Compound optimized rhizosphere strain composition and application thereof in improving nitrogen utilization efficiency of crops
By combining and optimizing compound biological agents, including three Acinetobacter strains—Acinetobacter1, Acinetobacter2, and Acinetobacter3—the problem of fertilizer dependence in corn production has been solved, the efficiency of nitrogen use in corn has been improved, and fertilizer reduction and efficiency enhancement have been achieved, as well as green agricultural production increases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-14
- Publication Date
- 2026-04-14
AI Technical Summary
In existing technologies, corn production relies excessively on chemical nitrogen fertilizers, leading to increased production costs and a heightened risk of environmental pollution. There is a lack of corn-specific microbial resources that combine efficient nitrogen fixation, good rhizosphere adaptability, and field stability, especially complex microbial communities with synergistic effects.
The compound biological agent, which is optimized by compounding, includes three Acinetobacter strains: Acinetobacter1, Acinetobacter2, and Acinetobacter3. Through seed treatment, root-targeted application, and soil/planting substrate improvement, the nitrogen use efficiency of crops is improved.
It significantly improves nitrogen use efficiency in maize, promotes maize plant biomass and root indicators, achieves reduced fertilizer use and increased efficiency, and promotes green agricultural production.
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Figure CN121844783A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of specialized biological agents, and in particular to an optimized combination of rhizosphere bacterial strains and its application in improving crop nitrogen use efficiency. Background Technology
[0002] With the continuous development of green agricultural technologies centered on environmental friendliness and resource conservation, research on new green bio-fertilizers is becoming increasingly in-depth. Traditional agricultural production has long relied on chemical fertilizers, which, while significantly increasing crop yields, has also caused a series of ecological problems such as soil degradation, environmental pollution, and increased carbon emissions. Against this backdrop, microbial fertilizers, as an important green agricultural technology, can promote soil nutrient cycling, improve soil ecology, and enhance crop stress resistance through multiple mechanisms, including nitrogen fixation, phosphorus solubilization, nutrient transformation promotion, and the secretion of growth-promoting substances, thereby achieving both fertilizer reduction and increased efficiency, and green yield increases.
[0003] The rhizosphere, as the core interface of soil-plant interaction, is rich in beneficial microorganisms that are naturally adapted to the rhizosphere environment, have strong colonization capabilities, and diverse functions, providing important resources for the development of high-efficiency microbial fertilizers. In particular, the original population of wild rice—Dongxiang wild rice—exists only in China, has evolved a unique and abundant rhizosphere microbial resource over a long period in its natural environment, preserving a group of "ancient" but highly functional microbial groups. These microorganisms not only play a key role in nutrient acquisition and environmental adaptation in wild rice itself, but also hold promise as a "functional reservoir" that can be introduced into other crop systems to improve nutrient cycling efficiency and stress resistance. It is noteworthy that, compared to single microbial agents, compound microbial agents composed of synergistic and functionally complementary strains are more likely to form stable communities in the rhizosphere, exerting a synergistic effect of "1+1>2," significantly improving the stability and environmental adaptability of field applications. Based on this, the construction of high-efficiency compound microbial agents has significant application prospects and practical significance for promoting the green transformation of agriculture and ensuring sustainable agricultural development.
[0004] As the most widely planted and highest-yielding grain crop in my country, corn holds irreplaceable strategic significance for national food security, feed supply, and industrial raw material security. Given the limited arable land resources, continuously increasing corn yield per unit area and total output is a core task for ensuring basic grain self-sufficiency. However, current corn production relies excessively on chemical nitrogen fertilizers, leading to increased production costs and heightened environmental pollution risks. Therefore, promoting fertilizer reduction and achieving efficient nitrogen fertilizer utilization while stabilizing and increasing yields has become a critical issue urgently needing to be addressed for my country's agricultural modernization and food security. Utilizing biological nitrogen fixation technology, especially rhizosphere nitrogen-fixing bacteria that can establish a symbiotic relationship with corn, can convert atmospheric nitrogen into a usable nitrogen source, representing an ideal way to reduce reliance on chemical fertilizers at the source.
[0005] However, there is a lack of maize-specific microbial resources that combine high nitrogen fixation capacity, good rhizosphere adaptability and field stability, especially complex microbial communities with synergistic effects. This is a key factor restricting the development of the industry and also a key and hot issue that urgently needs to be addressed in scientific research. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to provide a compounded and optimized combination of rhizosphere strains and its application in improving crop nitrogen use efficiency.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.
[0008] In a first aspect, the present invention includes a biological method for improving crop nitrogen use efficiency by applying a compounded and optimized biological agent; the compounded biological agent includes any one of the following strains, or is composed of three groups of strains A, B, and C mixed in a certain proportion: Strain A: Its name is Acinetobacter1, which is deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No.37047; Strain B: Its name is Acinetobacter2, which is deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with the accession number CGMCC No.37048; Strain C: Its name is Acinetobacter3, which is deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with the accession number CGMCC No.37049.
[0009] As a preferred embodiment of the present invention, the method of applying the compound biological agent is selected from any of the following options: A. Seed treatment; B. Targeted application to the root; C. Soil / planting substrate improvement; D. Composting and fermentation combined with application, substrate mixing and application.
[0010] As a preferred embodiment of the present invention, the seed treatment includes: seed dressing; seed soaking; seed coating; seed fertilizer dressing; the root-targeted application includes: root dipping, root irrigation, hole application, furrow application, drip irrigation root dipping, and hole irrigation with mulch; the soil improvement includes: fertigation, drip / micro-sprinkler irrigation, broadcasting and tillage, fertilizer-mixed basal application, fertilizer-mixed topdressing, and soil flooding.
[0011] As a preferred technical solution of the present invention, the compound biological agent is prepared by using a 1:1:1 ratio of bacterial strains as a basis and adjusting the amount of strains as needed.
[0012] As a preferred technical solution of the present invention, the method of preparing the compound biological agent includes: ① separately fermenting and culturing each component strain to obtain the fermentation broth or cell of each strain; then taking the strain fermentation broth or cell for further compounding; ② taking the three groups of strains for compound fermentation to obtain the compound biological agent.
[0013] As a preferred embodiment of the present invention, the crop includes: corn; crops of the same genus as corn; grass crops that are C4 plants like corn; grass crops.
[0014] On the other hand, the present invention also includes strain A for improving nitrogen use efficiency in maize: its name is Acinetobacter1, deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37047; its main biological characteristics are: Acinetobacter antiviralis species, Gram-negative. - The cells are rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, catalase-positive, nitrate-reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, and starch hydrolysis-positive. The colonies are grayish-white, opaque, and round.
[0015] On the other hand, the present invention also includes a strain B for improving nitrogen use efficiency in maize: its name is Acinetobacter2, deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37048; its main biological characteristics are: Acinetobacter antiviralis species, Gram-negative. - The cells are rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, catalase-positive, nitrate-reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, and starch hydrolysis-positive. The colony morphology is grayish-white and mucous-like.
[0016] On the other hand, the present invention also includes a C strain for improving nitrogen use efficiency in maize: its name is Acinetobacter3, deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37049; its main biological characteristics are: Acinetobacter antiviralis species, Gram-negative. -The cells are rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, catalase-positive, nitrate-reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, and starch hydrolysis-positive. The colonies are grayish-white, opaque, and round.
[0017] Finally, the present invention also includes a compound microbial agent comprising strains A, B, C, or any combination thereof as described in any of the preceding claims.
[0018] The beneficial effects of adopting the above technical solution are as follows: Overall, the research results of this invention construct a new type of green microbial fertilizer with efficient nitrogen-fixing bacteria as the core, which has important scientific value and application prospects for breaking through the bottleneck of nitrogen nutrition management, promoting the reduction and efficiency of chemical fertilizers, and realizing green yield increase of corn.
[0019] The three Acinetobacter strains of this invention, Acinetobacter 1, Acinetobacter 2, and Acinetobacter 3, can all grow colonies in 50% TSB medium for 24 hours and form clear colonies with obvious transparent zones in Assoube nitrogen-free solid medium for 24 hours. The three strains exhibit a synergistic effect after being mixed, significantly improving the nitrogenase activity of the compound bacterial agent. The compound bacterial agent was inoculated into the rhizosphere of maize plants of variety Dongdan 1331. Based on the evaluation of the physicochemical indicators of maize plants and the nitrogen element indicators of rhizosphere soil, its effect on improving nitrogen use efficiency and promoting growth in maize was further confirmed, providing a basic support for further application in production.
[0020] In this study on improving nitrogen utilization efficiency in maize, rhizosphere soil from a well-developed original population of wild rice from Dongxiang County, exhibiting significant nitrogen-efficient capabilities, was collected. A high-throughput microbial library construction technique was used to build a wild rice rhizosphere bacterial library. Bioinformatics analysis and reinfection verification were then performed on the wild rice rhizosphere bacterial community. After repeated screening and verification, three Acinetobacter strains (Acinetobacter 1, Acinetobacter 2, and Acinetobacter 3) were finally screened and verified from a large number of bacterial communities. Notably, the combination of these three strains promoted maize plant biomass and root indicators and improved nitrogen use efficiency, significantly outperforming their individual effects. This suggests that the three strains not only have individual nitrogen-promoting effects but also exhibit synergistic symbiotic effects at the evolutionary and physiological levels. Detailed experimental and production data are provided in the examples below. Attached Figure Description
[0021] Figure 1 The colony morphology of three Acinetobacter strains, Acinetobacter1, Acinetobacter2 and Acinetobacter3, is observed on a 50% TSB plate. Figure 2 Phylogenetic tree of three Acinetobacter strains: Acinetobacter1, Acinetobacter2, and Acinetobacter3; Figure 3 The colony morphology of three Acinetobacter strains, Acinetobacter1, Acinetobacter2 and Acinetobacter3, cultured on Ashube nitrogen-free solid medium is shown in the figure. Figure 4 Figure 1 shows the observation of a plate confrontation experiment of three Acinetobacter strains, Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3. Figure 5 Nitrogenase activity of three Acinetobacter strains (Acinetobacter 1, Acinetobacter 2, and Acinetobacter 3) and a compound bacterial agent was measured. Figure 6 The nitrogen-efficient and growth-promoting effects of three Acinetobacter strains (Acinetobacter 1, Acinetobacter 2, and Acinetobacter 3) and a compound bacterial agent were studied. Figure 7 To compare the growth of corn inoculated with three strains of Acinetobacter, Acinetobacter 1, Acinetobacter 2, and Acinetobacter 3, as well as a compound bacterial agent, with that of uninoculated corn.
[0022] Preservation Instructions Strain A: Its name is Acinetobacter1, which is deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No.37047; Strain B: Its name is Acinetobacter2, deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37048; its main biological characteristics are: Acinetobacter antiviralis species; Strain C: Its name is Acinetobacter3, which is deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with the accession number CGMCC No.37049. Detailed Implementation
[0023] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase.
[0024] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.
[0025] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.
[0026] As used in this application specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."
[0027] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.
[0028] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.
[0029] The strains of this invention were collected from natto and obtained through screening using the single-strain protein fiber plate transparency method.
[0030] In this study on improving nitrogen utilization efficiency in maize, rhizosphere soil from a well-developed original population of wild rice from Dongxiang County, exhibiting significant nitrogen-efficient capacity, was collected. A high-throughput microbial library construction technique was used to construct a rhizosphere bacterial library of wild rice. Bioinformatics analysis and reinfection verification were then performed on the wild rice rhizosphere bacterial community. After repeated screening and verification, three Acinetobacter strains—Acinetobacter1, Acinetobacter2, and Acinetobacter3—were finally screened and verified from a large number of bacterial communities. The research data suggests that these three strains exhibit a synergistic symbiotic effect in the maize rhizosphere, both evolutionarily and physiologically. Example 1
[0031] The bacteria used in this embodiment are three Acinetobacter strains, Acinetobacter1, Acinetobacter2, and Acinetobacter3, which are deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession numbers CGMCC No. 37047, CGMCC No. 37048, and CGMCC No. 37049.
[0032] The methods for obtaining Acinetobacter baumannii (Acinetobacter 1), Acinetobacter antiviralis (Acinetobacter 2), and Acinetobacter populi (Acinetobacter 3) in this embodiment are as follows: Constructing a microbial bank: Select wild rice plants that have grown normally to harvest time, take fresh rhizosphere soil, and mix at a ratio of 10... -3 10 -4 10 -5 The cells were diluted according to the specified dilution ratio and then incubated in 96-well cell culture plates containing 10% TSB at room temperature (25°C) for 2 weeks. At the end of the culture, a concentration of 10... -5 The 96-well cell culture plates were used for subsequent bacterial bank construction.
[0033] First, take 10 µL of bacterial culture from a 96-well cell culture plate, add 16.6 µL of alkaline lysis buffer (25 mM NaOH and 0.2 mM Na2-EDTA (pH = 12)) and lyse at 95°C for 30 min. Then, add 16.6 µL of neutralization buffer (40 mM Tris-HCl (pH = 7.5)) and mix well to obtain the bacterial DNA. Store at -20°C.
[0034] The first round of PCR amplification was performed using universal bacterial primers 799F and 1193R. 799F was used for the first round of amplification. The amplification system consisted of: 3 µL template, 3 µL 10× Buffer, 2.4 µL dNTPs (2.5 mM each), 0.3 µL 799F (10 µM), 0.3 µL 1193R (10 µM), and 0.15 µL HS taq (5 U / µL). The amplification program was as follows: pre-denaturation at 95℃ for 5 min; 25 cycles of 95℃ for 10 s, 55℃ for 15 s, and 72℃ for 1 min; final incubation at 72℃ for 5 min; and storage at 4℃.
[0035] In the second round of PCR, the template was a 40-fold dilution of the first-round PCR product. The primers used were 799F and 1193R primers with tags (barcodes) and the adapters required for sequencing. The amplification system and procedure were the same as in the first round of PCR. After both rounds of PCR amplification, the obtained DNA samples were mixed and subjected to agarose gel electrophoresis. The gel was then recovered according to the kit instructions. Finally, the concentration of the recovered samples was checked using Nanodrop. Qualified samples were then used for Illumina sequencing.
[0036] Bioinformatics analysis to identify isolated and cultured bacteria: Data analysis was performed according to the method of "High-throughput isolation, culture and identification of plant root bacteria".
[0037] Isolation and Purification: Bacteria identified through bioinformatics analysis were isolated and purified. Identified bacterial cultures were selected from wells of a 96-well cell culture plate, and 10 µL of bacterial culture was streaked onto a 50% TSB plate for purification, with three replicates per well. The strains were cultured at 28°C for 3-5 days for further purification, repeated twice until pure colonies were obtained. The pure strains were then cultured in 50% TSB liquid medium at 28°C and 180 rpm for 1-2 days for 16S sequencing confirmation.
[0038] TSB liquid culture medium formula: tryptone 17.0 g / L, sodium chloride 5.0 g / L, soybean papain hydrolysate 3.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, ultrapure water added to 1L.
[0039] TSB plate formulation: tryptone 17.0 g / L, sodium chloride 5.0 g / L, soybean papain hydrolysate 3.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, agar 1.5%-2%. Example 2
[0040] 1. Identification of strains Acinetobacter1, Acinetobacter2 and Acinetobacter3 Physiological and biochemical identification of strains Acinetobacter1, Acinetobacter2, and Acinetobacter3 was performed according to the *Handbook of Systematic Identification of Common Bacteria*. Strains Acinetobacter1, Acinetobacter2, and Acinetobacter3 are Gram-negative bacteria. Their colonies are grayish-yellow and opaque, with smooth surfaces and regular edges. They can grow on TSB medium and grow relatively quickly. Morphological images of strains Acinetobacter1, Acinetobacter2, and Acinetobacter3 on 50% TSB plates are shown below. Figure 1 As shown.
[0041] 2. Molecular identification of strains Acinetobacter1, Acinetobacter2 and Acinetobacter3 DNA was extracted from strain E2 using alkaline lysis buffer and buffer. The V5-V7 region of the 16S rRNA gene was selected for detection using universal primers (799F and 1193R sequences). The reaction mixture consisted of: 3 µL template, 3 µL 10× Buffer, 2.4 µL dNTPs (2.5 mM each), 0.3 µL 799F (10 µM), 0.3 µL 1193R (10 µM), and 0.15 µL HS taq (5 U / µL). The amplification program was as follows: pre-denaturation at 95℃ for 5 min; 25 cycles of 95℃ for 10 s, 55℃ for 15 s, and 72℃ for 1 min; final amplification at 72℃ for 5 min; and storage at 4℃.
[0042] After PCR amplification, the products were examined by 1.5% agarose gel electrophoresis, showing distinct characteristic bands. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequences were uploaded to NCBI. Homology comparisons were performed using BLAST analysis with sequences in gene banks, and a phylogenetic tree was constructed using MEGA11 software. Figure 2 As shown.
[0043] The strain Acinetobacter1 showed the highest homology with Acinetobacter baumannii, reaching 99.81%. Combined with the identification results of bacterial morphology characteristics and growth conditions, it was determined that the strain Acinetobacter1 of the present invention is Acinetobacter baumannii.
[0044] The strain Acinetobacter2 showed the highest homology with Acinetobacter antiviralis, reaching 96.61%. Combined with the identification results of bacterial morphology characteristics and growth conditions, it was determined that the strain Acinetobacter2 of the present invention is Acinetobacter antiviralis.
[0045] The strain Acinetobacter3 showed the highest homology with Acinetobacter populi, reaching 96.78%. Combined with the identification results of bacterial morphology characteristics and growth conditions, it was determined that the strain Acinetobacter3 of this invention is Acinetobacter populi. Example 3
[0046] Verification of nitrogen fixation function of Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3 The isolated and purified strains Acinetobacter1, Acinetobacter2, and Acinetobacter3 were inoculated onto Ashby nitrogen-free solid medium and cultured at 28°C for 24-48 hours to verify their nitrogen fixation function. The nitrogen fixation performance of Acinetobacter 1, Acinetobacter2, and Acinetobacter3 was verified as follows: Figure 3 , Figure 3 The results showed that Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3 could all grow on Ashby nitrogen-free solid medium and have a clear zone, indicating that they all have nitrogen fixation function. Example 4
[0047] Verification of the antagonistic effects of Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3 in Acinetobacter Three immobile stem cells (Acinetobacter 1, Acinetobacter 2, and Acinetobacter 3) were inoculated at equidistant points on 50% TSB solid plates and cultured for 24 hours. A plate confrontation experiment was then conducted to verify the antagonistic effects among the three cells. Figure 4 , Figure 4 The results showed that the bacterial strains did not form an inhibition zone or growth inhibition zone after they met, and the colonies were able to intertwine and grow normally after contact. Therefore, these three strains showed no antagonistic effect under the tested conditions, exhibiting good compatibility and enabling co-culture. Example 5
[0048] Verification of the nitrogen fixation performance improvement of Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3 Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3 were inoculated into 100 mL of Ashby nitrogen-free liquid medium and cultured for 48 hours. The nitrogen fixation synergistic effect (nitrogenase activity) of the compound was then measured. Figure 5 The results showed that the nitrogenase activity of the three strains was low when cultured alone, but significantly increased when cultured in pairs. The nitrogenase activity reached its highest level when all three strains were cultured together, significantly superior to all single and pair combinations. This indicates that the combination of the three strains can produce a significant synergistic effect, greatly enhancing the nitrogen-fixing function of the complex bacterial community. Example 6
[0049] Preparation and application of a compound inoculant containing Acinetobacter 1, Acinetobacter 2 and Acinetobacter 3 Pretreatment of bacterial culture: Single colonies of the isolated and purified strains Acinetobacter1, Acinetobacter2 and Acinetobacter3 were picked and inoculated into 100 mL of 50% TSB liquid medium and cultured at 28℃ and 180 rpm for 24-48 h. The OD600 of the strains was measured (the minimum value of 1.0 was used to judge the growth status of the strains). The cultured bacterial suspensions were transferred to sterile 50 mL centrifuge tubes and centrifuged at 5000 rpm. The suspensions were resuspended in sterile water and centrifuged again. This process was repeated twice to remove the culture medium. Then, sterile water was added to adjust the OD of the bacterial suspensions to approximately 0.5. The three bacterial suspensions were then diluted with sterile water at a volume ratio of 1:100 (bacterial suspension: sterile water). The diluted suspensions were then dispensed at a ratio of 1 mL:1 g (approximately 100 viable cells per gram of soil). 6 Sterilized corn planting soil (black soil to vermiculite volume ratio of 1:1) was added, and the planting soil with added bacterial solution was finally filled into small planting pots (60 ± 5g / pot). A control group without bacterial solution was set up with the same weight.
[0050] The corn seedlings were further cultivated in an indoor greenhouse (25℃, 14 h light, 10 h darkness) for 45 days, with normal watering (sterile water) throughout the process. Example 7
[0051] Effects of applying compound microbial agents on promoting growth and improving nitrogen use efficiency in maize seedlings The corn seedlings were treated with compound microbial agent and then cultivated in an indoor greenhouse (25℃, 14 h light, 10 h darkness) for 45 days. After 45 days, the corn plants were sampled.
[0052] Fresh weight weighing: Pour out the soil from the planting pot, remove the whole corn plant, and separate the roots from the surrounding soil. Weigh the whole plant for fresh weight.
[0053] Measure plant height: Use a ruler to measure the height of the above-ground part of the corn plant.
[0054] Measure the root length: Use a ruler to weigh the length of the root system of the corn plant.
[0055] Measure the diameter at ground level: Use vernier calipers to measure the diameter of the corn stalk at a point 0.5 mm from the ground.
[0056] Chlorophyll content determination: The chlorophyll content of well-developed leaves of plants was measured using a chlorophyll meter.
[0057] Determination of total nitrogen in plants: Accurately weigh 0.3000 g of finely ground sample into an Erlenmeyer flask, add 1.8 g of catalyst (selenium powder: copper sulfate: potassium sulfate = 1:10:100), add 4 mL of concentrated sulfuric acid, and digest on a hot plate until the solution is clear. After cooling, transfer to a 100 mL volumetric flask, dilute to volume with distilled water, shake well, and determine using a continuous flow chemistry analyzer (SKALAR SAN++).
[0058] Determination of total nitrogen in soil: Accurately weigh 1.0000 g of air-dried soil sample that has passed through a 0.149 mm sieve into an Erlenmeyer flask, add 1.8 g of catalyst (selenium powder: copper sulfate: potassium sulfate = 1:10:100), add 4 mL of concentrated sulfuric acid, and digest on a hot plate until the soil particles turn grayish-white, the solution is slightly greenish, and there are no black particles on the digestion solution and the flask wall. After cooling, transfer to a 100 mL volumetric flask, dilute to volume with distilled water, shake well, and determine using a continuous flow chemistry analyzer (SKALAR SAN++).
[0059] Determination of NH4-N and NO3-N in soil: Weigh 5.00 g of fresh soil sample that has passed through a 2 mm sieve into a plastic bottle, add 25 mL of 2MKCl, shake and extract for 30 minutes, remove and filter, and determine the NH4-N and NO3-N content of the filtrate using a continuous flow chemistry analyzer (SKALAR SAN++).
[0060] The effects of compound microbial agents on promoting growth and improving nitrogen use efficiency in maize seedlings are as follows: Figure 6 , Figure 7 As shown. Figure 6 , Figure 7 The results showed that compound microbial agents can help corn seedlings grow faster and improve their nitrogen use efficiency.
[0061] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, preferred embodiments are described in this invention. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of this invention, and should all be included within the protection scope of this invention.
Claims
1. A biological method for improving crop nitrogen use efficiency, characterized by: By applying a compounded and optimized biological agent, crop nitrogen use efficiency can be improved; the compound biological agent includes any of the following strains, or is composed of the following three groups of strains A, B, and C in a certain proportion: Strain A: Its name is Acinetobacter 1. Deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37047; Strain B: Its name is Acinetobacter 2. Deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37048; Strain C: Its name is Acinetobacter 3. Deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37049.
2. The biological method for improving crop nitrogen use efficiency according to claim 1, characterized in that: The method of application of the compound biological agent is selected from any of the following options: A. Seed treatment; B. Targeted application to the root; C. Soil / planting substrate improvement; D. Composting and fermentation combined with application, substrate mixing and application.
3. The biological method for improving crop nitrogen use efficiency according to claim 2, characterized in that: The seed treatments include: seed dressing; seed soaking; seed coating; and seed fertilizer dressing. The root-targeted application includes: root dipping, root irrigation, hole application, furrow application, drip irrigation root dipping, and hole irrigation with mulch. The soil improvement includes: fertigation, drip / micro-sprinkler irrigation, broadcasting and tillage, fertilizer-mixed basal application, fertilizer-mixed topdressing, and soil flooding.
4. The biological method for improving crop nitrogen use efficiency according to claim 1, characterized in that: The compound biological agent is prepared by using a 1:1:1 ratio of bacterial strains as a baseline and adjusting the amount of strains as needed.
5. The biological method for improving crop nitrogen use efficiency according to claim 1, characterized in that: The formulation method of the compound biological agent includes: ① fermenting and culturing each component strain separately to obtain the fermentation broth or cell of each strain; then taking the strain fermentation broth or cell for compounding; ② taking the three groups of strains for compound fermentation to obtain the compound biological agent.
6. The biological method for improving crop nitrogen use efficiency according to any one of claims 1-5, characterized in that: The crops include: maize; crops of the same genus as maize; grass crops that are C4 plants like maize; and grass crops.
7. Strain A for improving nitrogen use efficiency in maize: its name is Acinetobacter 1. Deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37047; its main biological characteristics are: Acinetobacter antiviralis species, Gram-negative G - The cells are rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, catalase-positive, nitrate-reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, and starch hydrolysis-positive. The colonies are grayish-white, opaque, and round.
8. B strain for improving nitrogen use efficiency in maize: its name is Acinetobacter 2. Deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37048; its main biological characteristics are: Acinetobacter antiviralis species, Gram-negative G - The cells are rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, catalase-positive, nitrate-reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, and starch hydrolysis-positive. The colony morphology is grayish-white and mucous-like.
9. C strain for improving nitrogen use efficiency in maize: its name is Acinetobacter 3. Deposited at the China General Microbiological Culture Collection Center on December 12, 2025, with accession number CGMCC No. 37049; its main biological characteristics are: Acinetobacter antiviralis species, Gram-negative G - The cells are rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, catalase-positive, nitrate-reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, and starch hydrolysis-positive. The colonies are grayish-white, opaque, and round.
10. Compound microbial agent: comprising the strains described in any one of claims 7-9 or any combination thereof.