Compound microbial inoculant for promoting nitrogen absorption and utilization of peanuts and application thereof

Abstract

The application discloses a compound microbial inoculum capable of significantly promoting nitrogen absorption and utilization of peanuts and application thereof, and belongs to the technical field of microorganisms. The active bacteria in the compound microbial inoculum are composed of Sphingomonas melonis SPZ1 CCTCC No:M20241884 and Arthrobacter oryzae ARZ1 CCTCC No:M20241883. The two kinds of bacteria promote dry matter accumulation of peanuts through synergistic effect, and the nitrogen content of the aboveground and underground parts is obviously improved, especially the nitrogen accumulation amount of the whole peanut plant can be improved, and the utilization efficiency of nitrogen of the peanuts can be greatly improved when the compound microbial inoculum is applied to the peanut planting process.

Description

Technical Field

[0001] This invention relates to a compound microbial agent that can significantly promote the absorption and utilization of nitrogen in peanuts, and more particularly to a compound microbial agent composed of Sphingomonas melonis SPZ1 and Arthrobacter oryzae and their metabolites, belonging to the field of microbial technology. Background Technology

[0002] Peanuts are an important oilseed and economic crop in my country, with both edible and oil-producing value. Their total output ranks first among oilseed crops in the country, and they play an important role in ensuring the safety of edible oil in my country.

[0003] Nitrogen is one of the essential nutrients for peanuts and a major factor limiting their growth and yield. Within a certain application range, nitrogen has a positive effect on peanut growth, promoting the development of lateral branches and roots, improving root absorption capacity, increasing photosynthetic efficiency of leaves, and promoting nutrient accumulation and translocation, thereby increasing the dry matter accumulation of various organs and promoting pod formation. However, in the context of intensive agriculture, excessive nitrogen application severely inhibits nitrogen fixation by peanut rhizobia, reduces nitrogen fertilizer utilization, causes excessive vegetative growth and premature aging, and lowers peanut yield and quality. Furthermore, the ineffective absorption and utilization of nitrogen fertilizer applied to the soil leads to its accumulation, further causing gaseous nitrogen loss, such as ammonia volatilization and nitrous oxide emissions, resulting in environmental pollution.

[0004] Therefore, improving nitrogen utilization efficiency in peanuts without increasing nitrogen fertilizer application is crucial for achieving the green and sustainable development of the peanut industry. Previous studies have primarily focused on reducing nitrogen fertilizer application, improving fertilization methods, and optimizing fertilizer properties (CN116789502A) to enhance nitrogen fertilizer utilization in peanuts, but have overlooked the important role of microorganisms in the efficient utilization of nitrogen in peanuts. Soil nitrogen turnover is mainly driven by microorganisms; therefore, screening effective microorganisms to regulate soil nitrogen turnover and enhance the absorption and utilization capacity of nitrogen in peanuts may be an important method for improving nitrogen fertilizer utilization efficiency. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a compound microbial agent that can significantly promote the absorption and utilization of nitrogen in peanuts and its application in peanut production.

[0006] To achieve the above objectives, the inventors conducted extensive experiments to investigate the effects of different bacterial strains on the dry matter accumulation, nitrogen content, nitrogen accumulation amount, and nitrogen utilization efficiency of two peanut varieties. The final result was the following technical solution: a compound microbial agent that promotes nitrogen absorption and utilization in peanuts. The active bacteria in this compound microbial agent consist of *Sphingomonas melonis* SPZ1 and *Arthrobacter oryzae* ARZ1. The strain preservation number of *Sphingomonas melonis* SPZ1 is CCTCC No: M20241884, and the strain preservation number of *Arthrobacter oryzae* ARZ1 is CCTCC No: M20241883.

[0007] It should be noted that the *Arthrobacter oryzae* ARZ1 used in this invention was isolated from peanut rhizosphere soil and grows well under pH 6.0-9.0 conditions. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) on September 2, 2024, with accession number CCTCC No: M20241883. The *Sphingomonas melonis* SPZ1 used in this invention was also isolated from peanut rhizosphere soil and grows well under pH 6.0-9.0 conditions. This strain was deposited at the CCTC on September 2, 2024, with accession number CCTCC No: M20241884.

[0008] More preferably, the compound microbial agent for promoting the absorption and utilization of nitrogen in peanuts as described above, wherein the active bacteria are composed of Sphingomonas melonis SPZ1 and Arthrobacter oryzae ARZ1 in a live bacteria ratio of 2:(1-2).

[0009] While screening strains to improve nitrogen use efficiency in peanuts, the inventors unexpectedly discovered that a compound bacterial solution containing *Sphingomonas melonis* SPZ1 and *Arthrobacter oryzae* ARZ1 significantly improved the dry matter accumulation of peanut plants and markedly increased the nitrogen content and accumulation in both the aboveground and underground parts, thereby enhancing the overall nitrogen use efficiency of the peanut plant. Based on this discovery, the inventors prepared a compound bacterial agent by culturing and fermenting these two bacteria. Therefore, this invention provides applications of the above-mentioned compound bacterial agent, namely: its application in promoting peanut dry matter accumulation; and its application in promoting nitrogen accumulation and improving nitrogen use efficiency in peanuts.

[0010] In addition, the compound microbial agent involved in this invention can be prepared by the following method: Sphingomonas melonis SPZ1 bacterial suspension cultured to the logarithmic growth phase and Arthrobacter oryzae ARZ1 bacterial suspension are mixed thoroughly, and a protectant is added to the mixed bacterial suspension to adjust the viable cell count to (1.0–2.0) × 10⁻⁶. 10 cfu / mL, pH 6-9; the protective agent consists of: 90-120 g / L skim milk powder, 25-35 mL / L glycerol, 90-120 g / L maltodextrin, 120-180 g / L laccase and 8-13 g / L L-glutamate sodium.

[0011] The fermentation culture method for *Sphingomonas melonis* SPZ1 is as follows: *Sphingomonas melonis* SPZ1 is inoculated into beef extract peptone broth at an inoculum size of 4%-10%, and cultured at 25-27℃ for 24-72 hours. After centrifugation and washing, a mixture containing 0.5 g / L yeast extract, 20 g / L mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L agar is added to each bacterial precipitate. The pH is adjusted accordingly. 6-9, and adjust the viable count of *Sphingomonas melonis* SPZ1 (a type of cucurbit) to (1.0–2.0) × 10⁻⁹. 10 CFU / mL, mix well and pour into culture.

[0012] The fermentation culture method for Arthrobacter oryzae ARZ1 is as follows: Arthrobacter oryzae ARZ1 is fermented... Arthrobacter oryzae ARZ1 was inoculated at a rate of 4%-10% into beef extract peptone broth and incubated at 25-27℃ for 24-72 hours. After centrifugation and washing, a mixture containing 0.5 g / L Yeast Extract, 20 g / L Mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L Agar was added to each bacterial precipitate. The pH was adjusted to 6-9, and the viable count of Arthrobacter oryzae ARZ1 was adjusted to (1.0-2.0) × 10⁻⁶. 10 CFU / mL, mix well and pour into culture.

[0013] In addition, the present invention also provides a planting method for promoting the absorption and utilization of nitrogen in peanuts using the above-mentioned compound microbial agent, the method comprising preparing the compound microbial agent to a total viable count of (1.0~2.0)×10⁻⁶. 10 The bacterial solution with cfu / mL was applied for the first time at the beginning of peanut flowering, at a rate of 3L / 667m³. 2 The second application is during the peanut pod-setting stage, at a rate of 3L / 667m³. 2 .

[0014] More preferably, in the planting method for promoting nitrogen absorption and utilization in peanuts as described above, if manual application is used, the solution is diluted with water and sprayed onto the peanut roots using an agricultural sprayer; if drone spraying is used, the bacterial solution is directly diluted with water and placed in a spray tank for spraying before rain.

[0015] More preferably, in the planting method for promoting nitrogen absorption and utilization in peanuts as described above, when peanuts are planted under mulch, drip irrigation with bacterial solution is used, and the irrigation depth is sufficient to moisten the soil to a depth of 8-10 cm.

[0016] More preferably, in the planting method for promoting the absorption and utilization of nitrogen in peanuts as described above, it should be noted that, regardless of the application method, the applied bacterial solution and pesticides should not be used together, and the time interval between their application should be more than 2 days.

[0017] Compared with the prior art, the active bacteria in the compound microbial agent of this invention are composed of Arthrobacter oryzae ARZ1 and Sphingomonas melonis SPZ1. The two bacteria in this compound microbial agent promote the accumulation of dry matter in peanuts through synergistic effects, and significantly increase the nitrogen content in both above-ground and underground parts, especially the nitrogen accumulation of the whole peanut plant. When applied to peanut cultivation, it can greatly improve the efficiency of nitrogen utilization in peanuts. Attached Figure Description

[0018] Figure 1 Plate culture and scanning electron microscope image of Arthrobacter oryzae ARZ1;

[0019] Figure 2 Plate culture and scanning electron microscope image of Sphingomonas melonis SPZ1;

[0020] Figure 3 Effects of different treatments on peanut dry matter accumulation;

[0021] Figure 4 Effects of different treatments on nitrogen content in peanut plants;

[0022] Figure 5 Effects of each treatment on nitrogen accumulation in peanut plants;

[0023] Figure 6 The effects of applying different strains on soil nitrogen use efficiency;

[0024] Figures 3-6 Different letters in the text represent significant differences between different treatments within the same group (p<0.05). Detailed Implementation

[0025] The following embodiments further illustrate the above-described content of the present invention in detail. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Furthermore, unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0026] Example 1: Screening and isolation of ARZ1 and SPZ1 strains

[0027] (1) Discovery, screening, isolation, and identification of bacterial strains. Based on the screening experiment of adding specific root exudates in maize-peanut intercropping, the inventors found that adding root exudates can increase the nitrogen content in peanut rhizosphere soil and stimulate nitrogen absorption by peanut plants. Therefore, we speculate that the addition of root exudates reassembles the soil microbial community, thereby promoting the absorption and utilization of soil nitrogen by peanuts. On this basis, we prepared peanut rhizosphere soil samples under the above root exudate treatment into suspensions, spread the bacterial suspension on bacterial culture medium, and isolated and purified the bacterial strains. The 16S rRNA sequence of the isolated strains was obtained using high-throughput sequencing technology. The 16S rRNA sequence was compared with the NCBI nr database. Sequence clustering analysis was performed based on the degree of comparison and similarity to construct a phylogenetic tree. The isolated and purified strains were compared with known strains, and comprehensive analysis of morphology, physiology, and biochemistry was performed to determine the bacterial species. To further verify the function of the strains, peanut cultivation addition experiments were carried out using the isolated and purified strains.

[0028] (2) Microbiological properties of the microorganisms. Arthrobacter oryzae ARZ1 colonies are white, round, and moist. Figure 1 It can grow on nitrogen-fixing medium, with an optimal growth pH of 6-9 and a temperature of 25-27℃. *Sphingomonas melonis* SPZ1 colonies are yellow, round, with small, moist, smooth, opaque mycelial growths, regular edges, and no halo. Figure 2 It can grow on nitrogen-fixing medium, with a suitable pH of 6-9 and a temperature of 25-27℃.

[0029] Example 2: Preparation of ARZ1, SPZ1 inoculants and compound inoculants

[0030] (1) Arthrobacter oryzae ARZ1 was inoculated into beef extract peptone broth (3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 15 g / L agar) at an inoculation rate of 4%-10%, pH 10. 7.4-7.6, incubate at 25-27℃ for 24-72 hours. After centrifugation and washing, add a nitrogen-fixing medium containing 0.5 g / L Yeast Extract, 20 g / L Mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L Agar to the bacterial precipitate. Adjust the pH to 6-9 and adjust the viable count of *Arthrobacter oryzae* ARZ1 to approximately 1.0 × 10⁻⁶. 10 CFU / mL, mix well and then pour culture; the specific operation method of pour culture is as follows: dilute the Arthrobacter oryzae ARZ1 suspension and add it to sterile nitrogen-fixing medium. After solidification, invert the culture and culture. A single strain will proliferate to form a colony. Take a single colony to make a suspension. Repeat the process multiple times to obtain a pure culture.

[0031] (2) Inoculate *Sphingomonas melonis* SPZ1 at a concentration of 4%-10% into beef extract peptone broth (3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 15 g / L agar), and incubate at 25-27℃ for 24-72 hours. After centrifugation and washing, add a mixture containing 0.5 g / L Yeast Extract, 20 g / L Mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L Agar to each bacterial precipitate. The pH is adjusted accordingly. 6-9, and adjust the viable count of Sphingomonas melonis SPZ1 to approximately 1.0 × 10⁻⁹. 10cfu / mL, mix well and then pour culture; the specific operation method of pour culture is as follows: dilute the suspension of Sphingomonas melonis SPZ1 and add it to sterile nitrogen-fixing medium. After solidification, invert the culture and culture. A single strain will proliferate to form a colony. Take a single colony to make a suspension. Repeat the process multiple times to obtain a pure culture.

[0032] (3) Prepare a protective agent with the following composition: 100g / L skim milk powder, 30mL / L glycerol, 100g / L maltodextrin, 150g / L sea saccharide and 10g / L L-glutamate sodium, pH 6-9.

[0033] (4) Add the protectant prepared in step (3) to the Arthrobacter plantarum ARZ1 bacterial suspension obtained in step (1) to adjust the viable count to approximately 1.0 × 10⁻⁶. 10 cfu / mL ARZ1 bacterial agent was obtained. Add the protectant prepared in step (3) to the Sphingomonas cucurbita SPZ1 bacterial suspension obtained in step (2) to adjust the viable count to approximately 1.0 × 10⁻⁶. 10 cfu / mL SPZ1 bacterial agent was obtained. .

[0034] The bacterial solutions obtained in steps (1) and (2) were mixed at a viable cell ratio of 2:(1-2) for *Sphingomonas cucurbita* SPZ1 and *Arthrobacter plantarum* ARZ1. The protective agent prepared in step (3) was then added to the mixed bacterial solution to adjust the total effective viable cell count to approximately 1.0 × 10⁻⁶. 10 cfu / mL To obtain compound microbial agents .

[0035] Example 3: Trial of ARZ1 inoculant in potted peanut cultivation

[0036] Experiment type: Pot experiment

[0037] Experimental location: Qingdao Agricultural University (Qingdao, China)

[0038] Experiment period: May 2023 - September 2023

[0039] Experimental crop: Peanuts (varieties: Qinghua 6 and Qinghua 22)

[0040] Test soil: Sterilized soil (soil pH 6.70, organic matter 11.92 g / kg, available nitrogen 99.86 mg / kg, available phosphorus 35.22 mg / kg, available potassium 89.79 mg / kg).

[0041] Experimental groups: CK was a blank aqueous solution control, ARZ1-F and ARZ1-T were both treated with Arthrobacter plantarum ARZ1 inoculum, with 3 replicates for each treatment, arranged in randomized block design.

[0042] Experimental design: PVC pots with an inner diameter of 16cm and a height of 23.5cm were used for potted cultivation. Two peanut plants were planted in each pot. Bacterial solution was added 25 days after peanut sowing, and samples were taken at harvest time after the addition of the bacterial solution.

[0043] Dosage and method: For ARZ1-F, apply 0.05 mL / pot of ARZ1 inoculant prepared in Example 2; for ARZ1-T, apply 0.1 mL / pot of ARZ1 inoculant prepared in Example 2. Dilute the inoculant to 200 mL and pour it onto the peanut roots. CK is 200 mL of blank aqueous solution.

[0044] Table 1. Dry matter accumulation of plants treated with Arthrobacter phytosporum ARZ1

[0045]

[0046] Note: Different letters indicate significant differences (p<0.05) between different treatments within the same group. The same applies below.

[0047] As shown in Table 1, the aboveground dry matter accumulation of Qinghua No. 6 was significantly increased by 22.52% and 16.28% under ARZ1-F treatment compared to CK and ARZ1-T, respectively. The underground dry matter accumulation was significantly increased by 11.90% and 25.33% under ARZ1-T treatment compared to CK and ARZ1-F, respectively. The aboveground dry matter accumulation of Qinghua No. 22 was significantly increased by 17.33% and 20.79% under ARZ1-F and ARZ1-T treatments compared to CK, respectively. However, there was no significant difference between the two concentration treatments, ARZ1-F and ARZ1-T. The underground dry matter accumulation of Qinghua No. 22 was significantly increased by 57.14% and 41.03% under ARZ1-F treatment compared to CK and ARZ1-T, respectively. In conclusion, both ARZ1-F and ARZ1-T treatments with Arthrobacter oryzae ARZ1 significantly enhanced the dry matter accumulation of peanut plants.

[0048] Example 4: Trial of SPZ1 inoculant in potted peanut cultivation

[0049] Experiment type: Pot experiment

[0050] Experimental location: Qingdao Agricultural University (Qingdao, China)

[0051] Experiment period: May 2023 - September 2023

[0052] Experimental crop: Peanuts (varieties: Qinghua 6 and Qinghua 22)

[0053] Test soil: Sterilized soil (soil pH 6.70, organic matter 11.92 g / kg, available nitrogen 99.86 mg / kg, available phosphorus 35.22 mg / kg, available potassium 89.79 mg / kg).

[0054] Experimental groups: CK was a blank aqueous solution control, SPZ1-F and SPZ1-T were both treated with SPZ1 inoculum of Sphingomonas cucurbita, with 3 replicates per treatment, arranged in randomized block design.

[0055] Experimental design: PVC pots with an inner diameter of 16 cm and a height of 23.5 cm were used for potted cultivation. Two peanut plants were planted in each pot. The bacterial strain was added 25 days after peanut sowing, and samples were taken at harvest time after the addition of the bacterial solution.

[0056] Dosage and method: SPZ1-F was treated with 0.05 mL of SPZ1 bacterial agent prepared in Example 2 per pot, and SPZ1-T was treated with 0.1 mL of SPZ1 bacterial agent prepared in Example 2 per pot. The bacterial agents were diluted to 200 mL and poured onto the peanut roots. CK was 200 mL of blank aqueous solution.

[0057] Table 2. Dry matter accumulation of plants under treatment with Sphingomonas sp. SPZ1 in cucurbitaceous organisms

[0058]

[0059] The experimental results in Table 2 show that the SPZ1-T treatment significantly improved the dry matter accumulation of both peanut varieties. Under the SPZ1-T treatment, compared with the control (CK) and SPZ1-F, the aboveground dry matter accumulation of Qinghua 6 increased significantly by 14.21% and 9.23%, respectively, and the underground dry matter accumulation increased significantly by 27.38% and 59.70%, respectively. For Qinghua 22, the aboveground dry matter accumulation increased significantly by 20.54% and 61.00%, respectively, and the underground dry matter accumulation increased significantly by 47.14% and 24.10%, respectively. In conclusion, the SPZ1-T treatment with *Sphingomonas melonis* SPZ1 can promote peanut plant growth and increase the dry matter accumulation of peanut plants.

[0060] Example 5: Trial of Compound Microbial Agent in Field Peanut Cultivation

[0061] Test location: Pingdu City, Qingdao, Shandong Province

[0062] Trial period: May 2024 - September 2024

[0063] Experimental crop: Peanuts (varieties: Qinghua 6 and Qinghua 22)

[0064] Test soil: The soil was sandy black soil (soil pH 7.06, organic matter 16.18 g / kg, available nitrogen 77.76 mg / kg, available phosphorus 45.58 mg / kg, available potassium 78.69 mg / kg).

[0065] Experimental groups (each group occupies 1 mu):

[0066] CK is a blank aqueous solution control;

[0067] The ARZ1-F group was treated with the ARZ1 bacterial agent prepared in Example 2 at a dosage of 1.5 L / 667 m³. 2 ;

[0068] The ARZ1-T group was treated with the ARZ1 bacterial agent prepared in Example 2 at a dosage of 3 L / 667 m³. 2 ;

[0069] Group SPZ1-F was treated with the SPZ1 bacterial agent prepared in Example 2 at a dosage of 1.5 L / 667 m³. 2 ;

[0070] The SPZ1-T group was treated with the SPZ1 bacterial agent prepared in Example 2 at a dosage of 3 L / 667 m³. 2 ;

[0071] The ST+AF group was treated with the compound microbial agent prepared in Example 2, which was a mixture of *Sphingomonas cucurbitae* SPZ1 and *Arthrobacter plantarum* ARZ1 in a 2:1 ratio, at a dosage of 3 L / 667 m³. 2 ;

[0072] The ST+AT group was treated with the compound microbial agent prepared in Example 2, which was a 1:1 mixture of *Sphingomonas cucurbitae* SPZ1 and *Arthrobacter plantarum* ARZ1, at a dosage of 3 L / 667 m³. 2 .

[0073] Application method: For the first application, at the initial flowering stage of peanuts, dilute the microbial agent mentioned above with water to 15L and apply manually using an agricultural sprayer to the peanut roots. For the second application, at the pod-setting stage of peanuts, use the same dosage and method as the first application. Note that the microbial agent should not be used with pesticides, and the application time for both should be at least 2 days apart.

[0074] Planting Management: Peanuts are planted using the mulching and ridging method, with a ridge spacing of 90cm, 2 rows per ridge, a small row spacing of 35cm on the ridge, and a hole spacing of 11cm (2 seeds per hole). Apply 50 kg of compound fertilizer (N-P2O5-K2O: 15-15-15) per mu, and no further topdressing is required later.

[0075] Experimental results:

[0076] 1) Effects on peanut dry matter accumulation

[0077] pass Figure 3 The experimental results showed that, compared with the CK group, the ST+AT compound microbial agent treatment significantly increased the dry matter accumulation of the aboveground peanuts of Qinghua 6 by 39.95% and the underground peanuts by 46.43%, while the treatment of Qinghua 22 significantly increased the dry matter accumulation of the aboveground peanuts by 33.66% and the underground peanuts by 85.71%. Furthermore, statistical comparisons showed that, in terms of dry matter accumulation for both peanut varieties, the ST+AT group significantly improved the accumulation compared with the SPZ1-F, SPZ1-T, ARZ1-F, and ARZ1-T groups; and the ST+AF group significantly improved the dry matter accumulation of the underground peanuts compared with the CK, SPZ1-F, SPZ1-T, ARZ1-F, and ARZ1-T groups.

[0078] 2) Effects on nitrogen content in peanut plants

[0079] like Figure 4 As shown, statistical comparisons revealed that, compared to the control group (CK) and the four single-strain groups, the ST+AT compound inoculant group significantly increased the nitrogen content in both aboveground and underground parts of the two peanut varieties, while the ST+AF compound inoculant group significantly increased the nitrogen content in the underground parts of both peanut varieties. Specifically, compared to the control group (CK), the ST+AT compound inoculant group significantly increased the nitrogen content in the aboveground parts of Qinghua 6 by 73.86% and in the underground parts by 115.50%, while the ST+AT compound inoculant group significantly increased the nitrogen content in the aboveground parts of Qinghua 22 by 48.31% and in the underground parts by 83.89%.

[0080] 3) Effects on nitrogen accumulation in peanut plants

[0081] like Figure 5As shown, statistical comparison revealed that, compared to the control (CK) and the four single-strain groups, the combined microbial agents ST+AF and ST+AT significantly increased nitrogen accumulation in both aboveground and underground parts of the two peanut varieties. Specifically, compared to the CK treatment group, the combined microbial agent ST+AT treatment group significantly increased nitrogen accumulation in the aboveground and underground parts of Qinghua 6 by 143.32% and 217.81%, respectively, and in Qinghua 22 by 93.11% and 239.49%, respectively. In the combined microbial agent ST+AF treatment group, the nitrogen accumulation in the aboveground and underground parts of Qinghua 6 significantly increased by 130.13% and 212.36%, respectively, and in Qinghua 22 by 78.55% and 231.53%, respectively.

[0082] 4) Impact on soil nitrogen use efficiency

[0083] pass Figure 6 The comparative results of the experiments showed that, compared with the control (CK) and each single strain group, the ST+AT compound microbial agent group significantly reduced the dependence of peanut plants on soil nitrogen, and significantly increased the nitrogen contribution rate, nitrogen fertilizer agronomic efficiency, and nitrogen fertilizer partial productivity. The ST+AF compound microbial agent group significantly reduced the dependence of peanut plants on soil nitrogen and significantly increased nitrogen fertilizer partial productivity. Specifically, compared with the CK group, under the ST+AT compound microbial agent treatment, the soil nitrogen dependence rate of Qinghua 6 and Qinghua 22 was significantly reduced by 2.83 and 2.38 percentage points, respectively; the nitrogen contribution rate was significantly increased by 54.54% and 42.00%, respectively; the nitrogen fertilizer agronomic efficiency was significantly increased by 134.73% and 60.39%, respectively; and the nitrogen fertilizer partial productivity was significantly increased by 233 and 173 percentage points, respectively.

[0084] In summary, statistical comparisons showed that the ST+AT compound microbial agent treatment significantly improved nitrogen absorption and utilization indicators in peanuts compared to the single strain and the blank control. The ST+AF compound microbial agent also significantly improved nitrogen fertilizer partial productivity, nitrogen accumulation, and the accumulation of dry matter and nitrogen content in underground parts. This indicates that the compound microbial agent prepared by mixing *Sphingomonas melonis* SPZ1 and *Arthrobacter oryzae* ARZ1 at a ratio of 2:(1-2) (with a total effective viable count of (1.0–2.0) × 10⁻⁶) effectively enhances these indicators. 10 cfu / mL), at 3L / 667m 2 Applying the appropriate amount of nitrogen at the initial flowering stage and the pod-setting stage of peanuts can significantly promote the accumulation of dry matter in peanut plants, increase the nitrogen content, nitrogen accumulation, and nitrogen use efficiency, and improve the absorption and utilization of nitrogen in peanut plants.

Claims

1. A complex microbial agent for promoting nitrogen absorption and utilization of peanuts, characterized in that, The active bacteria in this compound microbial agent consist of Sphingomonas cucurbita (… Sphingomonas melonis SPZ1 and Plantar Arthrobacter Arthrobacter oryzae ARZ1 composition, the aforementioned Sphingosine monocytogenes of Cucurbita ( Sphingomonas melonis The strain preservation number of SPZ1 is CCTCC No: M20241884, and the described plant arthrobacter is... Arthrobacter oryzae The strain preservation number of ARZ1 is CCTCC No:M20241883.

2. The compound microbial inoculant for promoting nitrogen absorption and utilization of peanuts according to claim 1, characterized in that, The active bacteria in the complex microbial agent are composed of guava sphingomonas (Sphingomonas nigrifum) Sphingomonas melonis ) SPZ1 and plant arthrobacter (Arthrobacter Arthrobacter oryzae ARZ1 in the ratio of 2: (1-2) of the number of active bacteria.

3. The application of the complex microbial agent of claim 1 or 2 in promoting peanut dry matter accumulation.

4. The application of the complex microbial agent of claim 1 or 2 in promoting peanut nitrogen accumulation.

5. The application of the complex microbial agent of claim 1 or 2 in improving nitrogen use efficiency.

6. A method for preparing the complex microbial agent according to claim 1 or 2, characterized by, The method comprises the following steps: mixing Sphingomonas melonis (SPZ1) bacterial solution and plant Arthrobacter ARZ1 bacterial solution which are cultured to logarithmic growth phase, and adding a protective agent into the mixed bacterial solution, adjusting the viable count to (1.0-2.0)×10 10 cfu / mL, pH 6-9; the protective agent comprises 90-120 g / L skimmed milk powder, 25-35 mL / L glycerol, 90-120 g / L malt dextrin, 120-180 g / L honey locust sugar and 8-13 g / L L-glutamic acid sodium. Sphingomonas melonis Arthrobacter oryzae ​​ 7. A planting method for promoting nitrogen absorption and utilization of peanuts by using the complex microbial inoculant according to claim 2, characterized in that, The method comprises formulating the complex microbial agent into a bacterial solution with a total number of viable bacteria of (1.0-2.0)×10 10 cfu / mL, applying the complex microbial agent to peanuts at the flowering stage for the first time, with a dosage of 2.5-3.5 L / 667 m 2 ; and applying the complex microbial agent to peanuts at the podding stage for the second time, with a dosage of 2.5-3.5 L / 667 m 2 .

8. The method for promoting nitrogen uptake and utilization of peanut according to claim 7, wherein, If artificial application is adopted, after dilution with clean water, the peanut roots are sprayed with an agricultural sprayer; if unmanned aerial vehicle spraying is used, the microbial liquid diluted with clean water is directly placed in a pesticide tank and sprayed before rain.

9. The method for promoting nitrogen uptake and utilization of peanut according to claim 7, wherein, When the peanut is planted in a film mulching mode, the microbial liquid is drip irrigated under the film, and the irrigation depth is 8-10 cm to wet the soil.

10. The method for promoting nitrogen uptake and utilization of peanut according to any one of claims 7-9, wherein, The microbial liquid cannot be used together with pesticides, and the interval between the use of the two is more than 2 days.

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