Compound microbial inoculant for promoting nitrogen utilization of corn

Abstract

The present application relates to the technical field of microorganism, and discloses a compound microbial inoculant for promoting nitrogen utilization of corn, which comprises a functional bacterial inoculant and a liquid Trichoderma harzianum inoculant, and the functional bacterial inoculant is mixed by Klebsiella variicola powder, Bacillus amyloliquefaciens powder and Enterobacter asburiae powder according to the active bacteria number ratio of 3:1:1.The present application promotes the growth of corn, improves the nitrogen utilization efficiency and yield of corn, reduces the application of chemical nitrogen fertilizer, and realizes cost reduction and income increase.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a compound microbial agent that promotes nitrogen utilization in maize. Background Technology

[0002] In current agricultural production practices, the excessive application of chemical nitrogen fertilizers is prevalent in black soil regions in pursuit of short-term increases in corn yield. Although the external input of chemical nitrogen fertilizers can increase corn production in the short term, long-term excessive nitrogen application has triggered a series of serious chain problems: the rapid conversion of chemical nitrogen fertilizers in the soil directly leads to a continuous decline in the nitrogen use efficiency of farmland, and a large amount of nitrogen is lost through ammonia volatilization, leaching, and other pathways, which not only exacerbates greenhouse gas emissions but also promotes soil acidification, severely depleting the health of black soil farmland; and the continuous decline in black soil fertility will further restrict the realization of corn yield potential, ultimately forming a vicious cycle of ecological degradation and declining production efficiency.

[0003] A deeper investigation into the core causes of these problems reveals that the long-term, excessive application of chemical nitrogen fertilizers significantly inhibits soil microbial activity, disrupting the microbial-dominated soil nitrogen cycle system. This makes the nitrogen supply required for corn growth highly dependent on the external input of chemical nitrogen fertilizers, thus creating a vicious cycle of excessive nitrogen application—reduced soil microbial activity—nitrogen cycle imbalance—further increased nitrogen fertilizer dependence. To break this industry predicament, an attempt was made to launch a zero-growth action plan for fertilizer use, with core measures including directly reducing the application of chemical nitrogen fertilizers and increasing the proportion of straw and organic fertilizers returned to the field. However, production practice shows that such measures have significant limitations: on the one hand, directly reducing the application of chemical fertilizers will directly reduce the supply of readily available nutrients that can be quickly absorbed and utilized during the corn growth cycle, which can easily lead to a reduction in corn yield; on the other hand, although straw and organic fertilizer contain rich nutrients, they generally have a high carbon-to-nitrogen ratio. During the decomposition and transformation process in the soil, they need to consume the original nitrogen in the soil, which can easily lead to competition with corn plants for nitrogen. This results in limited regional applicability of the scheme, poor field operability, and high implementation costs, making it difficult to promote and apply it on a large scale in the corn planting system of the black soil region of Northeast China.

[0004] In contrast, biological improvement technologies based on functional microbial combinations offer significant advantages such as environmental friendliness, ease of operation, and low cost, demonstrating immense application potential in improving black soil health, enhancing its intrinsic production potential, and achieving efficient nitrogen utilization in crops. In the soil-microbe-crop continuous system, microorganisms, through their physiological functions and interspecific synergistic interactions, regulate the entire process of nitrogen fixation, transformation, and supply in the farmland ecosystem, directly impacting soil health and maize growth and development. Existing production practices have confirmed that applying compound microbial agents can enhance soil nitrogen supply capacity and fertilizer nutrient longevity through multiple pathways, including strengthening biological nitrogen fixation, inhibiting soil urease activity to slow urea decomposition, and promoting crop root growth. This improves maize root traits and ultimately achieves the synergistic goal of efficient nitrogen utilization and yield increase in maize in an economical, efficient, and environmentally friendly manner.

[0005] However, a review of relevant research and applications both domestically and internationally reveals that current research on compound microbial agents for farmland soil largely focuses on the exploration of microbial resources, improvement of organic matter decomposition efficiency, and soil carbon enrichment and fertilization effects. Research on the development of specialized microbial formulations for efficient nitrogen utilization in maize, as well as large-scale field application practices, remains relatively scarce. Furthermore, existing compound microbial agents generally suffer from overlapping ecological niches, intense interspecific competition, weak soil adaptability, and poor rhizosphere colonization. They struggle to simultaneously achieve the synergistic effects of biological nitrogen fixation, urease activity inhibition, growth promotion and nitrogen uptake, and disease control. Consequently, they cannot stably exert nitrogen regulation effects throughout the entire maize growth cycle, failing to achieve the synergistic goal of nitrogen reduction and efficiency enhancement in maize while protecting the black soil ecosystem. This has become a core technical bottleneck restricting the large-scale application of functional microbial agents in the maize planting system of the Northeast black soil region. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent that promotes nitrogen utilization in maize, so as to solve the above-mentioned technical problems.

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

[0008] On one hand, the present invention provides a compound microbial agent for promoting nitrogen utilization in maize, comprising:

[0009] Functional bacterial inoculants and Trichoderma harzianum liquid inoculants;

[0010] The functional bacterial agent is composed of Klebsiella variicola bacterial powder, Bacillus amyloliquefaciens bacterial powder, and Enterobacter huaxiensis bacterial powder in a live bacteria ratio of 3:1:1.

[0011] The *Klebsiella pneumoniae* species are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.15640 and accession date April 10, 2016; the *Bacillus amyloliquefaciens* species are deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC AB No. 2022279 and accession date June 3, 2022; the *Enterobacter spp.* species are deposited at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with accession date March 29, 2023 and accession number GDMCC No. 813095; and the *Trichoderma harzianum* species are deposited at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with accession date September 13, 2018 and accession number GDMCC No. 3.667.

[0012] To improve the stability and adaptability of the composite microbial agent system during the construction process, this invention combines species from multiple genera during the strain selection process. This avoids internal competition caused by overlapping ecological niches and resource utilization in the rhizosphere environment, and the interaction of multiple species enhances environmental adaptability. In the microbial composition of this invention, the nitrogen-fixing bacterium is *Klebsiella heterotrophus*, isolated from maize roots, exhibiting significant nitrogen-fixing activity; the biocontrol bacterium is *Bacillus amyloliquefaciens* isolated from soil, possessing biological disease control functions; the growth-promoting bacterium is *Enterobacter schwanniferum* isolated from potato roots, capable of dissolving organophosphates and producing IAA; and the fungal biocontrol bacterium is *Trichoderma harzianum*, which has antagonistic effects against various fungi and effectively controls fungal diseases.

[0013] Preferably, the functional bacterial agent is mixed with Trichoderma harzianum liquid agent at a ratio of 1g:5mL to obtain a compound microbial agent; the viable count of the Klebsiella heterotrophus powder is ≥1.0×10⁻⁶. 11 CFU / g, the viable counts of both the *Bacillus amyloliquefaciens* powder and the *Enterobacter xanthipes* powder are ≥1.0 × 10⁻⁶. 10 CFU / g.

[0014] Preferably, the spore content of the Trichoderma harzianum liquid inoculant is 10. 6 -10 9 CFU / mL, root colonization rate ≥95%.

[0015] On the other hand, the present invention also provides a method for preparing a compound microbial agent that promotes nitrogen utilization in maize, comprising the following steps:

[0016] (1) Preparation of single bacterial powder: Single bacterial powders of Klebsiella pneumoniae, Bacillus amyloliquefaciens and Enterobacter spp. were prepared respectively. The preparation method of the single bacterial powder is as follows: the target strain is subjected to liquid deep fermentation, the fermentation broth is continuously centrifuged, and the precipitate after centrifugation is spray-dried to obtain the corresponding single bacterial powder.

[0017] (2) Preparation of functional bacterial agent: Klebsiella pneumoniae powder, Bacillus amyloliquefaciens powder and Enterobacter schwannii powder obtained in step (1) are mixed evenly at a live count ratio of 3:1:1 to obtain functional bacterial agent;

[0018] (3) Preparation of Trichoderma harzianum liquid inoculant;

[0019] (4) Preparation of compound microbial agent: The functional bacterial agent and Trichoderma harzianum liquid agent are mixed at a ratio of 1g:5mL to obtain the compound microbial agent that promotes nitrogen utilization in maize.

[0020] Preferably, in step (1), the viable count of the prepared Klebsiella pneumoniae powder is ≥1.0×10⁻⁶. 11 The CFU / g of both *Bacillus amyloliquefaciens* powder and *Enterobacter xanthipes* powder were ≥1.0 × 10⁻⁶. 10 CFU / g.

[0021] Preferably, in step (3), the preparation method of the Trichoderma harzianum liquid inoculant includes the following steps:

[0022] S1. Preparation of host plant seedlings: Corn was selected as the host plant. The corn seeds were disinfected, rinsed and germinated. At the same time, Trichoderma harzianum inoculum was sprinkled into the culture medium, which was a mixture of sand and vermiculite in a volume ratio of 3:1. The germinated corn seeds were then evenly sown into the culture medium, watered and irrigated with Hogland nutrient solution every 4 weeks. After 14 days of cultivation, the root colonization rate of the host plant was tested. When the root colonization rate was ≥5%, the preparation of the host plant seedlings was completed.

[0023] S2. Liquid culture expansion: The host plant seedlings prepared in step S1 are placed in the culture medium of a rotating culture vessel for 8 weeks of liquid culture. After the culture is completed, the above-ground parts of the plant are removed, and the plant roots are left. The roots are broken up and mixed with the culture medium to obtain Trichoderma harzianum liquid inoculant.

[0024] Preferably, the Trichoderma harzianum liquid inoculant obtained in step S2 has a spore content of 10%. 6 -10 9 CFU / mL, root colonization rate ≥95%.

[0025] Furthermore, this invention also provides the application of a compound microbial agent that promotes nitrogen utilization in maize in promoting maize growth, improving nitrogen utilization rate, and increasing maize yield.

[0026] Preferably, the compound microbial agent is applied during the corn seedling stage.

[0027] Preferably, the compound microbial agent is applied by root irrigation of corn; the dosage per acre is: 200g of functional bacterial agent diluted 500 times, mixed evenly with 1000mL of Trichoderma harzianum liquid agent and then applied.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The compound microbial agent of this invention can synergistically achieve biological nitrogen fixation, inhibit urease activity to reduce nitrogen volatilization and leaching loss, promote maize root growth and enhance nitrogen absorption, and also has the function of biological control of fungal diseases. It can significantly promote maize growth, enhance photosynthetic intensity, effectively improve maize nitrogen utilization rate and grain and plant nitrogen content, and greatly increase maize yield. It can reduce the application of chemical nitrogen fertilizer, reduce agricultural production costs, and achieve cost reduction and income increase. The agent uses a compound of multiple genera of fungi, with no ecological niche and resource utilization competition among the strains. It has strong adaptability to soil environment, and the functional strains can stably colonize in the rhizosphere of maize and play a continuous and stable role throughout the entire growth period of maize. It is suitable for large-scale field production and application, and is environmentally friendly and easy to operate. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The diagram shows the growth-promoting effect of the compound microbial agent provided in the embodiments of the present invention;

[0032] Figure 2 The image shows the results of a potted plant experiment provided in an embodiment of the present invention.

[0033] Figure 3 This is a diagram showing the results of a field trial of maize provided in an embodiment of the present invention;

[0034] Figure 4 The response diagram of maize yield in the experimental field to the application of microbial agents is provided for an embodiment of the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] The microbial composition of this invention embodiment is as follows:

[0037] The associative nitrogen-fixing bacterium was *Klebsiella variicola*, purchased from the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC 1.15640 and accession date of April 10, 2016. This bacterium was isolated from maize roots and exhibits significant associative nitrogen-fixing activity.

[0038] The biocontrol bacterium is Bacillus amyloliquefaciens, which was purchased from the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. The accession number is CCTCC AB 2022279, and the accession date is June 3, 2022. It was isolated from soil and has the function of controlling biological diseases.

[0039] The growth-promoting bacterium was *Enterobacter huaxiensis*, with accession number GDMCC 813095. This bacterium was purchased from the Guangdong Provincial Microbial Culture Collection Center, located at No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, and was deposited on March 29, 2023. It was isolated from potato roots and possesses the ability to dissolve organophosphates and produce IAA (inorganic acid).

[0040] The fungal biocontrol agent is *Trichoderma harzianum*, which was purchased from the Guangdong Provincial Center for Microbial Culture Collection (address as above), deposited on September 13, 2018, with accession number GDMCC 3.667. It has antagonistic effects against a variety of fungi and is effective in controlling fungal diseases.

[0041] Preparation method of compound microbial inoculant according to embodiments of the present invention:

[0042] Klebsiella variicola, Bacillus amyloliquefaciens, and Enterobacter huaxiensis bacterial powders were prepared separately. The preparation method involved: first, liquid submerged fermentation; then, continuous centrifugation of the resulting fermentation broth; and finally, spray drying of the centrifuged precipitate. The Klebsiella variicola bacterial powder achieved a viable count of 1.0 × 10⁻⁶. 11 The CFU / g, Bacillus amyloliquefaciens, and Enterobacter wilfordii bacterial counts reached 1.0 × 10⁻⁶. 10 CFU / g. Subsequently, Klebsiella pneumoniae powder, Bacillus amyloliquefaciens powder, and Enterobacter huaxiensis powder were mixed at a live count ratio of 3:1:1.

[0043] The preparation method of Trichoderma harzianum liquid inoculant is as follows: First, select corn as the host plant. Disinfect and rinse the seeds, then germinate them. Simultaneously, add the fungal biocontrol agent (Trichoderma harzianum fungal strain) to the culture medium (a mixture of sand and vermiculite in a 3:1 volume ratio). Then, evenly sow the germinated host seeds into this culture medium. Water regularly, and apply a 1 / 2 strength Hoagland nutrient solution every 4 weeks. After 14 days of cultivation, check the root colonization rate of the host plant. When the root colonization rate is ≥5%, the host plant seedlings are ready. Then, place the prepared host plant seedlings in the above-mentioned culture medium in a rotary culture tank for 8 weeks of liquid culture (rotary culture tank speed set to 60-80 r / min, culture temperature 25℃, photoperiod 12h light / 12h dark). After cultivation, remove the above-ground parts of the plant, leaving the roots. Crush the roots and mix them with the culture medium to obtain a spore content of 10... 6 -10 9 A liquid inoculant for *Trichoderma harzianum* with a CFU / mL concentration and a root colonization rate ≥95%. The root colonization rate (%) is calculated as follows: (Number of root segments with *Trichoderma harzianum* colonization detected ÷ Total number of root segments detected) × 100%.

[0044] Experimental Example 1: Potted Plant Experiment

[0045] Soil samples were collected from the topsoil of black soil farmland that had been subjected to long-term chemical fertilizer application. After air drying, the soil was sieved through a 5 mm sieve. Pots with a depth of 20 cm and an inner diameter of approximately 22 cm were used for the experiment. The pots were filled with 20 cm of soil, with all pots containing the same amount of soil. Different nitrogen application gradients were set up for the pot experiment: no nitrogen fertilizer (N0), 20% nitrogen fertilizer (N20), 40% nitrogen fertilizer (N40), 60% nitrogen fertilizer (N60), 80% nitrogen fertilizer (N80), and 100% nitrogen fertilizer (N100). Phosphorus and potassium fertilizer application rates were the same as conventional management. For each nitrogen application rate, a treatment group (M) with added compound microbial inoculant and a control group without added compound microbial inoculant were set up, for a total of 12 treatments, with 3 replicates for each treatment. Chemical fertilizer was applied to a depth of 7 cm in all pots. Plump and similarly sized corn seeds were selected for sowing. To ensure germination, 3 seeds were sown per pot. After germination, 2 seedlings were randomly removed manually. After sowing, spray each pot with 400mL of water to thoroughly moisten the soil. Dilute the bacterial inoculant to 1.0×10⁻⁶. 7 CFU / mL was mixed with an equal volume of Trichoderma harzianum liquid inoculant to form a compound microbial inoculant. The treatment group received 50 mL of the compound microbial inoculant as a root drench using a syringe, while the control group received an equal volume of sterile water. Forty days after application of the compound microbial inoculant, maize plant height, stem diameter, SPAD (spider plant density), aboveground and belowground dry weight, and the number of nitrogen-fixing bacteria in the soil were measured. The growth-promoting effect of the compound microbial inoculant was also recorded (see attached image). Figure 1 The left side of the figure shows corn seeds treated with compound microbial inoculant under 80% nitrogen fertilizer, while the right side shows control group seeds treated with sterile water under 80% nitrogen fertilizer. The pot experiment design is shown in Table 1, and the results are shown in Table 2. Figure 2 As shown. Figure 2 middle, For p < 0.05, For p < 0.01, The p value is less than 0.001.

[0046] Table 1 Pot Experiment Design

[0047] N0 0kg / ha —— N20 27.6 kg / ha —— N40 55.2 kg / ha —— N60 82.8 kg / ha —— N80 110.4 kg / ha —— N100 138kg / ha —— M-N0 0kg / ha 50mL M-N20 27.6 kg / ha 50mL M-N40 55.2 kg / ha 50mL M-N60 82.8 kg / ha 50mL M-N80 110.4 kg / ha 50mL M-N100 138kg / ha 50mL

[0048] Table 2 Results of pot experiment

[0049] N0 63.91±2.21 7.95±0.68 1.33±0.12 0.21±0.02 39.28±1.64 <![CDATA[1.41×10 4 ]]> N20 70.30±3.95 8.21±0.85 1.37±0.06 0.22±0.03 42.61±2.14 <![CDATA[1.53×10 4 ]]> N40 75.38±1.45 8.34±0.43 1.39±0.14 0.23±0.05 43.95±1.46 <![CDATA[1.79×10 4 ]]> N60 76.28±0.95 10.56±0.94 1.75±0.32 0.29±0.04 45.86±0.68 <![CDATA[1.81×10 4 ]]> N80 80.23±2.48 11.01±0.61 1.84±0.28 0.30±0.01 46.81±1.49 <![CDATA[2.01×10 5 ]]> N100 82.65±3.65 11.95±0.89 1.91±0.09 0.32±0.03 46.76±2.48 <![CDATA[2.32×10 5 ]]> M-N0 73.67±1.18 8.23±0.87 1.37±0.21 0.22±0.03 43.33±1.85 <![CDATA[3.46×10 4 ]]> M-N20 80.02±2.46 10.03±1.02 1.81±0.19 0.30±0.04 45.86±1.98 <![CDATA[5.96×10 4 ]]> M-N40 81.22±1.96 10.91±0.96 1.99±0.11 0.32±0.01 46.73±2.94 <![CDATA[3.84×10 5 ]]> M-N60 85.81±2.85 13.56±1.64 2.34±0.28 0.37±0.01 49.19±1.52 <![CDATA[2.45×10 6 ]]> M-N80 94.45±2.95 15.24±1.76 2.68±0.45 0.41±0.02 52.41±0.96 <![CDATA[5.24×10 6 ]]> M-N100 95.60±1.08 15.85±1.24 2.66±0.29 0.43±0.04 52.69±1.82 <![CDATA[5.18×10 6 ]]>

[0050] Note: The data in the table are mean ± standard error, n=3.

[0051] As shown in Table 2, regardless of the application of compound microbial agents, all maize indicators and the number of nitrogen-fixing bacteria in the soil increased with the amount of nitrogen fertilizer applied. Comparing the effects of compound microbial agents at the same nitrogen application rate revealed that all maize growth indicators significantly increased after application. Under all nitrogen application levels, plant height increased by 13.79%, SPAD by 9.36%, and aboveground biomass by 25.99%. However, the increase in underground biomass was as high as 32.82%, and the aboveground nitrogen content increased by 29.81%, both exceeding the increase in aboveground biomass. The increase in the number of nitrogen-fixing bacteria in the soil also increased with the increase in nitrogen application rate. It should be noted that there were no significant differences in any of the indicators under the two nitrogen application rates of N80 and N100. The above results demonstrate that the compound microbial agent of this invention can enhance the nitrogen absorption capacity and overall growth and development indicators of maize by promoting root growth, which is beneficial for the accumulation of substances during the vegetative growth stage. The number of nitrogen-fixing bacteria in the soil is increased by more than two orders of magnitude, and by more than one order of magnitude under medium and high nitrogen application levels, which significantly improves the abundance of nitrogen-fixing bacteria in the soil. It also proves that nitrogen-fixing bacteria in the compound microbial agent can colonize in complex soil environments and play a stable role throughout the maize growing season.

[0052] Depend on Figure 2 It was found that with increasing nitrogen application, the plant height, aboveground and belowground biomass of maize in both groups showed a significant upward trend. At the same nitrogen application level, the plant height in the inoculant group was significantly higher than that in the control group, with highly significant differences in N0, N60, N80, and N100 levels (p<0.001). Regarding aboveground biomass, the inoculant group showed significant differences in N20 and N40 levels (p<0.05), and highly significant differences in N60-N100 levels (p<0.001). The nitrogen content in the aboveground parts of maize continued to increase with increasing nitrogen application. The inoculant group showed significantly higher N20, N40, and N60 levels than the control group (p<0.05), and highly significant differences in N80 and N100 levels (p<0.001). The number of rhizosphere nitrogen-fixing bacteria also showed an upward trend with increasing nitrogen application, and the inoculant group was highly significantly higher than the control group at all nitrogen application levels (p<0.001). The order of nitrogen-fixing bacteria increased from that in the control group to that in the control group. CFU / g increased to the bacterial agent group CFU / g.

[0053] The above results demonstrate that the application of this microbial agent can reduce nitrogen fertilizer application and has broad application potential. The maize plant height, biomass, and nitrogen accumulation in the microbial agent group at the N60 level reached or even exceeded the control group at the N80-N100 level, indicating that the application of this microbial agent can reduce chemical fertilizer nitrogen input by 20%-40% while maintaining normal maize growth and nitrogen nutrition levels, providing a feasible technical path for green nitrogen-reduced maize cultivation. After treatment with the microbial agent, the number of nitrogen-fixing bacteria in the maize rhizosphere increased by 1-2 orders of magnitude, maintaining a high abundance of nitrogen-fixing bacteria even under low nitrogen conditions. This provides an additional nitrogen source for maize by enhancing biological nitrogen fixation, while improving the root growth environment and increasing nitrogen absorption efficiency, thus ensuring maize growth performance under nitrogen-reduced conditions from a microecological perspective.

[0054] Experimental Example 2: Field Experiment

[0055] Selected farmland with long-term chemical fertilizer application was used, and nitrogen fertilizer application gradients were set up (N60, N80, and N100). Phosphorus and potassium fertilizers were applied normally. Each treatment included application of compound microbial inoculant (M) and no application of compound microbial inoculant, for a total of 6 treatments, with 3 replicates per treatment, resulting in 18 plots. Each plot was 10 rows wide and 10 meters long. Basal fertilizer was applied and maize was sown in mid-May at a planting density of 65,000 plants / ha. The compound microbial inoculant was applied after maize emergence. The inoculation amount of the compound microbial inoculant per acre in the field trial was: 200g of compound bacterial powder + 1000ml of Trichoderma harzianum liquid. The dosage of the compound microbial inoculant per acre was 200g of functional bacterial inoculant diluted 500 times, then mixed evenly with 1000ml of Trichoderma harzianum liquid inoculant. Other field management measures were carried out according to local management practices. During the autumn harvest in October, 10 representative maize plants were continuously collected from each plot to determine plant height, aboveground biomass, yield, grain nitrogen content, and plant nitrogen content. The test results are shown in Table 3. Figure 3 As shown. Figure 3 middle, This means p < 0.05. This means p < 0.01. This means p < 0.001.

[0056] Table 3 Results of maize field trials

[0057] N60 2.44±0.21 156.58±8.95 16.21±0.94 8281.22±859.24 11.21±0.56 8.51±0.06 N80 2.62±0.15 172.12±10.12 22.19±1.16 12053.04±751.95 12.98±0.24 9.96±0.14 N100 2.79±0.25 175.65±11.58 23.34±2.86 12301.12±1251.89 13.15±0.87 10.04±0.17 M-N60 2.67±0.16 183.84±4.61 20.15±1.53 10948.55±895.76 12.94±0.55 10.25±0.21 M-N80 2.86±0.18 206.83±10.95 26.84±2.27 14231.12±1369.15 15.86±0.09 12.41±0.35 M-N100 2.91±0.22 202.03±8.59 26.98±2.19 14112.43±1416.27 15.69±0.24 12.16±0.18

[0058] Note: The data in the table are mean ± standard error, n=3.

[0059] As shown in Table 3, in the treatment without compound microbial inoculant application, directly reducing nitrogen fertilizer application did indeed cause a significant reduction in maize yield. Compared with the conventional nitrogen application rate (N100), the yields of N80 and N60 decreased by 2.01% and 32.68%, respectively. After the application of compound microbial inoculant, the biomass of maize plants under N100, N80, and N60 treatments increased by 15.31%, 20.56%, and 20.86%, respectively; and the maize yield increased by 14.72%, 18.07%, and 32.21%, respectively. Moreover, the nitrogen content of maize grains and plants increased significantly by 18.97% and 22.05%, respectively. Combined with the pot experiment results, it can be concluded that the application of compound microbial inoculant during the vegetative growth stage of maize increases nitrogen supply by increasing the number of nitrogen-fixing bacteria in the soil and reduces the application of chemical nitrogen fertilizer. In addition, Trichoderma harzianum and growth-promoting bacteria stimulate root growth, ultimately achieving the simultaneous reduction of chemical fertilizer application, improvement of nitrogen use efficiency, and increase of grain yield.

[0060] Depend on Figure 3 The results of the field trials show that, under the three nitrogen application levels of N60, N80, and N100, the compound microbial agent treatment significantly promoted maize plant height, stem and leaf biomass, root biomass, yield, and nitrogen accumulation. The plant height in the microbial agent group was significantly higher than that in the control group at the N80 level (p<0.05). The stem and leaf biomass was extremely significantly increased at the N60 and N80 levels (p<0.001) and significantly increased at the N100 level (p<0.05). The root biomass was also extremely significantly increased at the N60 and N80 levels (p<0.001) and significantly increased at the N100 level (p<0.05). The maize yield was significantly or extremely significantly higher than that in the control group at all nitrogen application levels. The yield of the microbial agent group at the N60 level was close to that of the control group at the N100 level, while the yield of the microbial agent group at the N80 level was 1930 kg / ha higher than that of the control group at the N100 level, an increase of 15.7%. The nitrogen content of grains and plants was significantly or extremely significantly increased at all nitrogen application levels, showing a clear nitrogen reduction and efficiency enhancement characteristic. In summary, this compound microbial agent has stable effects on promoting growth, increasing yield, and regulating nitrogen in maize under field conditions. It can significantly improve maize plant height, aboveground / underground biomass, yield, and nitrogen accumulation. It can also achieve "reduced nitrogen without reduced yield" by enhancing root growth and nitrogen absorption efficiency, reducing fertilizer nitrogen input by about 20%. It has the potential to be promoted and applied in green and efficient maize cultivation to achieve fertilizer saving and efficiency improvement.

[0061] Figure 4 The graph shows the response of maize yield in the experimental field to the application of microbial inoculants, where p < 0.05. Figure 4It was found that under conventional fertilization, the average plant height of maize at the jointing stage was 69.5 cm, which significantly increased to 77.3 cm after the application of microbial inoculants. The SPAD value of maize was 42.5 under conventional fertilization, increasing to 49.6 after the application of microbial inoculants. These results demonstrate the promoting effect of microbial inoculants on the vegetative growth stage of maize and the enhancement of nitrogen utilization in the plants. Autumn harvest yield measurements showed that the maize yield under conventional management was 12301 kg / ha, which increased to 14231 kg / ha after the application of microbial inoculants, significantly improving maize yield.

[0062] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A compound microbial agent for promoting nitrogen utilization in maize, characterized in that, include: Functional bacterial inoculants and Trichoderma harzianum liquid inoculants; The functional bacterial agent is composed of Klebsiella variicola bacterial powder, Bacillus amyloliquefaciens bacterial powder, and Enterobacter huaxiensis bacterial powder in a live bacteria ratio of 3:1:

1. The *Klebsiella pneumoniae* species are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.15640 and accession date April 10, 2016; the *Bacillus amyloliquefaciens* species are deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC AB No. 2022279 and accession date June 3, 2022; the *Enterobacter spp.* species are deposited at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with accession date March 29, 2023 and accession number GDMCC No. 813095; and the *Trichoderma harzianum* species are deposited at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with accession date September 13, 2018 and accession number GDMCC No. 3.

667.

2. The compound microbial agent for promoting nitrogen utilization in maize according to claim 1, characterized in that, The functional bacterial agent and Trichoderma harzianum liquid agent were mixed at a ratio of 1g:5mL to obtain a compound microbial agent; The viable count of the Klebsiella pneumoniae powder is ≥1.0×10¹¹ CFU / g, and the viable counts of the Bacillus amyloliquefaciens powder and the Enterobacter schwanniferum powder are both ≥1.0×10¹⁰ CFU / g.

3. The compound microbial agent for promoting nitrogen utilization in maize according to claim 1, characterized in that, The Trichoderma harzianum liquid inoculant has a spore content of 10⁶-10⁹ CFU / mL and a root colonization rate of ≥95%.

4. A method for preparing a compound microbial agent for promoting nitrogen utilization in maize as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of single bacterial powder: Single bacterial powders of Klebsiella pneumoniae, Bacillus amyloliquefaciens and Enterobacter schreberi were prepared respectively. The preparation method of the single bacterial powder is as follows: the target strain is subjected to liquid deep fermentation, the fermentation broth is continuously centrifuged, and the precipitate after centrifugation is spray-dried to obtain the corresponding single bacterial powder. (2) Preparation of functional bacterial agent: Klebsiella pneumoniae powder, Bacillus amyloliquefaciens powder and Enterobacter schwannii powder obtained in step (1) are mixed evenly at a live count ratio of 3:1:1 to obtain functional bacterial agent; (3) Preparation of Trichoderma harzianum liquid inoculant; (4) Preparation of compound microbial agent: The functional bacterial agent and Trichoderma harzianum liquid agent are mixed at a ratio of 1g:5mL to obtain the compound microbial agent that promotes nitrogen utilization in maize.

5. The method for preparing the compound microbial agent for promoting nitrogen utilization in maize according to claim 4, characterized in that, In step (1), the viable count of the prepared Klebsiella pneumoniae powder is ≥1.0×10¹¹ CFU / g, and the viable counts of Bacillus amyloliquefaciens powder and Enterobacter schwanniferum powder are both ≥1.0×10¹⁰ CFU / g.

6. The method for preparing the compound microbial agent for promoting nitrogen utilization in maize according to claim 4, characterized in that, In step (3), the preparation method of the Trichoderma harzianum liquid inoculant includes the following steps: S1. Preparation of host plant seedlings: Corn was selected as the host plant. The corn seeds were disinfected, rinsed and germinated. At the same time, Trichoderma harzianum inoculum was sprinkled into the culture medium, which was a mixture of sand and vermiculite in a volume ratio of 3:

1. The germinated corn seeds were then evenly sown into the culture medium, watered and irrigated with Hogland nutrient solution every 4 weeks. After 14 days of cultivation, the root colonization rate of the host plant was tested. When the root colonization rate was ≥5%, the preparation of the host plant seedlings was completed. S2. Liquid culture expansion: The host plant seedlings prepared in step S1 are placed in Hogrange culture medium in a rotating culture device for 8 weeks of liquid culture. After the culture is completed, the above-ground parts of the plant are removed, and the plant roots are left. The roots are crushed and mixed with the culture medium to obtain Trichoderma harzianum liquid inoculum.

7. The method for preparing the compound microbial agent for promoting nitrogen utilization in maize according to claim 6, characterized in that, The Trichoderma harzianum liquid inoculant prepared in step S2 has a spore content of 106-109 CFU / mL and a root colonization rate of ≥95%.

8. The application of the compound microbial agent for promoting nitrogen utilization in maize as described in any one of claims 1-3 in promoting maize growth, improving maize nitrogen utilization rate and maize yield.

9. The application according to claim 8, characterized in that, The compound microbial agent is applied during the corn seedling stage.

10. The application according to claim 8, characterized in that, The compound microbial agent is applied by root irrigation of corn roots; the dosage per acre is: dilute 200g of functional bacterial agent 500 times, mix it evenly with 1000mL of Trichoderma harzianum liquid agent, and then apply.

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