A compound microbial inoculant for enhancing nitrogen fixation, carbon fixation, efficiency and stress resistance and its application

Abstract

The present invention discloses a compound microbial inoculant with nitrogen fixation, carbon fixation, efficiency enhancement and stress resistance and its application, belonging to the technical field of functional microbial preparations. The compound microbial inoculant provided by the present invention takes associative 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. On the one hand, the compound microbial inoculant provided by the present invention can efficiently awaken the nitrogen-fixing bacterial community in the roots, accelerate the conversion of atmospheric nitrogen, and play the role of nitrogen fixation and efficiency enhancement. On the other hand, it can increase the content of soil organic carbon, improve soil fertility, thereby improving the quality of agricultural products and achieving the purpose of increasing production and income. In addition, the compound microbial inoculant also has the function of stress resistance and disease prevention, can relieve the abiotic stress caused by the saline-alkali soil environment, prevent plant diseases caused by bacteria or fungi, improve the survival rate of crop cuttings, and enhance the anti-consecutive cropping ability of crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microbial agents, and particularly relates to a composite microbial agent with nitrogen fixation, carbon fixation, efficiency enhancement and stress resistance and its application. Background Art

[0002] Nitrogen is the mineral nutrient element with the largest demand by plants. The lack of nitrogen nutrients in the soil is the main factor restricting plant growth. In agricultural production, applying nitrogen fertilizers can meet the growth needs of crops. However, unreasonable and excessive application of nitrogen fertilizers will lead to nitrogen loss and trigger a series of environmental problems such as acid rain, soil compaction, and water eutrophication, seriously restricting 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 - 14 days. Urease inhibitors can inhibit the activity of urease and slow down the hydrolysis rate and ammonification rate of urea, thereby reducing nitrogen loss. However, the potential safety problems of compound urease inhibitors and the process cost problems of plant extract urease inhibitors force researchers to seek a new way to obtain urease inhibitors.

[0004] Nitrogen-fixing microorganisms provide a new solution for achieving an efficient and pollution-free nitrogen application method. In nature, certain prokaryotic microorganisms can convert nitrogen in the air into ammonia, and this process is 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 associative nitrogen fixation, which are also the two microbial nitrogen fixation methods that can be applied to agricultural production so far. Compared with the former, associative nitrogen-fixing bacteria have advantages such as a wide host range and strong adaptability, so they have a broader application prospect.

[0005] In recent years, reports on urease inhibitor-producing microorganisms have emerged one after another, including both natural strains such as Bacillus subtilis and Bacillus coagulans, and engineered strains such as Escherichia coli and yeast that have been artificially modified. The application fields 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 "associative nitrogen fixation" and "microbial urease inhibitors" can be effectively combined to solve the problems of low utilization rate and environmental pollution in the use of traditional chemical nitrogen fertilizers at the same time, then the significance of this technology in reducing nitrogen fertilizer substitution and reducing costs and emissions in agriculture will be self-evident.

[0006] The application of microbial inoculants has played an important role in achieving sustainable agricultural development. CN 118285403A provides an ARC microbial inoculant with controlled toxicity and nitrogen fixation coupling effects (Bacillus laterosporus + Bacillus amyloliquefaciens + Bacillus mucilaginosus + Enterobacter ludwigii), which can promote early nodulation and nitrogen fixation in soybeans, early flowering and podding, increase the abundance of rhizobia in the rhizosphere of leguminous crops, and increase the number of nodules in leguminous crops. CN 118266480A provides a microbial inoculant with acid- and alkali-tolerant nitrogen-fixing Bacillus tropicus as the core. In addition to promoting plant growth, it can also improve the soil microbial community structure, increase the richness, diversity, evenness and species diversity of the microbial community, and increase the contents of total nitrogen, available phosphorus and available potassium in the soil. By improving the activities of high-yield ACC deaminase and nitrogenase, the efficiency of nitrogen fixation to ammonia is increased, and the highest nitrogenase activity can reach 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 various complex interaction relationships such as antagonism, synergy and symbiosis between nitrogen-fixing bacteria and other functional strains. Therefore, the development of composite microbial inoculants with better nitrogen fixation ability or new functions is still the current research focus and difficulty. Summary of the Invention

[0007] Aiming at the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a composite microbial inoculant with nitrogen fixation, carbon fixation, efficiency enhancement and stress resistance and its application. The composite microbial inoculant prepared by combining nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, and compounding salt-tolerant, biocontrol, growth-promoting, and carbon-fixing bacteria realizes the efficient utilization of nitrogen fertilizer, improves the quality and yield of agricultural products, significantly enhances the ability to resist abiotic stress caused by saline-alkali soil environment, and has important significance for agricultural production.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] In the first aspect of the present invention, a composite microbial inoculant is provided, comprising: nitrogen-fixing bacteria in combination, high-yield urease inhibitor bacteria, salt-tolerant bacteria, biocontrol bacteria, growth-promoting bacteria and carbon-fixing bacteria.

[0010] The nitrogen-fixing bacteria in combination are Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) NBL-B11002; the high-yield urease inhibitor bacteria are Brevibacillus laterosporus ( Brevibacillus laterosporus ) BLCC1-1219; the salt-tolerant bacteria are Halobacillus dabanensis ( Halobacillus dabanensis strain ) NBL-BS214; the biocontrol bacteria are Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) NBL-AP73; the growth-promoting bacteria are Bacillus subtilis ( Bacillus subtilis ) NBL-B12004; the carbon-fixing bacteria are Priestia megaterium ( Priestia megaterium) C17.

[0011] Among them, the preservation number of Bacillus amyloliquefaciens NBL-B11002 is CCTCC NO: M 20232184, and this strain has been recorded in the published patent CN 117660243 A;

[0012] Brevibacillus laterosporus BLCC1-1219 has been deposited in the China Center for Type Culture Collection (CCTCC, address: Culture Collection Center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), and the deposit date is: September 29, 2024, and the deposit number is CCTCC NO: M 20242118. This strain was isolated from fresh livestock and poultry manure samples and has significant urease inhibitory activity. The urease activity inhibition rate of the strain was measured by the phenol-hypochlorite method at different times. The test results showed that the inhibition rate of the strain on urease reached 92.52% within 30 minutes, 92.61% within 60 minutes, and 97.07% within 120 minutes.

[0013] The preservation number of Halobacillus dabanensis NBL-BS214 is CCTCC NO: M 20221370, and this strain has been recorded in the published patent CN 116286461 B;

[0014] Bacillus amyloliquefaciens NBL-AP73 has been deposited in the China Center for Type Culture Collection (CCTCC, address: Culture Collection Center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), and the deposit date is: June 19, 2024, and the deposit number is CCTCC NO: M 20241295, and this strain has been recorded in the published patent CN 118931780 A. This strain was isolated from tomato plants newly infected with tomato crown rot and root rot. The strain NBL-AP73 was inoculated onto a plate containing the pathogen Fusarium oxysporum f. sp. radicis-lycopersici, and the plate was streaked without inoculating the NBL-AP73 bacteria as a control group. According to the formula: (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100%, the inhibition rate was calculated. The test results of the inhibition rate detection showed that the antibacterial ability of this strain against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) reached 93.38%, showing a good antibacterial effect. At the same time, the growth conditions of tomato seedlings watered with the Bacillus amyloliquefaciens NBL-AP73 bacterial solution were compared with those of tomato seedlings not treated with the Bacillus amyloliquefaciens NBL-AP73 bacterial solution. The results showed that applying the Bacillus amyloliquefaciens NBL-AP73 bacterial solution could effectively promote the increase in plant height and stem thickness of tomato seedlings. The results showed that Bacillus amyloliquefaciens NBL-AP73 also had a good growth-promoting effect on tomato seedlings.

[0015] The preservation number of Bacillus subtilis NBL-B12004 is CCTCC NO: M 20231506, and this strain has been recorded in the published patent CN 117487699 B;

[0016] The preservation number of Priestia megaterium C17 is CCTCC NO: M 20241724, and this strain has been recorded in the published patent CN118853507A;

[0017] Improving the growth and health of crops by inoculating microbial inoculants is essentially a process of artificially constructing a microbial community, that is, a synthetic microbial community. In the ecosystem composed of plants, soil, and microorganisms, the species diversity of the synthetic microbial community directly determines the resistance stability of this system. Therefore, preparing a compound microbial inoculant based on the niche theory is of great significance for improving the environmental adaptability and effect stability of products and realizing the overall regulation of community functions.

[0018] Plant secretions (roots, leaves, phloem, etc.) provide nutrients for symbiotic microorganisms and have a shaping effect on their community structure. When inoculating a single microbial inoculant, it is easy to increase the metabolic load of crops. The overlap rate of the utilization of various strain resources described in this application is low, and the established functional partitions conform to the microscopic regulation of crop growth by microorganisms under the background of the large 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 on the premise of outputting sufficient nitrogen nutrition, which is beneficial to maintaining soil fertility and fertility improvement and promoting crop growth; on the other hand, through biocontrol and salt-tolerant bacteria activating the biological stress / abiotic stress response mechanism, the adaptability of crops to adversity is significantly enhanced, which is beneficial to crops maintaining a healthy growth trend, thereby improving the quality of agricultural products and achieving the purpose of increasing production and income.

[0019] The viable count ratio of associative nitrogen-fixing bacteria, high-yield urease inhibitor bacteria, salt-tolerant bacteria, biocontrol bacteria, growth-promoting bacteria, and carbon-fixing bacteria in the compound microbial inoculant is (5-6):(1-2):1:1:1:1.

[0020] Preferably, the total viable count in the compound microbial inoculant ≥ 10 billion / g.

[0021] Furthermore, when preparing the compound microbial inoculant, a carrier can also be added according to the needs of the dosage form.

[0022] Preferably, the dosage form of the compound microbial inoculant is any one of granule, wettable powder, suspending agent, and water dispersible granule.

[0023] In the second aspect of the present invention, there is provided the application of the above compound microbial inoculant in at least one of the following (1)-(6):

[0024] (1)Nitrogen fixation;

[0025] (2)Carbon fixation;

[0026] (3)Promote the growth of crops;

[0027] (4)Enhance the stress resistance of crops;

[0028] (5)Enhance the disease prevention ability of crops;

[0029] (6)Improve the ability of crops to resist continuous cropping.

[0030] The nitrogen fixation is specifically manifested as: 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 nodulation number of leguminous crops.

[0031] The carbon fixation is specifically manifested as: increasing the organic carbon content in the soil.

[0032] The promotion of crop growth is specifically manifested as: promoting the growth of the above-ground and underground parts of crops, increasing the dry matter accumulation of crops and / or increasing the crop yield.

[0033] The stress resistance is specifically manifested as: resisting the abiotic stress caused by the saline-alkali soil environment. The above-mentioned compound microbial inoculant can reduce the ethylene accumulation caused by moderate salt stress and significantly increase the antioxidant enzyme activity in crops.

[0034] The disease resistance is specifically manifested as: preventing plant diseases caused by bacteria or fungi.

[0035] Further, the plant diseases caused by bacteria or fungi are bacterial soft rot or potato wilt.

[0036] Advantages of the present invention:

[0037] (1)The compound microbial inoculant provided by the present invention is prepared with associative 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 compound microbial inoculant obtained by innovating the component ratio in the components has good application effects and is of great significance to agricultural production.

[0038] (2) The compound microbial inoculant provided by the present invention, on the one hand, can efficiently awaken the nitrogen-fixing flora in the roots, accelerate the conversion of atmospheric nitrogen, alleviate the loss of soil nitrogen fertilizer, establish an efficient associative nitrogen-fixing system with crops, achieve the efficient utilization and reduction of nitrogen fertilizer and increase efficiency, significantly improve the nitrogen-fixing efficiency, play the role of nitrogen-fixing and increasing efficiency, and can also increase the content of soil organic carbon, 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 functions of stress resistance and disease prevention, resisting abiotic stress caused by saline-alkali soil environment, reducing the ethylene accumulation caused by moderate salt stress, significantly increasing the antioxidant enzyme activity in crops, preventing plant diseases caused by bacteria or fungi, and playing the role of improving the continuous cropping resistance of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Comparison of the growth of corn plants between Formula Four and the control group.

[0040] Figure 2 Growth of wheat plants under different treatments.

[0041] Figure 3 Comparison of the growth of wheat plants between Formula Four and the control group; among them, Figure 3 A in it is the growth comparison diagram of the plant height of wheat plants between Formula Four and the control group, Figure 3 B in it is the growth comparison diagram of the root length of wheat plants between Formula Four and the control group.

[0042] Figure 4 Comparison of the growth of peanut plants between Formula Four and the control group.

[0043] Figure 5 Comparison of the disease conditions of Chinese cabbage between Formula Four and the control group.

[0044] Figure 6 Comparison of the disease conditions of potatoes ("Lucinda V7") between Formula Four and the control group.

[0045] Figure 7 Comparison of the survival conditions of strawberries between Formula Four and the control group.

[0046] Figure 8 Comparison of the roots of Formula Four and the control group in the early stage of soybean maturity. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0048] The specific embodiments of the present invention will be further described in detail below in conjunction with the embodiments. The following detailed description is illustrative and is intended to provide further explanation of the present application rather than limiting the scope of the present invention.

[0049] In the embodiment of the present invention, the preservation number of Bacillus amyloliquefaciens NBL-B11002 is CCTCC NO: M 20232184, and this strain has been recorded in the published patent CN 117660243 A;

[0050] The preservation number of Halobacillus dabanensis NBL-BS214 is CCTCC NO: M 20221370, and this strain has been recorded in the published patent CN 116286461 B;

[0051] The preservation number of Bacillus subtilis NBL-B12004 is CCTCC NO: M 20231506, and this strain has been recorded in the published patent CN 117487699 B;

[0052] In the embodiment of the present invention, Brevibacillus laterosporus BLCC1-1219 has been deposited in the China Center for Type Culture Collection (CCTCC, address: Culture Collection Center, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), the deposit date is: September 29, 2024, and the preservation number is CCTCC NO: M 20242118.

[0053] In the embodiment of the present invention, Bacillus amyloliquefaciens NBL-AP73 has been deposited in the China Center for Type Culture Collection (CCTCC, address: Culture Collection Center, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China), the deposit date is: June 19, 2024, and the preservation number is CCTCC NO: M 20241295. This strain has been recorded in the published patent CN118931780 A.

[0054] The preservation number of Priestia megaterium C17 is CCTCC NO: M 20241724, and this strain has been recorded in the published patent CN118853507A;

[0055] Example 1: Preparation of compound microbial inoculum

[0056] Bacillus amyloliquefaciens NBL-B11002, Brevibacillus laterosporus BLCC1-1219, Halobacillus dabanensis NBL-BS214, Bacillus amyloliquefaciens NBL-AP73, Bacillus subtilis NBL-B12004 and Priestia megaterium C17 were separately prepared into bacterial powders. The preparation method of the bacterial powders was as follows: first, liquid submerged fermentation was carried out, and the obtained fermentation broth was continuously centrifuged, and finally the precipitate obtained by centrifugation was spray-dried. Produced according to this process, the viable count of the bacterial powders of Bacillus amyloliquefaciens NBL-B11002, Bacillus amyloliquefaciens NBL-AP73, Bacillus subtilis NBL-B12004 and Priestia megaterium C17 can reach 1.0×10 11 CFU / g, the viable count of the bacterial powder of Halobacillus dabanensis NBL-BS214 can reach 5.0×10 10 CFU / g, and the viable count of the bacterial powder of Brevibacillus laterosporus BLCC1-1219 can reach 1.0×10 10 CFU / g.

[0057] The bacterial powders of Bacillus amyloliquefaciens NBL-B11002, Brevibacillus laterosporus BLCC1-1219, Halobacillus dabanensis NBL-BS214, Bacillus amyloliquefaciens NBL-AP73, Bacillus subtilis NBL-B12004 and Priestia megaterium C17 obtained after spray drying were mixed according to the viable count ratio of 5:2:1:1:1:1 respectively. After mixing, the total viable count in the compound microbial inoculant was ≥10 billion / g.

[0058] Example 2: Maize pot experiment for promoting growth

[0059] 1. Maize variety: The maize variety for the test was "Lainong 14".

[0060] 2. Test method: Flowerpots with an inner diameter of 23.5 cm and a depth of 20 cm were selected for the test. Each flowerpot was filled with 3.0 kg of sandy loam. The experimental design of the potted maize is shown in Table 1 (the inoculant compositions of Formulation Group 1 to Formulation Group 4 are also applicable to Example 3). All treatments were set with 3 replicates. In the control group and each formulation group, 0.8 g of urea was applied at about 8 cm from the soil surface. Maize seeds with the same grain size and plumpness were selected for sowing (4 seeds per pot). After sowing, the inoculant was topdressed in each formulation group. The inoculant in each formulation group was diluted to a total viable count of 2.0×10 6 cfu / mL, and 400 mL was topdressed per pot. The water group and the control group were topdressed with an equal amount of water. 14 days after sowing, the urease activity of the soil at a depth of about 8 cm was measured. 45 days after sowing, the plant height and stem diameter of the maize were measured. At the same time, the total number of soil bacteria and the number of nitrogen-fixing bacteria were measured, and the average value was statistically analyzed. The measurement and detection methods of each index are as follows:

[0061] (1) Plant height: Measure the height from the soil surface to the growth point at the top of the plant using a meter stick.

[0062] (2) Stem diameter: Measure using a vernier caliper.

[0063] (3) Determination of total nitrogen content in plants: After measuring the plant height and stem diameter, cut off the above-ground part of the corn plant and divide it into two parts: leaves and stems. After the samples are packed according to the treatment, put them into an oven at 105°C for 30 minutes, then lower the temperature to 60 - 80°C and continue to dry until constant weight. Grind the dried samples and sieve them, digest them using the H2SO4–H2O2 method, and use the Kjeldahl method to determine the total nitrogen content in the leaves and stems of the corn plant respectively.

[0064] (4) Soil urease activity: Determine according to the method of the kit (Solarbio, BC0120).

[0065] (5) Determination of viable bacteria count in soil: Use nutrient agar (NA, Haibo Biotech) and Ashby's medium (Haibo Biotech) to determine the total number of bacteria and the viable count of nitrogen-fixing bacteria in the soil.

[0066] Table 1 Design of pot experiment for corn

[0067]

[0068] 3. Experimental results

[0069] Table 2 Determination of corn plant and soil indicators

[0070]

[0071] Note: Data are mean ± standard error. Different lowercase letters in the same column indicate significant differences ( P <0.05), and the same letters in the same column indicate no significant differences ( P >0.05).

[0072] From Table 2, Figure 1It can be seen that the growth of corn in each formula group is higher than that of the control group to varying degrees. Among them, the plant height and stem diameter of the corn plants in Formula Group 4 increased by 41.53% and 30.38% respectively compared with the control group. The total nitrogen content in the leaves of the 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 in 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 the accumulation of nitrogen in plants, and play a synergistic effect with other functional bacteria. The soil urease activity of Formula Group 2 and Formula Group 4 is 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 ammonification rate of chemical nitrogen fertilizers, delay the release and volatilization of urea, and reduce the loss of chemical nitrogen fertilizers. After adding the compound microbial inoculant of Formula 4, the total number of soil bacteria increased significantly, and the number of nitrogen-fixing bacteria increased by two orders of magnitude compared with the control group, indicating that each strain can colonize and multiply in the soil and better play a synergistic effect.

[0073] Example 3: Wheat Pot Experiment for Promoting Growth

[0074] 1. Test materials: The tested wheat variety is "Jimai 22".

[0075] 2. Test method: Select a seedling cup with an inner diameter of 8 cm and a depth of 12 cm for the experiment. Fill 400 g of sandy soil into each seedling cup. The control group and each formula group apply 0.1 g of urea at about 8 cm. Select wheat seeds with the same grain size and plumpness, wash them several times with distilled water, then disinfect them with 1% sodium hypochlorite for 10 minutes, and then wash them several times with distilled water. After rinsing off the sodium hypochlorite attached to the seeds, sow them. Sow 50 seeds in each pot, and set 6 replicates for each group. After sowing, apply the inoculant to each formula group once, dilute the inoculant of each formula group to a total viable count of 2.0×10 6 cfu / mL, apply 100 mL to each pot, and the control group applies an equal amount of water. The specific application types of each treatment group are shown in Table 3. After 28 days of sowing, measure the plant height, root length, dry weight, total nitrogen content in the leaves and soil organic carbon content of wheat, and calculate the average value. The measurement methods are as follows:

[0076] (1) Plant height: Measure the height from the soil surface to the growth point at the top of the plant with a meter stick.

[0077] (2) Root length: Carefully dig out the wheat plants with their roots, try to keep the integrity of the roots, gently shake off the soil attached to the roots of the dug-out plants and wash them. Use a meter stick to measure from the base of the roots until the end of the roots.

[0078] (3) Dry weight: After sample collection, the samples were divided into aboveground and underground parts. After the samples were packed according to the treatments, they were placed in an oven at 105 °C for 30 minutes, then the temperature was reduced to 60 - 80 °C and drying continued until constant weight was achieved.

[0079] (4) Determination of total nitrogen content in plants: The dried aboveground samples were crushed and sieved, digested by the H2SO4–H2O2 method, and the total nitrogen content in the aboveground parts of wheat plants was determined by the Kjeldahl method.

[0080] (5) Determination of soil organic carbon content: The determination was carried out according to the K2Cr2O7―H2SO4 volumetric method.

[0081] Table 3 Design of pot experiment for wheat

[0082]

[0083] 3. Experimental results

[0084] Table 4 Determination of wheat growth indicators

[0085]

[0086] Note: Data are mean ± standard error. Different lowercase letters in the same column indicate significant differences ( P <0.05), and the same letters in the same column indicate no significant differences ( P >0.05).

[0087] From the growth diagrams of wheat plants in different treatment groups ( Figure 2 ), the comparison diagram of the growth of wheat plants in the fourth formula group and the control group ( Figure 3 ) and the data statistically analyzed in Table 4, it can be seen that applying the fourth formula can significantly promote the growth of the aboveground and underground parts of wheat plants.

[0088] Further, it can be seen from Table 4 that formulas one, two, three, and four can all promote the growth of the aboveground and underground parts of wheat plants to varying degrees, increase dry matter accumulation, and at the same time increase the total nitrogen content in the aboveground parts. Especially for the fourth formula, its plant height, root length, dry weight, and total nitrogen content in the aboveground parts increased significantly compared with the control group, increasing by 38.64%, 40.64%, 39.19%, and 27.31% respectively compared with the control group. The soil organic carbon content before the experiment was 0.55%, at a relatively low level. Formulas three and four can promote the conversion of soil inorganic carbon into organic carbon, supplement soil organic matter, and make the soil organic carbon content increase 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 substances, the soil organic carbon content in the control group, formula one group, and formula two group all decreased to varying degrees after the experiment.

[0089] Based on the test results of Example 2 and Example 3, Formula Four (i.e., the compound microbial inoculant of Example 1 of the present invention) was preferably selected for subsequent tests.

[0090] Example 4: Evaluation of the stress resistance effect of the compound microbial inoculant on peanuts

[0091] 1. Test materials: The peanut variety to be tested was "Shanhua No. 9".

[0092] 2. Test method: Flowerpots with an inner diameter of 23.5 cm and a depth of 20 cm were selected for the test. Peanut seeds with consistent grain size and plumpness were sown (3 seeds per pot), and 3.0 kg of sandy, moderately saline-alkali soil (pH = 8.5, salt content 3.5‰) was filled into each flowerpot. Peanut seeds needed to be germinated before sowing. When the radicle broke through the seed coat and grew to 3 - 5 mm, sowing was carried out. There were 6 pots in each of the control group and the treatment group. 7 days after the peanuts emerged, the compound microbial inoculant of Example 1 (Formula Four inoculant) was diluted to a total viable count of 2.0×10 6 cfu / mL. 400 mL was irrigated into each pot in the treatment group, and an equal amount of clear water was irrigated in the control group. The ethylene release amount of peanuts was measured 20 days after irrigating the Formula Four inoculant, and the growth indexes and leaf antioxidant enzyme activities of peanuts were measured about 60 days after irrigating the Formula Four inoculant, and the average values were statistically calculated.

[0093] 3. Index determination

[0094] (1) Determination of peanut ethylene release amount: Peanut leaves with equal fresh weight were taken from the control group and the treatment group, immediately sealed and stored in the dark, and the ethylene release amount of peanut leaves was measured by gas chromatography. Each group had 3 replicates. Gas chromatography conditions: GC-2010 gas chromatograph (Shimadzu), hydrogen flame (FID) detector, column temperature 45°C, HP-55% Phenyl MethylSiloxane chromatographic column, detector temperature 250°C, fuel gas H2 flow rate 40 mL / min, combustion-supporting air flow rate 450 mL / min, carrier gas N2 flow rate 30 mL / min, splitless injection, injection volume retention time 5 min. The injection volume was 0.1 mL, and injection was repeated 3 times.

[0095] (2) Determination of peanut leaf antioxidant enzyme activity: 60 days after irrigating the Formula Four inoculant, peanut leaves were randomly cut from 6 replicates of the control group and the treatment group. The leaves were fully mixed by taking 2 pots each as a replicate, and there were 3 replicates in both the control group and the treatment group. The cut leaves were immediately transferred to liquid nitrogen for use, and the activities of SOD superoxide dismutase (Solarbio, BC0170), POD peroxidase (Solarbio, BC0090), and CAT catalase (Solarbio, BC0200) were measured by the kit method.

[0096] (3) Plant height: The straight height from the soil surface to the growth point at the top of the peanut plant was measured with a meter stick.

[0097] (4)Length of the first pair of lateral branches: Use a meter stick to measure the lengths of the first and second primary branches growing from the axils of the cotyledon nodes of the peanut main stem, and take the average value.

[0098] (5)Aboveground dry weight: Collect fresh peanut plants, cut off the roots, put them into an oven at 105 °C for 30 minutes for fixation, then reduce the temperature to 80 °C and continue drying until constant weight, and then weigh them.

[0099] 4. Test results

[0100] Table 5 Growth indexes, antioxidant enzyme activities in leaves and ethylene release amounts of peanut plants

[0101]

[0102] Note: The 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 Figure 4 can be seen, compared with the control group, the roots of peanut plants in the treatment group are thicker, and the aboveground growth is more lush.

[0104] As can be seen from Table 5, applying the four-bacteria agent formula by irrigation can significantly reduce the ethylene release amount in peanut leaves, alleviate the ethylene accumulation caused by moderate salt stress, and relieve the inhibitory effect of high-concentration ethylene on peanut growth. The ethylene release amount in peanut leaves of the treatment group is only 45.57% of that of the control group, while the plant height, length of the first pair of lateral branches and aboveground dry weight are increased by 21.92%, 29.48% and 61.41% respectively compared with the control group.

[0105] Moreover, applying the four-bacteria agent formula by irrigation can also significantly improve the antioxidant enzyme activities in peanut leaves and activate the stress response mechanism of the plant centered on the scavenging of reactive oxygen species (ROS). Compared with the control group, the activities of SOD, POD and CAT in peanut leaves of the treatment group are increased by 38.39%, 21.98% and 65.37% respectively.

[0106] The test results of Example 4 show that the compound microbial inoculant has obvious effects on improving the stress resistance of crops, especially the abiotic stress caused by saline-alkali soil environment.

[0107] Example 5: Evaluation of the disease prevention effect of the compound microbial inoculant on Chinese cabbage

[0108] 1. Test materials: The tested Chinese cabbage variety is "Tiejia 75" (coarse-leaved Chinese cabbage).

[0109] 2. Test method: The test was carried out in Shanggao Manor, Taishan District, Tai'an City, Shandong Province. The experimental plot was 2 mu in total, divided into 2 groups (control group, treatment group), with 1 mu for each group. Chinese cabbage seeds with consistent grain size and plumpness were selected for sowing. After the Chinese cabbage emerged for 2 weeks, the treatment group was topdressed with the formula four-bacteria agent diluted 500 times, and the dosage of the bacteria agent was 1 kg / mu, while the control group was topdressed with an equal amount of clear water. The basic fertilization and field management measures of the control group and the treatment group (formula four-bacteria agent) were carried out according to the local management method and kept consistent.

[0110] 3. Control efficacy determination

[0111] (1) Disease grading: Seven days before the Chinese cabbage was harvested, according to the "Guidelines for Field Efficacy Trials of Pesticides", the disease was graded according to the proportion of the lesion area to the leaf area. 0 was grade 0; 1% - 5% was grade 1; 6% - 10% was grade 3; 11% - 25% was grade 5; 26% - 50% was grade 7; >50% was grade 9.

[0112] (2) Disease index: Disease index = ∑(number of diseased leaves at each level × disease level) / (total number of leaves surveyed × highest disease level) × 100.

[0113] (3) Control effect: Control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100.

[0114] 4. Test results

[0115] Table 6 Field control efficacy determination of the formula four-bacteria agent against Chinese cabbage soft rot

[0116]

[0117] As shown in Table 6, topdressing the formula four-bacteria agent at the seedling stage can effectively control Chinese cabbage bacterial soft rot, and the control effect is as high as 75.96%. One week before harvest, the old leaves of Chinese cabbage in the control group were widely covered with lesions, and the phenomenon of leaf soft rot and rupture was serious. While the Chinese cabbage in the bacteria agent topdressing group grew well, the new leaves were emerald green, and only occasional lesions were seen on the old leaves. The comparison of the disease conditions of Chinese cabbage with the formula four-bacteria agent topdressing and the control group is as Figure 5 shown.

[0118] It can be seen from this that the compound microbial bacteria agent provided by the present invention can effectively antagonize the pathogens of Chinese cabbage soft rot, which is beneficial to improving the quality and increasing the income of vegetables.

[0119] Example 6: Evaluation of the disease prevention effect of the compound microbial bacteria agent on potatoes

[0120] 1. Test materials: The tested potato variety was "Lucinda V7".

[0121] 2. Test method: The test was conducted at Shusheng Family Farm, Wangzhuang Town, Feicheng City, Tai'an City, Shandong Province. The experimental plot was 1.1 mu in total, divided into 2 groups (control group, treatment group), with each group being 0.55 mu. Potatoes with intact appearance and no pests and diseases were selected as seed potatoes, and the seed potatoes were cut into tubers of the same size for sowing. Two weeks after the potatoes emerged, the treatment group was topdressed with the formulated four-bacteria agent diluted 500 times, with the dosage of the bacteria agent being 1 kg / mu, while the control group was topdressed with the same amount of clear water. The basic fertilization and field management measures of the control group and the treatment group (formulated four-bacteria agent) were carried out according to the local management method and kept consistent.

[0122] 3. Test design

[0123] Table 7 Field test design of "Lucinda V7" potatoes

[0124]

[0125] Note: Conventional fertilization: Compound fertilizer (Hong Sifang, 160 kg / mu) + Organic fertilizer (Gengtianle, 25 kg / mu).

[0126] 4. Efficacy determination

[0127] (1) Disease grading: 14 days before potato harvest, grading was carried out according to the degree of yellowing and withering of the plant leaves. If there was no obvious yellowing and withering of the leaves, it was grade 0; if 0 - 25% of the leaves were yellowing and withering, it was grade 1; if 26% - 50% of the leaves were yellowing and withering, it was grade 2; if 51% - 75% of the leaves were yellowing and withering, it was grade 3; if more than 75% of the leaves were yellowing and withering or the whole plant was dead, it was grade 4.

[0128] (2) Disease index: Disease index = ∑(number of plants at each level × disease level) / (total number of plants surveyed × highest disease level) × 100.

[0129] (3) Control effect: Control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100.

[0130] 5. Test results

[0131] Table 8 Field efficacy determination of the formulated four-bacteria agent against potato fusarium wilt

[0132]

[0133] As shown in Table 8, topdressing with the formulated four-bacteria agent during the seedling stage can effectively avoid the occurrence and development of potato fusarium wilt, with the control effect reaching as high as 92.36%. Two weeks before harvest, a large number of the above-ground leaves of the control group had withered, seriously hindering photosynthesis and thus affecting the nutrient accumulation in tubers, while the potatoes in the group topdressed with the bacteria agent grew well and no withering phenomenon was observed in the leaves ( Figure 6 )

[0134] It can be seen from this that the compound microbial inoculant provided by the present invention can effectively antagonize the pathogens of potato wilt, which is beneficial to improving the quality and increasing the income of tuber crops.

[0135] Example 7: Evaluation of the effect of the compound microbial inoculant on preventing continuous cropping of strawberries

[0136] 1. Test materials: The tested strawberry variety is "Xiangye".

[0137] 2. Test method: The test was carried out in the Bafu Green Circular Agriculture Demonstration Park, Ningyang County, Tai'an City, Shandong Province. The experimental plot is 1 mu in total, divided into 2 groups (control group, treatment group), with 0.5 mu in each group. Strawberries have been continuously planted on the experimental plot for 5 years. The basic physical and chemical properties of the soil are as follows: total nitrogen 1.45 g / kg, available potassium 105.37 mg / kg, available phosphorus 138.12 mg / kg, and organic matter 23.27 g / kg. Due to continuous cropping for many years, a large amount of pathogenic microorganisms have accumulated in the experimental plot, and the survival rate of strawberry seedlings has been less than 85% for two consecutive years. Strawberries were planted in the experimental plot by transplanting cuttings, and the seedling age was 45 days. One week before strawberry transplantation and two weeks after transplantation, the treatment group was topdressed with the formula four-in-one microbial inoculant diluted 500 times, and the dosage of the inoculant was 1 kg / mu, while the control group was topdressed with an equal amount of clear water. The basic fertilization and field management measures of the control group and the treatment group (formula four-in-one microbial inoculant) were carried out according to the local management methods and kept consistent.

[0138] 3. Index determination: Ten weeks after transplantation, the survival rates of strawberry plants in the control group and the treatment group were counted.

[0139] 4. Test results

[0140] Table 9 Statistical results of the survival rate of strawberry cuttings

[0141]

[0142] Topdressing the formula four-in-one microbial inoculant before and after transplantation can significantly improve 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-in-one microbial inoculant) increased by 12.57%, effectively avoiding the problem of increased input costs caused by replanting seedlings. The imbalance of the soil microbial flora and the accumulation of pathogenic microorganisms are the main reasons for the death of strawberry cuttings. Topdressing the formula four-in-one microbial inoculant can antagonize the pathogens and propagules of strawberry soil-borne diseases and improve the microbial community structure in the rhizosphere of strawberries. In addition, the addition of functional strains can also effectively activate soil nutrients, improve the problem of soil nutrient imbalance caused by continuous cropping of protected crops for many years, and improve the survival rate of strawberry cuttings.

[0143] Example 8: Application test of the compound microbial inoculant on potatoes

[0144] 1. Test materials: The tested potato variety is "Holland 15".

[0145] 2. Test method: The test was carried out at the Shusheng Family Farm in Wangzhuang Town, Feicheng City, Tai'an City, Shandong Province. The experimental plot was 3 mu in total, divided into 2 groups (control group, treatment group), with 1.5 mu in each group. After the potato entered the tuber formation stage, the treatment group was topdressed with the formulated four-bacteria agent diluted 500 times, and the dosage of the bacteria agent was 1 kg / mu, while the control group was topdressed with the same amount of clear water. The basic fertilization and field management measures of the control group and the treatment group (formulated four-bacteria agent) were carried out according to the local management method and kept consistent.

[0146] 3. Test design

[0147] Table 10 Field test design of "Holland 15" potato

[0148]

[0149] Note: Conventional fertilization is: compound fertilizer (Red Square, 160 kg / mu) + organic fertilizer (Gengtianle, 25 kg / mu).

[0150] 4. Test results

[0151] Photos of on-site potato yield measurement are as Figure 8 shown. The yields and total yields of various types of potatoes in the control group and the treatment group were counted, and the yield increase rate was calculated. The results are shown in Table 11.

[0152] Table 11 Application effect of formula four on "Holland 15" potato

[0153]

[0154] Note: Potatoes with a single weight > 200 g and no green patches on the epidermis are high-quality potatoes, potatoes with a single weight > 200 g and green patches on the epidermis are green-headed potatoes, and potatoes with a single weight < 200 g are small potatoes.

[0155] As can be seen from Table 11, compared with the control group, the high-quality potatoes in the treatment group increased by 925.67 jin per mu, with an increase rate of 20.55%. It is estimated that the income per mu will increase by 1519.9 yuan (1.75 yuan / jin * 925.67 - 100 yuan of cost); the total yield (high-quality + green-headed + small potatoes) increased by 9.90% per mu. The above results show that applying the compound microbial inoculant during the tuber formation stage can establish an efficient combined nitrogen fixation system with potatoes, which is beneficial to the increase of potato yield and the increase of production and income.

[0156] Example 9: Application test of compound microbial inoculant on soybeans

[0157] 1. Test materials: The tested soybean variety was "Qihuang 34".

[0158] 2. Test method: The test was carried out at Jinhui Ecological Farm, Nanguan, Zhoucheng Town, Dongping County, Tai'an City, Shandong Province. The experimental plot was 2 mu in total, divided into 2 groups (control group, treatment group), 1 mu for each group, and 3 kg of soybean seeds were used per mu. The treatment group was topdressed with the formula four-bacteria agent diluted 500 times, and the dosage of the bacteria agent was 1 kg / mu, while the control group was topdressed with an equal amount of clear water. The basic fertilization and field management measures of the control group and the treatment group (formula four-bacteria agent) were carried out according to the local management methods and kept consistent. The detailed treatments are shown in Table 12.

[0159] 3. Experimental design

[0160] Table 12 Field soybean experimental design

[0161]

[0162] 4. Index determination

[0163] (1) Determination of growth indexes: 30 plants were randomly sampled from each group to investigate plant height, stem diameter, pod number, etc.

[0164] (2) Determination of yield and yield component indexes: At harvest, the number of plants per mu, the number of grains per plant, 100-grain weight, and moisture content were investigated, and the yield per mu was calculated.

[0165] Calculation method of field yield:

[0166] A. Number of plants per mu: 6 rows were investigated in each group to measure the row spacing. 10 m was selected for each row to investigate the number of plants, repeated 3 times, and the number of plants in the row was measured. The planting density (number of plants per mu) was calculated according to the row spacing and plant spacing.

[0167] B. Collection of plant samples: 10 representative plants were randomly and continuously collected from each group, repeated 3 times, for a total of 30 plants. After threshing, the number of grains per plant was counted, and the moisture content was measured and the 100-grain weight was weighed.

[0168] Theoretical yield (kg / mu) = number of plants per mu (plants) × number of grains per plant (grains) × 100-grain weight (grams) × 10 -5 × (1 - moisture content) / 0.865

[0169] 5. Test results

[0170] Table 13 Investigation of soybean growth indexes and yield components

[0171]

[0172] Note: The 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).

[0173] As can be seen from 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, indicating that the application of the formula four microbial agents can stimulate the rapid growth of plants, promote soybean branching and flowering, and increase fertilization and pod formation.

[0174] The number of seeds per plant, 100-seed weight and yield per mu of the treatment group were all higher than those of the control group, increasing by 16.69%, 5.91% and 23.32% respectively compared with the control group. Moreover, the root system and the number of root nodules were also significantly more than those of the control ( Figure 8 ), indicating that after the application of microbial agents, it is not only beneficial for plants to form more pods and seeds, but also can promote root growth and nodulation, improve nitrogen fixation, enhance dry matter accumulation, increase the weight of soybean grains, and is beneficial to yield increase.

[0175] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification

[0176] equivalent replacement, improvement, etc. should be included in the protection scope of the present 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 NBL-B11002; the high-yield urease inhibitor bacteria is Brevibacillus laterosporus BLCC1-1219; the salt-tolerant bacteria is Halobacillus dabanensis strain NBL-BS214; the biocontrol bacteria is Bacillus amyloliquefaciens NBL-AP73; the growth-promoting bacteria is Bacillus subtilis NBL-B12004; and the carbon-fixing bacteria is Priestiamegaterium C17; The Bacillus amyloliquefaciens NBL-B11002 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232184; the Brevibacillus laterosporus BLCC1-1219 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242118; the Daban Halophilic Bacillus NBL-BS214 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20221370; the Bacillus amyloliquefaciens NBL-AP73 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241295; the Bacillus subtilis NBL-B12004 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241295 NO: M20231506; the Priesteria gigantea C17 is deposited in China Center for Type Culture Collection with the deposit number CCTCCNO: M 20241724.

2. 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.

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

4. Use of the composite microbial agent for nitrogen and carbon fixation, synergistic and stress resistance according to claim 1 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; The stress resistance is to enhance the ability of peanut to resist abiotic stress caused by saline-alkali soil environment; The disease prevention is to effectively antagonize the pathogen of cabbage soft rot and / or the pathogen of potato wilt; The method of resisting repeated cropping is to improve the survival rate of strawberry cutting seedlings.

5. The use according to claim 4, 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.

6. The use according to claim 4, characterized in that: The carbon fixation is specifically manifested as increasing the organic carbon content in the soil.

7. The use according to claim 4, 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.

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