Figure 5 shown.

[0077] It can be seen that the composite microbial agent provided by the present invention can effectively antagonize the pathogens of cabbage soft rot, which is beneficial to improving the quality of vegetables and increasing income.

[0078] Example 6: Evaluation of the disease prevention effect of composite microbial agents on potatoes 1. Experimental materials: The potato variety used for the test was “Lucinda V7”.

[0079] 2. Experimental method: The experiment was conducted at Shusheng Family Farm, Wangzhuang Town, Feicheng City, Tai'an City, Shandong Province. The experimental plot is 1.1 acres in total, divided into 2 groups (control group and treatment group), each group is 0.55 acres. Potatoes with complete appearance and no pests and diseases were selected as seed potatoes, and the seed potatoes were cut into tubers of uniform size for sowing. Two weeks after the potato seedlings emerged, the treatment group was flushed with a 500-fold diluted formula four bacterial agent, and the dosage of the bacterial agent was 1kg / mu, while the control group was flushed with an equal amount of water. The basic fertilizer and field management measures of the control group and the treatment group (formula four bacterial agent) were carried out in accordance with the local management methods and remained consistent.

[0080] 3. Experimental Design Table 7 Field trial design of “Lucinda V7” potato Note: Conventional fertilization: compound fertilizer (Hong Sifang, 160kg / mu) + organic fertilizer (Gengtianle, 25kg / mu).

[0081] 4. Determination of protective efficacy (1) Disease classification: 14 days before potato harvest, the plants are graded according to the degree of yellowing of leaves. No obvious yellowing or wilting of leaves is graded as 0; 0-25% of leaves are yellowing or wilting is graded as 1; 26%-50% of leaves are yellowing or wilting is graded as 2; 51%-75% of leaves are yellowing or wilting is graded as 3; more than 75% of leaves are yellowing or wilting or the whole plant is dead is graded as 4.

[0082] (2) Disease index: Disease index = ∑ (number of plants at each level × disease level) / (total number of plants surveyed × highest disease level) × 100.

[0083] (3) Prevention and treatment effect: Prevention and treatment effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0084] 5. Test results Table 8 Field efficacy test of four bacterial agents against potato wilt As shown in Table 8, the application of the four-microbial agent at the seedling stage can effectively avoid the occurrence and development of potato wilt, with a prevention rate of up to 92.36%. Two weeks before harvest, the aboveground leaves of the control group had withered in large numbers, seriously hindering photosynthesis and thus affecting the accumulation of tuber nutrients, while the potatoes in the microbial agent-application group grew well and the leaves did not wither ( Figure 6 ).

[0085] It can be seen that the composite microbial agent provided by the present invention can effectively antagonize the pathogen of potato wilt, which is beneficial to improving the quality and increasing the income of tuber crops.

[0086] Example 7: Evaluation of the effect of composite microbial agent on strawberry against repeated cropping 1. Experimental materials: The strawberry variety used for the test was “Countryside”.

[0087] 2. Experimental method: The experiment was conducted in the Bafu Green Circular Agriculture Demonstration Park in Ningyang County, Tai'an City, Shandong Province. The experimental plot is 1 mu in total, divided into 2 groups (control group and treatment group), each group is 0.5 mu. The experimental plot has been planted with strawberries for 5 consecutive years, and the basic physical and chemical properties of the soil are: total nitrogen 1.45g / kg, available potassium 105.37mg / kg, available phosphorus 138.12mg / kg, and organic matter 23.27g / kg. Due to the long-term continuous cropping of the experimental site, pathogenic microorganisms have accumulated in large quantities, and the survival rate of strawberry seedlings has been less than 85% for two consecutive years. The experimental site uses the method of transplanting cuttings to plant strawberries, and the seedling age is 45d. One week before and two weeks after transplanting the strawberries, the treatment group was flushed with a 500-fold diluted formula four bacterial agent, and the dosage of the bacterial agent was 1kg / mu, and the control group was flushed with an equal amount of water. The basic fertilizer and field management measures of the control group and the treatment group (formula four bacterial agent) were carried out in accordance with the local management methods and remained consistent.

[0088] 3. Index determination: Ten weeks after transplanting, the survival rates of strawberry plants in the control group and the treatment group were counted.

[0089] 4. Test results Table 9 Statistical results of survival rate of strawberry cuttings Applying a four-microbial formulation before and after transplanting can significantly increase the survival rate of strawberry cuttings ( Figure 7 , Table 9), as shown in Table 9, compared with the control group, the survival rate of the treatment group (formula four bacteria agent) increased by 12.57%, effectively avoiding the problem of increased input costs caused by seedling replacement. The imbalance of soil microbial flora and the accumulation of pathogenic microorganisms are the main causes of death of strawberry cuttings. The flushing of formula four bacteria agent can antagonize the pathogens and propagules of strawberry soil-borne diseases and improve the structure of strawberry rhizosphere microbial community. In addition, the addition of functional strains can effectively activate soil nutrients, improve the soil nutrient imbalance caused by long-term continuous cropping of facility crops, and improve the survival rate of strawberry cuttings.

[0090] Example 8: Application test of composite microbial agent on potatoes 1. Experimental materials: The potato variety used for the experiment was “Holland 15”.

[0091] 2. Experimental method: The experiment was conducted at Shusheng Family Farm, Wangzhuang Town, Feicheng City, Tai'an City, Shandong Province. The experimental plots totaled 3 mu, divided into 2 groups (control group and treatment group), each group of 1.5 mu. After the potatoes entered the tuber formation period, the treatment group was fertilized with a 500-fold diluted formula four bacterial agent, the dosage of the bacterial agent was 1kg / mu, and the control group was fertilized with an equal amount of water. The basic fertilizer and field management measures of the control group and the treatment group (formula four bacterial agent) were carried out in accordance with the local management methods and remained consistent.

[0092] 3. Experimental Design Table 10 Design of the potato field trial “Holland 15” Note: Conventional fertilization is: compound fertilizer (Hong Sifang, 160kg / mu) + organic fertilizer (Gengtianle, 25kg / mu).

[0093] 4. Test results Potato yield measurement photos Figure 8 As shown, the yield and total yield of each type of potatoes in the control group and the treatment group were statistically analyzed, and the yield increase was calculated. The results are shown in Table 11.

[0094] Table 11 Effect of formula 4 on "Holland 15" potatoes Note: Potatoes with a single weight of >200g and no green spots on the skin are high-quality potatoes, potatoes with a single weight of >200g and green spots on the skin are green-headed potatoes, and potatoes with a single weight of <200g are small potatoes.

[0095] As shown in Table 11, compared with the control group, the high-quality potato yield in the treatment group increased by 925.67 catties per mu, an increase of 20.55%, and the estimated income per mu increased by 1519.9 yuan (1.75 yuan / catties*925.67-100 yuan cost); the total yield (high-quality + green head + small potatoes) increased by 9.90% per mu. The above results show that the application of composite microbial agents during the tuber formation period can establish an efficient combined nitrogen fixation system with potatoes, which is beneficial to increase potato yield and increase production and income.

[0096] Example 9: Application test of composite microbial agent on soybean 1. Experimental materials: The soybean variety used for the test was “Qihuang 34”.

[0097] 2. Experimental method: The experiment was conducted at Jinhui Ecological Farm, Nanguan, Zhoucheng Town, Dongping County, Tai'an City, Shandong Province. The experimental plot totaled 2 mu, divided into 2 groups (control group and treatment group), 1 mu in each group, and 3 kg of bean seeds per mu. The treatment group was fertilized with a 500-fold diluted formula four bacterial agent, with a dosage of 1 kg / mu, and the control group was fertilized with an equal amount of water. The basic fertilizer and field management measures of the control group and the treatment group (formula four bacterial agent) were carried out in accordance with the local management methods and remained consistent. See Table 12 for detailed treatment.

[0098] 3. Experimental Design Table 12 Field soybean experiment design 4. Index determination (1) Growth index determination: 30 trees were randomly sampled from each group to investigate plant height, stem thickness, number of pods, etc.

[0099] (2) Determination of yield and yield component indicators: During harvest, the number of plants per mu, number of grains per plant, 100-grain weight, and moisture content were investigated to calculate the yield per mu.

[0100] Field yield calculation method: A. Number of plants per mu: In each group, investigate 6 rows and calculate the row spacing. Select 10m from each row and investigate the number of plants. Repeat 3 times and calculate the number of plants per row. Calculate the planting density (number of plants per mu) based on the row spacing and plant spacing.

[0101] B. Plant sample collection: Randomly collect 10 representative plants from each group, repeat 3 times, a total of 30 plants. After threshing, count the number of grains per plant, determine the moisture content and weigh the 100-grain weight.

[0102] Theoretical yield (kg / mu) = number of plants per mu (plants) × number of grains per plant (grains) × weight of 100 grains (g) × 10 -5 × (1-water content) / 0.865 5. Test results Table 13 Survey on soybean growth indicators and yield components Note: Data are mean ± standard error. "*" indicates significant difference by T test (P<0.05), and "**" indicates extremely significant difference by T test (P<0.01).

[0103] As shown in Table 13, compared with the control group, the plant height, stem diameter and pod number of the treatment group increased by 4.09%, 5.54% and 8.62% respectively, which shows that the application of the four-bacterial agent can stimulate the rapid growth of plants, promote soybean branching and flowering, and multiple fertilization and pod setting.

[0104] The number of grains per plant, 100-grain weight and yield per mu of the treatment group were higher than those of the control group, increasing by 16.69%, 5.91% and 23.32% respectively. The number of roots and nodules was also significantly higher than that of the control group ( Figure 8 ), indicating that the application of the bacterial agent is not only beneficial for the plants to produce more pods and grains, but also can promote root growth and nodulation, improve nitrogen fixation, enhance dry matter accumulation, increase bean weight, and help increase production.

[0105] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principle of the present application shall be deemed to be appropriate. Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A composite microbial agent for nitrogen and carbon fixation, synergistic and stress resistance, characterized in that: Contains: combined nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, salt-tolerant bacteria, biocontrol bacteria, growth-promoting bacteria and carbon-fixing bacteria; The combined nitrogen-fixing bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )NBL-B11002; the high-yield urease inhibitor bacteria is Brevibacillus laterosporus ( Brevibacillus laterosporus )BLCC1-1219; the salt-tolerant bacteria is Daban Halobacillus ( Halobacillus dabanensis strain ) NBL-BS214; the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )NBL-AP73; the growth-promoting bacteria is Bacillus subtilis ( Bacillus subtilis )NBL-B12004; the carbon-fixing bacteria is Priesteria gigantea ( Priestia megaterium )C17.

2. The composite microbial agent for nitrogen and carbon fixation, synergistic and stress resistance according to claim 1, characterized in that: The deposit number of the Bacillus amyloliquefaciens NBL-B11002 is CCTCC NO: M 20232184; the deposit number of the Brevibacillus laterosporus BLCC1-1219 is CCTCC NO: M 20242118; the deposit number of the Daban Halophilic Bacillus NBL-BS214 is CCTCC NO: M 20221370; the deposit number of the Bacillus amyloliquefaciens NBL-AP73 is CCTCC NO: M20241295; the deposit number of the Bacillus subtilis NBL-B12004 is CCTCC NO: M 20231506; the deposit number of the Priesteria gigantea ( Priestia megaterium ) The deposit number of C17 is CCTCC NO: M 20241724.

3. The composite microbial agent for nitrogen and carbon fixation, synergistic and stress resistance according to claim 1, characterized in that: The ratio of the number of live bacteria of nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, salt-tolerant bacteria, biocontrol bacteria, growth-promoting bacteria and carbon-fixing bacteria in the composite microbial agent is 5:2:1:1:1:

1.

4. The composite microbial agent for nitrogen and carbon fixation and stress resistance according to any one of claims 1 to 3, characterized in that: The formulation of the composite microbial agent is any one of granules, wettable powders, suspensions and water-dispersible granules.

5. Use of the composite microbial agent for nitrogen and carbon fixation, synergistic resistance and stress resistance according to any one of claims 1 to 4 in at least one of the following (1) to (6): (1) Nitrogen fixation; (2) Carbon fixation; (3) Promote crop growth; (4) Enhance the stress resistance of crops; (5) Enhance the disease resistance of crops; (6) Improve the ability of crops to resist repeated cropping.

6. The use according to claim 5, characterized in that: The nitrogen fixation is specifically manifested in: increasing the total nitrogen content of crops, inhibiting the urease activity in the soil, increasing the number of nitrogen-fixing bacteria in the soil and / or increasing the number of nodules in leguminous crops.

7. The use according to claim 5, characterized in that: The carbon fixation is specifically manifested as increasing the organic carbon content in the soil.

8. The use according to claim 5, characterized in that: The promotion of crop growth is specifically manifested in: promoting the growth of above-ground and underground parts of crops, increasing crop dry matter accumulation and / or improving crop yield.

9. The use according to claim 5, characterized in that: The stress resistance is resistance to abiotic stress caused by saline-alkali soil environment.

10. The use according to claim 5, characterized in that: The disease prevention is to prevent plant diseases caused by bacteria or fungi.

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