A method and bacterial agent for regulating soil nitrogen transformation to promote efficient utilization of nitrogen in flue-cured tobacco based on LL-8 strain

Abstract

This invention belongs to the field of microbial agent application technology, and particularly relates to a method and agent for promoting efficient nitrogen utilization in flue-cured tobacco by regulating soil nitrogen transformation based on the LL-8 strain. The agent is prepared by inoculating the LL-8 strain into a culture medium. The preservation information of the LL-8 strain is as follows: Preservation unit: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Preservation date: January 28, 2026; Preservation number: CGMCC No. 37560. This invention, through the LL-8 strain and its corresponding application method, precisely addresses the core problems of ammonium nitrogen accumulation and nitrate nitrogen deficiency in dryland tobacco-growing soils, as well as the lack of specificity and unstable nitrogen regulation effect of ordinary microbial agents, thereby achieving efficient nitrogen utilization in flue-cured tobacco.

Description

Technical Field

[0001] This invention belongs to the field of microbial agent application technology, and in particular relates to a method and agent for regulating soil nitrogen transformation and promoting efficient nitrogen utilization in flue-cured tobacco based on the LL-8 strain. Background Technology

[0002] flue-cured tobacco ( Nicotiana tabacum L. Nitrogenous nitrogen (NO3) is an important economic crop in my country. Its growth, development, and quality formation are highly dependent on nitrogen nutrition. As a typical nitrate-loving crop, nitrate nitrogen (NO3) is crucial for its growth. - Ammonium nitrogen (NH4+) is a key nitrogen source supporting improved photosynthetic efficiency, dry matter accumulation, and the formation of high-quality tobacco leaves. Soil nitrogen forms and supply levels directly determine the nitrogen use efficiency of flue-cured tobacco, with ammonium nitrogen (NH4+) being the most abundant inorganic nitrogen. + The balance between ammonium nitrogen (NH3) and nitrate nitrogen is a core factor affecting the growth of flue-cured tobacco. In the dryland soils widely distributed in tobacco-growing areas of my country, due to natural conditions such as the adsorption characteristics of soil clay particles and the water-limited activity of nitrifying bacteria, nitrogen forms have long been dominated by ammonium nitrogen, with nitrate nitrogen content only 1 / 1.5 to 1 / 2.3 of that of ammonium nitrogen. Furthermore, nitrate nitrogen is easily lost through water leaching or denitrification, leading to a continuous shortage of nitrate nitrogen required for flue-cured tobacco growth, becoming a key bottleneck restricting the improvement of flue-cured tobacco yield and quality. Microorganisms, as the core drivers of soil nitrogen transformation, can mediate the conversion of ammonium nitrogen to nitrate nitrogen through ammonification and nitrification, providing a feasible path for regulating soil nitrogen forms and adapting to the nitrate-loving characteristics of flue-cured tobacco. This has become a research hotspot in the agricultural field for improving crop nitrogen nutrition.

[0003] In existing technologies, to alleviate the problem of insufficient nitrate nitrogen supply in flue-cured tobacco, the main methods are to increase the application of chemical nitrate nitrogen fertilizers or apply ordinary microbial agents. However, excessive application of chemical nitrate nitrogen fertilizers can easily lead to environmental problems such as nitrogen leaching and soil acidification, and cannot fundamentally improve the soil's own nitrogen conversion capacity. Ordinary microbial agents are mostly complex microbial communities with a lack of functional specificity. They have not been targeted at the core requirement of "ammonium nitrogen to nitrate nitrogen conversion," resulting in unstable regulation of soil nitrogen forms and difficulty in accurately matching the nitrogen requirements of flue-cured tobacco. At the same time, existing agent application technologies have not clarified the synergistic mechanism between strains and the rhizosphere microecology and plant nitrogen metabolism pathways of flue-cured tobacco. Some agents may even compete with flue-cured tobacco for nitrogen sources or disrupt the original balance of the soil microbial community. This not only fails to effectively improve the nitrogen use efficiency of flue-cured tobacco, but may also lead to an imbalance in nitrogen distribution among the roots, stems, and leaves of flue-cured tobacco, further restricting the improvement of flue-cured tobacco yield and quality. In summary, existing technologies are insufficient to resolve the core contradiction between the accumulation of ammonium nitrogen and the scarcity of nitrate nitrogen in dryland tobacco-growing soils, and cannot achieve the goal of efficient nitrogen utilization and simultaneous improvement in yield and quality of flue-cured tobacco.

[0004] Therefore, there is an urgent need for a microbial strain with specific ammonia conversion function and supporting application technology. This technology should be able to directionally drive the conversion of ammonium nitrogen to nitrate nitrogen in dryland tobacco-growing soil, accurately supplementing the nitrate nitrogen required for flue-cured tobacco growth; at the same time, it should be able to optimize the rhizosphere microbial community structure, activate the nitrogen metabolism pathway of flue-cured tobacco roots, and synergistically improve the soil nitrogen supply capacity and the nitrogen absorption and assimilation efficiency of flue-cured tobacco, fundamentally solving the dilemma of insufficient nitrate nitrogen supply and low nitrogen utilization efficiency in existing technologies, and providing a green and efficient nitrogen nutrition regulation solution for high-quality and high-yield flue-cured tobacco. Summary of the Invention

[0005] The purpose of this invention is to provide a method and microbial agent based on LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco, so as to solve the above-mentioned problems.

[0006] To achieve the above objectives, the present invention provides the following solution: A microbial agent for regulating soil nitrogen transformation and promoting efficient nitrogen utilization in flue-cured tobacco based on strain LL-8, comprising: the microbial agent is prepared by inoculating strain LL-8 in a culture medium, and the preservation information of strain LL-8 is as follows: Name of depositary institution: China General Microbiological Culture Collection Center (CGMCC); Address of depositary institution: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Date of deposit: January 28, 2026; Accession number: CGMCC No. 37560; Classification name: Priestia sp.

[0007] Preferably, the inoculation method for the LL-8 strain is as follows: S1. Seed culture: A single colony of strain LL-8 was inoculated into 10 mL of beef extract peptone culture medium and cultured for 24 h to obtain seed culture; S2. Expanded culture: Take 1 mL of the seed culture and add it to 100 mL of beef extract peptone culture medium. Expand the culture by 1% inoculum for 24 h. S3. Centrifugation and washing: Centrifuge the fermentation broth after expansion culture at 4000 rpm for 10 min, discard the supernatant, and wash the precipitate twice with an equal volume of sterile water. After each wash, centrifuge at 4000 rpm for 10 min and retain the precipitate. S4. Preparation of bacterial agent: The washed precipitate is suspended in an equal volume of sterile water to obtain the LL-8 bacterial agent, with a concentration of 10. 8 cfu / mL.

[0008] A method for promoting efficient nitrogen utilization in flue-cured tobacco by using the microbial agent based on the LL-8 strain to regulate soil nitrogen transformation involves applying the LL-8 microbial agent at an inoculum rate of 1% to flue-cured tobacco, thereby promoting efficient nitrogen utilization in flue-cured tobacco by regulating soil nitrogen transformation and nitrogen metabolism pathways.

[0009] Preferably, the application is soil-based root irrigation, which includes the following steps: after transplanting flue-cured tobacco seedlings that are 40-50 days old and have uniform growth, the LL-8 microbial agent is applied to the root zone of each flue-cured tobacco plant at the time of transplanting and 25-35 days after transplanting.

[0010] Preferably, the dosage of LL-8 bacteria per plant per irrigation is 20 mL. Before transplanting, the soil should be mixed with tobacco base fertilizer. After transplanting, tobacco top dressing should be applied on days 22 and 37. During the growth period of flue-cured tobacco, the soil field water holding capacity should be maintained at about 70%.

[0011] Preferably, the N-P2O5-K2O of the tobacco base fertilizer is 10-10-25, and the application rate is 91 grams per 10 kilograms of soil. The N-P2O5-K2O ratio of the tobacco topdressing is 15-0-30, and the application rate is 45.5 grams per 10 kilograms of soil.

[0012] Preferably, the application is hydroponic root soaking application, which includes the following steps: rinsing the roots of flue-cured tobacco seedlings that are 40-50 days old and have uniform growth twice with sterile water, then soaking the roots in Hogland nutrient solution containing bacteria for 1 hour, and then fixing and culturing. The Hogland nutrient solution containing bacteria is prepared by adding 3 mL of LL-8 bacterial agent to Hogland nutrient solution at a 1% inoculation rate and mixing them evenly.

[0013] Preferably, the ammonium-nitrate ratio of the bacterium-containing Hogland nutrient solution is 25:75.

[0014] Preferably, when soaking roots in hydroponics, the initial concentration of the Hogland nutrient solution containing bacteria is 1 / 2 of the original concentration. This concentration is maintained for the first week, and then replaced with the full concentration of the Hogland nutrient solution containing bacteria for the next two weeks. The nutrient solution is changed every 3 days, and the roots are exposed to air for 0.5 hours every day. The pH of the nutrient solution is adjusted to 6.0 every 2 days.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: In this invention, the LL-8 strain and its corresponding application method precisely address the core issues of ammonium nitrogen accumulation and nitrate nitrogen deficiency in dryland tobacco-growing soils, as well as the lack of specificity and unstable nitrogen regulation effects of ordinary microbial agents, thereby achieving efficient nitrogen utilization in flue-cured tobacco. Targeted optimization of soil nitrogen forms to match the nitrate-loving requirements of flue-cured tobacco significantly enhances the activity of nitrate reductase and nitrite reductase, efficiently drives the conversion of ammonium nitrogen to nitrate nitrogen, and improves the content of total nitrogen and alkaline available nitrogen, avoiding the environmental risks caused by excessive application of chemical nitrogen fertilizers; It specifically regulates the rhizosphere microecology, enriches nitrogen cycle-related functional bacteria, maintains the balance of microbial community diversity and evenness, and enhances the stability of soil nitrogen transformation, unlike ordinary bacterial agents that damage the original soil microbial community. It promotes root development in flue-cured tobacco, increases root length, root surface area and number of root tips, enhances the content of key organic acids in root exudates, and strengthens nitrogen absorption capacity; at the same time, it activates nitrogen metabolism-related genes, optimizes the distribution of nitrogen in roots, stems and leaves, significantly improves agronomic traits and biomass of flue-cured tobacco, and achieves efficient accumulation and utilization of nitrogen. Attached Figure Description

[0016] 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 described 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. Figure 1 The impact of ammonia-converting bacteria on soil nitrogen content; among them, Figure 1 In the table, A represents the total nitrogen content of soil in different experimental groups. Figure 1 B in the table represents the alkaline hydrolysate nitrogen content of different experimental groups. Figure 1 C in the table represents the ammonium nitrogen content of different experimental groups. Figure 1 D in the table represents the nitrate nitrogen content of different experimental groups. Figure 1 In the table, E represents the microbial biomass nitrogen content of different experimental groups. Figure 1 F in the table represents the total soluble nitrogen content of different experimental groups.

[0017] Figure 2 The effects of ammonia-converting bacteria on soil enzyme activity; among them, Figure 2 In the table, A represents the nitrate reductase content of different experimental groups. Figure 2 B in the table represents the nitrite reductase content of different experimental groups. Figure 2 The C in the table represents the dehydrogenase content of different experimental groups. Figure 2 D in the table represents the hydroxylamine reductase content of different experimental groups. Figure 2 E in the table represents the urease content of different experimental groups; Figure 3 The relative abundance at the phylum and genus levels, where, Figure 3 In this context, A represents the relative abundance at the bacterial phylum level. Figure 3 In this context, B represents the relative abundance at the bacterial genus level. Figure 4 Cluster analysis of ammonia-converting bacteria at the phylum and genus levels in soil bacteria, among which, Figure 4 In this context, A represents cluster analysis at the phylum level of soil bacteria. Figure 4 In this context, B represents cluster analysis at the genus level of soil bacteria. Figure 5 The impact of ammonia-converting bacteria on soil microbial α-diversity was analyzed; among them, Figure 5 Table A in the table shows the species richness correlation index (Chao1) for groups CK and LL-8. Figure 5 B in the table represents the Shannon diversity index for the CK and LL-8 groups. Figure 5 The table below shows the species richness correlation index (ACE) for groups CK and LL-8. Figure 5 The table below shows the phylogenetic diversity index (Faith_pd) for the CK and LL-8 groups. Figure 5 E in the table represents the number of observable species (Observed_features) in the CK and LL-8 groups. Figure 5 The table below shows the community evenness index (Enspie) for the CK group and the LL-8 group.

[0018] Figure 6 Analysis of soil bacterial community β diversity; Figure 7 Differential species analysis of rhizosphere microorganisms under LL-8 treatment; Figure 8 The effects of ammonia-converting bacteria on the root morphology of flue-cured tobacco; among them, Figure 8 In the table, A represents the root length statistics for groups CK and LL-8. Figure 8 B in the table represents the root tip count statistics for the CK and LL-8 groups. Figure 8 C in the table represents the root diameter statistics for groups CK and LL-8. Figure 8 D in the table represents the root surface area statistics for groups CK and LL-8. Figure 8 E in the table represents the root volume statistics for groups CK and LL-8. Figure 9 The effect of the ammonia-converting bacterium LL-8 on the clustering of nitrogen metabolism functional genes; Figure 10 The effect of ammonia-transforming functional bacterium LL-8 on intergroup gene clustering differences - Principal component analysis (PCA); Figure 11 Volcano plot showing differential expression of nitrogen metabolism genes between the LL-8 treatment and CK groups; Figure 12 The number of differentially expressed genes upregulated and downregulated in the LL-8 ammonia-converting bacterium treatment group compared to the CK group; Figure 13 The enrichment of differentially expressed genes in nitrogen metabolism in GO entries; Figure 14 Enrichment graph of module GO entries; Figure 15 This is a diagram of the LL-8 nitrogen metabolism pathway. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Example 1: This embodiment provides a method for preparing LL-8 bacterial agent on a small-scale laboratory basis, specifically including: S1. Preparation of test strains: The LL-8 strain was obtained from the high-temperature material of aerobic high-temperature composting, using ammonia-oxidizing bacteria enrichment and separation medium (both with ammonium sulfate as nitrogen source) and screened at 50°C. This strain has the ability to reproduce using ammonium nitrogen as a nitrogen source. S2. Seed culture: A single colony of strain LL-8 was inoculated into 10 mL of beef extract peptone culture medium and cultured for 24 h to obtain seed culture; S3. Expanded culture: Take 1 mL of the above seed culture and add it to 100 mL of beef extract peptone culture medium for expanded culture for 24 h (inoculum amount 1%). S4. Centrifugation and washing: Centrifuge the fermentation broth after expansion culture at 4000 rpm for 10 min, discard the supernatant, and wash the precipitate twice with an equal volume of sterile water. After each wash, centrifuge at 4000 rpm for 10 min and retain the precipitate. S5. Preparation of bacterial agent: The washed precipitate was suspended in an equal volume of sterile water to obtain the test LL-8 bacterial agent, the concentration of which was 10. 8 cfu / mL, ready for use.

[0022] Example 2: Hydroponic application method of LL-8 inoculant during the seedling stage of flue-cured tobacco This embodiment provides a method for hydroponic application of LL-8 inoculant during the seedling stage of flue-cured tobacco, specifically including: Experimental material preparation: The tested flue-cured tobacco variety was Yunyan 87, and seedlings of approximately 45 days age and uniform growth were selected; the tested inoculant was LL-8 inoculant prepared in Example 1, with a concentration of 10. 8 cfu / mL; the hydroponic nutrient solution used was a modified Hoagland nutrient solution with an ammonium-nitrate ratio of 25:75. Its formula was: N 140 mg / L (composed of (NH4)2SO4 165 mg / L and Ca(NO3)2... 4H2O885 mg / L (provided), P 40 mg / L (KH2PO4175.48 mg / L), K 200 mg / L (K2SO4333.9 mg / L), Mg 40mg / L (MgSO4 7H2O 410 mg / L), and trace elements such as Mn, Mo, Cu, Zn, B, and Fe (the specific content is as per the formula in Table 2.1); Container sterilization: Use 300mL plastic cups as hydroponic containers and sterilize them with ultraviolet light for 0.5 hours before use; Nutrient solution preparation and bacterial agent addition: Pour 297 mL of 1 / 2 concentration of modified Hogland nutrient solution into a sterilized plastic cup, add 3 mL of LL-8 bacterial agent at a 1% inoculation rate, and mix well; Tobacco seedling treatment and transplanting: Rinse the roots of the tobacco seedlings twice with sterile water, then soak the roots in the above-mentioned bacterial nutrient solution for 1 hour, and then fix them on a hard plastic cover with a sponge. Cultivate 1 tobacco seedling per cup. Hydroponic management: After transplanting, lift the cover to expose the roots to air for 0.5 hours every day; adjust the pH of the nutrient solution to 6.0 every 2 days using 0.1mol / L NaOH or 0.1mol / L HCl; maintain a 1 / 2 concentration of nutrient solution for the first week, and then replace it with the full concentration of the modified Hoagland nutrient solution for the next 2 weeks, changing the nutrient solution every 3 days.

[0023] Example 3: Application method of LL-8 inoculant in soil cultivation of flue-cured tobacco in pots in the field. This embodiment provides a method for applying LL-8 inoculant to potted flue-cured tobacco in soil cultivation, specifically including: Preparation of experimental materials: The test soil was yellow soil, collected in Huaxi District, Guiyang City, Guizhou Province (106°38′47.16″E, 26°27′37.54″N). Its basic physicochemical properties were as follows: clay loam texture, pH 6.48, organic matter 26.36 g / kg, available nitrogen 43.52 mg / kg, available phosphorus 23.46 mg / kg, available potassium 172.44 mg / kg, total nitrogen 1.53 g / kg, total phosphorus 1.21 g / kg, and total potassium 12.15 g / kg. The tested flue-cured tobacco variety was Yunyan 87, and strong seedlings were cultivated using floating seedling raising. Seedlings with uniform growth and an age of about 45 days were selected. The tested inoculum was LL-8 inoculum prepared in Example 1, with a concentration of 10. 8 cfu / mL; the fertilizers were tobacco-specific base fertilizer (N-P2O5-K2O 10-10-25) and tobacco-specific top dressing (N-P2O5-K2O 15-0-30). Disinfection and filling of pots: Plastic pots (upper radius 35cm, lower radius 20cm, height 26.3cm) were disinfected with ultraviolet light for 0.5h before use; the test soil was air-dried, large pieces were broken up and mixed evenly, and then mixed with tobacco-specific base fertilizer at a rate of 91g per pot before filling the pots with 10kg of soil per pot. Transplanting tobacco seedlings: Carefully wash the roots of the tobacco seedlings with clean water, then rinse them twice with sterile water, and transplant them into the potting soil described above, one seedling per pot; Inoculant application: Apply 20 mL of LL-8 inoculant (1% inoculum) to the root zone of each tobacco seedling at the time of transplanting and 30 days after transplanting. Field management: Topdressing should be applied twice in equal amounts, at a rate of 45.5g per pot, 22 days and 37 days after transplanting, respectively; pots should be arranged with a row spacing of 110 cm × 55 cm, and the soil field water holding capacity should be maintained at around 70% until the tobacco matures.

[0024] Experiment 1: Regulatory effect of LL-8 strain on nitrogen components and key nitrogen transformation enzyme activities in tobacco-growing soil S1. Preparation of test materials The tested soil was yellow soil, collected in Huaxi District, Guiyang City, Guizhou Province (106°38′47.16″E, 26°27′37.54″N). Its basic physicochemical properties were as follows: texture clay loam, pH 6.48, organic matter 26.36 g / kg, available nitrogen 43.52 mg / kg, available phosphorus 23.46 mg / kg, available potassium 172.44 mg / kg, total nitrogen 1.53 g / kg, total phosphorus 1.21 g / kg, and total potassium 12.15 g / kg. The tested flue-cured tobacco variety was Yunyan 87. Strong seedlings were cultivated using floating seedling raising, and seedlings with a seedling age of about 45 days and uniform growth were selected. Test bacterial agent: LL-8 bacterial agent prepared in Example 1, with a concentration of 10. 8 cfu / mL; Control reagent: Sterile water (equal volume to the bacterial agent); Fertilizers: Tobacco-specific base fertilizer (N-P2O5-K2O 10-10-25) and tobacco-specific top dressing (N-P2O5-K2O 15-0-30).

[0025] S2, Experimental group culture This experiment consisted of two treatment groups, each with three biological replicates, arranged in a randomized block design. The specific groupings are as follows: Control group (CK): The application method for potted soil cultivation in Example 3 was followed, except that the LL-8 inoculant was replaced with an equal volume of sterile water, and 20 mL was applied to the roots at the time of transplanting and 30 days after transplanting. LL-8 treatment group: The application method for potted soil cultivation in Example 3 was strictly followed, and the LL-8 inoculant prepared in Example 1 was applied.

[0026] The pot specifications, soil volume, fertilizer application, and field management measures (row spacing, soil field water holding capacity, etc.) of the two groups were completely identical until the tobacco matured.

[0027] S3. Soil sample collection and testing Fresh soil samples were collected during the ripening period of flue-cured tobacco. After being sieved through a 10-mesh sieve, the samples were divided into three portions. One portion was stored in a 4℃ refrigerator, another portion was stored in a -80℃ refrigerator, and the remaining portion was air-dried and then sieved through 20-mesh and 100-mesh sieves before storage. These samples were used for the determination of soil nitrogen components and enzyme activities.

[0028] Soil nitrogen component determination: Total nitrogen (TN) was determined by sulfuric acid and hydrogen peroxide digestion-Kjeldahl method; Soil ammonium nitrogen (NH4+) was determined. + Soil nitrate nitrogen (NO3-N) was determined using the indophenol blue colorimetric method; - Soil nitrogen (N) was determined by ultraviolet spectrophotometry; alkaline available nitrogen (AN) was determined by alkaline diffusion method; total soluble nitrogen (TSN) was determined by potassium persulfate oxidation-ultraviolet spectrophotometry (GB 11894-89); soil microbial biomass nitrogen (MBN) was determined by fumigation extraction-ninhydrin colorimetric method. Determination of key enzyme activities in soil nitrogen transformation: Soil nitrate reductase (NR) activity was determined by the sulfanilamide colorimetric method; soil nitrite reductase (NiR) activity was determined by the α-naphthylamine-p-aminobenzenesulfonic acid colorimetric method; soil dehydrogenase (S-DHA) activity was determined by the TTC spectrophotometric method; soil hydroxylamine reductase (S-HR) activity was determined by the ferric ammonium sulfate-o-phenanthroline method; and soil urease (UA) activity was determined by the indophenol blue colorimetric method.

[0029] Experimental results: Compared with the control group (CK), the soil nitrogen composition of the LL-8 treatment group showed significantly optimized characteristics (see...). Figure 1 ): Total nitrogen content: The LL-8 treatment group reached 1.99 g / kg, which was significantly increased by 32.7% compared with the CK, demonstrating the unique advantage of the LL-8 strain in promoting soil total nitrogen accumulation; Alkaline nitrogen content: The LL-8 treatment group was 59.10 mg / kg, which was 36.8% higher than the CK group and significantly higher than the CK group, indicating that it has a prominent ability to activate soil nitrogen. Nitrate nitrogen content: The LL-8 treatment group had a content of 54.32 mg / kg, which was 37.5% higher than that of the control group, confirming that the LL-8 strain can efficiently drive the conversion of ammonium nitrogen to nitrate nitrogen, matching the nitrate-loving requirements of flue-cured tobacco. Ammonium nitrogen content: The coefficient of variation among the groups was only 3.2%, and the increase in the LL-8 treatment group compared with the CK was less than 7.5%, which was not significant. This indicates that the main regulatory direction was the conversion of ammonium nitrogen to nitrate nitrogen, rather than simply increasing the accumulation of ammonium nitrogen. Microbial biomass nitrogen content: LL-8 treatment group was 6.76 mg / kg, an increase of 57.2% compared to CK; Total soluble nitrogen content: The LL-8 treatment group reached 12.74 mg / L, which was 17.6% higher than the CK group and significantly higher than the CK group, indicating that it has a positive regulatory effect on the migration and transformation of dissolved nitrogen.

[0030] The LL-8 treatment group significantly enhanced the activity of key enzymes for soil nitrogen transformation, as shown in the following results. Figure 2 As shown: Nitrate reductase activity: significantly increased by 73.50% compared to CK, providing key enzymatic support for nitrate nitrogen production; Nitrite reductase activity: significantly increased by 69.23% compared to CK, and synergistically enhanced with nitrate reductase activity, promoting the efficient operation of the nitrogen conversion pathway; Dehydrogenase activity: significantly increased by 12.42% compared with CK, indicating that LL-8 strain can enhance soil microbial metabolic activity and form a positive feedback loop for nitrogen transformation; Hydroxylamine reductase activity: significantly increased by 13.16% compared to CK, further enhancing nitrogen conversion efficiency; Urease activity was significantly increased by 37.42% compared to the control (CK), and was significantly correlated with the increase in microbial biomass nitrogen, promoting the release and activation of soil nitrogen.

[0031] In summary, strain LL-8 significantly enhances the activity of key enzymes in soil nitrogen transformation, efficiently drives the conversion of soil ammonium nitrogen to nitrate nitrogen, and optimizes the content of soil components such as total nitrogen, available nitrogen, and soluble total nitrogen, providing sufficient and suitable nitrogen supply for flue-cured tobacco, thus verifying its core role in soil nitrogen regulation.

[0032] Experiment 2: Effect of LL-8 strain on the rhizosphere microbial community structure of flue-cured tobacco The experimental materials, group design, and culture management methods in this experiment are completely identical to those in Experiment 1, with the only difference being: S3. Sample Collection and Measurement Rhizosphere soil samples were collected during the ripening period of flue-cured tobacco. The specific method was as follows: the roots of the tobacco seedlings were carefully dug out, the loose soil attached to them was gently shaken off, and the soil close to the root surface (≤2mm) was collected as rhizosphere soil samples. After being sieved through a 10-mesh sieve, the samples were quickly placed in a freezer at -80℃ for storage and used for the determination of microbial community structure.

[0033] The structure of the rhizosphere soil microbial community was determined using 16S rRNA high-throughput sequencing. The specific steps are as follows: Total DNA extraction from soil microorganisms: Total DNA was extracted from rhizosphere soil samples using a kit, and the purity and integrity of the DNA were tested. Amplification and sequencing: Primers were designed to target the V3-V4 variable region of the bacterial 16S rRNA gene for PCR amplification. After purification and quantification, the amplicon was sequenced in high throughput on the Illumina MiSeq platform (Illumina, San Diego, USA). Data analysis: The raw sequencing data is subjected to quality control, splicing, and redundancy removal to obtain operable taxa (OTUs). Microbial community composition, diversity, and species differences are analyzed based on the OTUs.

[0034] Experimental results: Figure 3 The relative abundance at the phylum (A) and genus (B) levels showed significant differences in rhizosphere microbial community composition between the LL-8 treatment group and the control group (CK): At the phylum level: the dominant phyla in both groups were Pseudomonadota, Bacteroidota, Patescibacteria, Actinomycetota, and Acidobacteriota, but their relative abundances differed significantly. The abundances of Pseudomonadota, Bacteroidota, Acidobacteriota, Gemmatimonadota, and Thermoproteota in the LL-8 treatment group were significantly higher than those in the CK group, while the abundances of Patescibacteria, Actinomycetota, and Chloroflexi in the CK group were significantly higher than those in the LL-8 treatment group, indicating that the LL-8 strain altered the distribution pattern of saprophytic bacterial communities. At the genus level: the proportion of unclassified sequences was higher in the CK group, and Chujaibacter , Arachidicoccus (Related to the degradation of plant residues) Reyranella The relative abundance of genera such as (oligotrophic bacteria) was significantly higher in the LL-8 treatment group than in the LL-8 treatment group; in the LL-8 treatment group Sphingomonas (Typical beneficial growth-promoting bacteria, associated with the degradation of organic matter and stress resistance) Flavisolibacter (Participating in the carbon cycle) Lysobacter (Typical biocontrol bacteria) Devosia (Participates in nitrogen fixation and pesticide degradation) Gemmatimonas (Participates in the phosphorus cycle and photosynthesis) Rhodopseudomonas The relative abundance of genera such as photosynthetic bacteria (involved in nitrogen and carbon fixation) was significantly enriched, while the proportion of unclassified bacteria decreased, indicating that the LL-8 treatment targeted and improved the rhizosphere environment, constructing a rhizosphere microbial community with disease resistance, growth promotion, and efficient nutrient cycling as its core functions.

[0035] Figure 4 Cluster analysis of ammonia-converting bacteria at the phylum (A) and genus (B) levels in soil bacteria showed that the microbial community structure of the LL-8 treatment group and the CK group was significantly different: Phylogenetic clustering heatmap: The abundance of Proteobacteria in the LL-8 treatment group remained at a high level, the abundance of Chloroflexi increased significantly, while the abundance of Actinobacteria and Acidobacteria underwent adaptive changes, indicating that the LL-8 strain optimized the community structure by activating nitrogen metabolism pathways; Horizontal clustering heatmap: LL-8 processing group Mesorhizobium , Rhodanobacter , ReyranellaThe abundance of certain genera was significantly enriched, and these genera are closely related to nitrification and nitrate uptake. In the CK group, the abundance of some indigenous genera was even higher, confirming that the LL-8 strain was directionally enriched with nitrogen cycle-related functional genera.

[0036] α Diversity analysis: Figure 5 The study of the effects of ammonia-converting bacteria on soil microbial α-diversity showed that, compared with the control group, the LL-8 treatment group had significantly higher species diversity (Shannon index) and phylogenetic diversity (Faith_pd index), indicating that the LL-8 strain can maintain the balance between microbial community diversity and evenness and improve the balance of resource allocation. The Chao1 index, ACE index, and Observed_features index of the LL-8 treatment group were significantly higher than those of the control group, reflecting its positive regulatory effect on species richness. β-diversity analysis: The principal component analysis results of Figure 6 (β-diversity analysis of soil bacterial community) show that PC1 and PC2 cumulatively explained 32.51% of the total data variation. The distribution of the LL-8 treatment group and the CK group in the PC1-P2 two-dimensional space was significantly different. The LL-8 treatment group samples were concentrated in the higher positive value region of the PC1 axis, while the CK group was concentrated in the lower value region. This indicates that the regulatory effect of the LL-8 strain on the rhizosphere microbial community structure is significantly different from that of the control, and this regulation is closely related to key nitrogen metabolism factors.

[0037] Figure 7A (Differential species analysis of rhizosphere microorganisms under LL-8 treatment) shows that 138 bacterial strains were screened from the tobacco-growing soil treated with LL-8, of which 9 strains were significantly upregulated and 16 strains were significantly downregulated. After removing overlapping upregulated bacteria, the significantly upregulated bacteria included... Roseateles , Parasegitibacter , Pelomonas , Pseudomonas These significantly upregulated bacteria mainly promote the growth of flue-cured tobacco and nitrogen utilization in synergy with the LL-8 strain by producing plant hormones, participating in the decomposition and cycling of organic matter, or enhancing the plant's environmental adaptability.

[0038] In summary, strain LL-8 significantly regulated the rhizosphere microbial community structure of flue-cured tobacco by targeted enrichment of nitrogen-cycle-related functional genera, optimization of microbial community diversity and evenness, and reshaping community clustering characteristics, thus providing a favorable micro-ecological environment for soil nitrogen transformation and flue-cured tobacco nitrogen absorption.

[0039] Experiment 3: Regulatory effect of LL-8 strain on root morphology and root exudates in flue-cured tobacco S1. Preparation of test materials The tested flue-cured tobacco variety was Yunyan 87. Strong seedlings were cultivated using floating seedling raising, and seedlings with a seedling age of about 45 days and uniform growth were selected. Test bacterial agent: LL-8 bacterial agent prepared in Example 1, with a concentration of 10. 8 cfu / mL; Control reagent: Sterile water (equal volume to the bacterial agent); Hydroponic nutrient solution: Modified Hogland solution, with an ammonium-nitrate ratio of 25:75, and a formula of: N 140 mg / L (composed of (NH4)2SO4 165 mg / L and Ca(NO3)2 4H2O 885 mg / L (supplied), P 40 mg / L (KH2PO4175.48 mg / L), K 200 mg / L (K2SO4333.9 mg / L), Mg 40 mg / L (MgSO4 7H2O 410 mg / L), and trace elements such as Mn, Mo, Cu, Zn, B, and Fe (the specific content is as per the formula in Table 2.1); Hydroponic container: 300 mL plastic cup.

[0040] S2, Experimental group culture This experiment consisted of two treatment groups, each with five biological replicates, arranged in a completely randomized manner. The specific groupings are as follows: Control group (CK): An equal volume of sterile water was added to the hydroponic system, and all other conditions were the same as those of the treatment group; LL-8 treatment group: The hydroponic application method of Example 2 was followed, and the LL-8 bacterial agent prepared in Example 1 was added; Container sterilization: Disinfect 300 mL plastic cups with ultraviolet light for 0.5 hours before use; Nutrient solution and bacterial agent addition: Fill each cup with 297 mL of 1 / 2 concentration modified Hogland nutrient solution, add 3 mL of the corresponding bacterial agent or sterile water at a 1% inoculation rate, and mix well; Tobacco seedling treatment and transplanting: Rinse the roots of the tobacco seedlings twice with sterile water, soak the roots in sterile water or bacterial nutrient solution for 1 hour according to the corresponding treatment, and then fix them on a hard plastic cover with a sponge. One seedling is cultivated per cup. Hydroponic management: After transplanting, lift the cover to expose the roots to air for 0.5 hours every day; adjust the pH of the nutrient solution to 6.0 every 2 days with 0.1mol / L NaOH or 0.1mol / L HCl; maintain 1 / 2 concentration of nutrient solution for the first week, and replace it with full concentration of modified Hoagland nutrient solution for the next 2 weeks, changing the nutrient solution every 3 days until the end of the experimental period.

[0041] S3. Soil sample collection and testing Root sample collection: After the culture is completed, carefully remove the tobacco seedlings, gently rinse the residual nutrient solution on the root surface with sterile water, and absorb the surface moisture with filter paper for root morphology measurement. Root exudate collection: Root exudates of flue-cured tobacco from each treatment group were collected by calcium chloride soaking method. After collection, the exudates were immediately refrigerated and stored for high performance liquid chromatography detection.

[0042] Root morphology measurement: The root system was scanned using an EPSON scanner, and the root length, root diameter, root surface area, number of root tips, root volume and other indicators were calculated using image analysis software, and the root length density was also calculated. Root exudate determination: The contents of oxalic acid, fumaric acid, tartaric acid and malic acid in the exudate were detected and analyzed using a high performance liquid chromatograph (Agilent 1260Ⅱ).

[0043] Experimental results: Figure 8 The effects of ammonia-converting bacteria on the root morphology of flue-cured tobacco were shown to be significant, with the LL-8 treatment group exhibiting a comprehensive optimization effect. Root length: The root length of the LL-8 treatment group reached 358.22 cm, which was significantly increased by 41.5% compared with the control (253.07 cm); Root diameter: The root diameter of the LL-8 treatment group was 0.38 mm, which was significantly increased by 35.7% compared with the CK. Root surface area: The root surface area of ​​the LL-8 treatment group was 34.85 cm², which was significantly increased by 40.3% compared with the CK. Root tip count: The number of root tips in the LL-8 treatment group was 1559.5, an increase of 15.1% compared with the CK, which was significantly higher than the CK. Root volume: The LL-8 treatment group (8.41 cm³) increased significantly by 44.7% compared with the control group (5.82 cm³), with no significant difference between the two groups; Table 1: Effects of ammonia-converting bacteria on root exudates of hydroponically grown flue-cured tobacco

[0044] Note: The data in the table represent the mean plus the standard deviation (n=3). Different lowercase letters in the same column indicate significant differences between treatments (p<0.05), and the same applies below.

[0045] Table 1 (Effects of ammonia-converting bacteria on root exudates of hydroponically grown tobacco) shows that the organic acid content in the root exudates of the LL-8 treatment group and the control group (CK) differed significantly, specifically as follows: Oxalic acid: The LL-8 treatment group had a concentration of 7.24 ug / mL, which was significantly higher than the control (5.12 ug / mL) by 41.41%. Fumaric acid: The content in the LL-8 treatment group was 0.006 ug / mL, which was significantly increased by 200% compared with the CK (0.002 ug / mL); Tartaric acid: The LL-8 treatment group had a content of 817.75 ug / mL, which was significantly higher than the control group (657.61 ug / mL) by 24.36%. Malic acid: The content in the LL-8 treatment group was 3.31 ug / mL, which was significantly higher than that in the control group (2.46 ug / mL) by 34.55%.

[0046] In summary, strain LL-8 specifically promotes the growth of root length, root diameter, and root surface area in flue-cured tobacco, optimizes the three-dimensional root architecture to expand the nitrogen absorption area, and significantly increases the secretion of various key organic acids, thus providing support for rhizosphere nitrogen activation.

[0047] Experiment 4: Regulation of the nitrogen metabolism gene network in flue-cured tobacco roots by strain LL-8 The experimental materials, group design, and culture management methods in this experiment are completely identical to those in Experiment 3, with the only difference being: S3. Soil sample collection and testing After cultivation, the tobacco seedlings were carefully removed, and the residual nutrient solution on the root surface was gently rinsed with sterile water. The surface moisture was absorbed with filter paper, and fresh root tissue was quickly cut off, flash-frozen in liquid nitrogen, and then transferred to a -80°C freezer for storage for root transcriptomics analysis.

[0048] The gene network for nitrogen metabolism in flue-cured tobacco roots was analyzed using high-throughput transcriptome sequencing. The specific steps are as follows: Total RNA extraction: Total RNA was extracted from root samples using a kit, and the purity, integrity, and concentration of RNA were tested. Library construction and sequencing: Qualified RNA samples were sent to Shenzhen Vicomm Technology Co., Ltd., where cDNA libraries were constructed and then subjected to high-throughput sequencing. Gene expression level quantification: The readings mapped to each gene were calculated using Stringtie software, and the FPKM (Fragment Per Kbps) value for each gene was calculated based on gene length; Differential gene expression analysis: DESeq2 software (1.16.1) was used to analyze the differential expression between the two groups and screen for differentially expressed genes (DEGs). Differential gene enrichment analysis: GO enrichment analysis of differentially expressed genes was performed using cluster Profiler (3.4.4) software to screen for significantly enriched GO terms (P<0.05); KEGG nitrogen metabolism pathway analysis was also performed to identify the core metabolic pathways involved by differentially expressed genes.

[0049] Figure 9 (The effect of ammonia-converting bacteria on the clustering of nitrogen metabolism functional genes) showed that there was a significant difference in the expression patterns of nitrogen metabolism-related genes between the LL-8 treatment group and the CK group: The genes in the LL-8 group and the CK group formed independent clusters and were clearly separated, indicating that the strain has specific regulation of nitrogen metabolism genes; The LOC107789070, LOC107781744, and LOC10779369 gene clusters were specifically enriched in the LL-8 group, and their expression levels increased by 3.8-4.6 times compared with the CK group. These genes correspond to ammonia oxidation, nitrate transport, and nitrogen assimilation (GS / GOGAT) functions, respectively. Gene expression in the CK group showed a dispersed pattern with no significant clustering characteristics, and the expression level fluctuated by 1.5-2.3.

[0050] Figure 10 (The effect of ammonia-converting bacteria on differences in gene clustering between groups - Principal component analysis) showed that the two groups of samples exhibited a clear separation trend in the PCA plot: The PC1 axis contribution rate was 26.51%, mainly associated with the core regulatory effect of ammonia-converting bacteria on the expression of nitrogen metabolism genes. The LL-8 group scored significantly higher than the CK group on the PC1 axis. The PC2 axis contribution rate was 22.51%, but the distribution of the LL-8 group and the CK group was not significantly different.

[0051] Figure 11 (volcano plot of differential expression of nitrogen metabolism genes between LL-8 and CK treatments) shows that the nitrogen metabolism genes upregulated in the LL-8 group compared to the CK group include LOC107789070 (nitrate transporter-related), LOC107825623 (glutamine synthase-related), and LOC107802035 (nitrogen signal transduction-related), while LOC107804047 (negative regulator of nitrogen metabolism) is downregulated, thus relieving the inhibition of nitrogen assimilation. Figure 12 (intersection analysis of differentially expressed genes in flue-cured tobacco roots by ammonia-converting bacteria) shows that 1268 differentially expressed genes were identified between the LL-8 and CK groups, with 544 upregulated and 699 downregulated, indicating that the LL-8 strain has a wide-ranging effect on the root transcriptome. Figure 13 (Enrichment of differentially expressed genes in nitrogen metabolism in GO entries - GO GSEA) shows that differentially expressed genes are significantly enriched in entries such as "Nitrogen compound metabolism" (GO:0006807, 414 genes, accounting for 72.6% of the total genes in the component), "Cell elongation regulation" (GO:0051510, 37 genes), "Lateral root development" (GO:0048527, 35 genes), "Oxidoreductase activity" (GO:0016491, 25 genes), "Plasma membrane" (GO:0005886, 35 genes), and "Cell wall" (GO:0005618, 37 genes), which corresponds to the root morphology optimization (root length +41.5%, root diameter +35.7%) in the LL-8 treatment group at the molecular level. Figure 14 (Module GO Enrichment) shows that the gene enrichment of "oxidoreductase activity", "metal ion binding" and "nucleotide binding" items are the highest in the molecular function category, and the "nitrate response" and "cellular nitrate response" items are strongly enriched in the biological process category, which confirms that differentially expressed genes drive the efficient utilization of nitrogen in flue-cured tobacco by regulating key steps of nitrogen metabolism (nitrate reduction and ammonia assimilation) and signaling pathways.

[0052] Figure 15 (LL-8 nitrogen metabolism pathway diagram) shows that the LL-8 treatment group significantly activated the core nitrogen metabolism pathway: Nitrate (NO3) - Nitrate is reduced to nitrite (NO2) by nitrate reductase (1.7.2.5). - The process of reducing nitrite to ammonia (NH3) by nitrite reductase (1.7.2.4) is enhanced; The gene expression of glutamine synthase (GS, 6.3.1.2), a key enzyme in ammonia assimilation, was significantly upregulated. This enzyme catalyzes the synthesis of glutamine from glutamate and ammonia, which is the core step in the conversion of inorganic nitrogen into organic nitrogen. Its activation significantly promotes glutamate synthesis and improves the nitrogen assimilation efficiency of flue-cured tobacco. In this pathway, genes related to nitrogen uptake (nitrate transporter) and nitrogen assimilation (GS / GOGAT) are co-expressed, forming a complete and efficient nitrogen metabolism network.

[0053] In summary, strain LL-8 constructed an efficient root nitrogen metabolism gene network by specifically activating nitrogen uptake and assimilation-related genes, inhibiting negative regulatory factors, enriching nitrogen metabolism-related GO entries, and activating the KEGG nitrogen metabolism core pathway, thus providing molecular support for the efficient utilization of nitrogen in flue-cured tobacco.

[0054] Experimental Example 5: Effects of LL-8 inoculant on agronomic traits of flue-cured tobacco The experimental materials, group design, and culture management methods in this experiment are completely identical to those in Experiment 1, with the only difference being: S3. Sample Collection and Measurement Agronomic traits were measured at the tobacco plant's clump stage, vigorous growth stage, and maturity stage. Biomass was measured from whole tobacco plants collected at maturity. 1. Agronomic traits determination: The agronomic traits of flue-cured tobacco were determined in accordance with the "Standard of the Tobacco Industry of the People's Republic of China - Methods for Survey and Measurement of Agronomic Traits of Flue-cured Tobacco" (YC / T142-2010), including plant height, stem circumference, leaf length and width of the largest leaf; the relative chlorophyll content (SPAD value) of the leaves was determined using a chlorophyll meter. 2. Biomass determination: Tobacco root, stem and leaf samples were collected at maturity, first blanched at 105℃, then dried at 80℃ to constant weight, weighed separately and the dry matter of roots, stems and leaves was calculated.

[0055] Table 2. Effects of ammonia-converting bacteria on agronomic traits of flue-cured tobacco

[0056] Table 2 (Effects of ammonia-converting bacteria on agronomic traits of flue-cured tobacco) shows that the LL-8 treatment group was significantly better than the control group (CK) in core agronomic traits at the seedling stage, vigorous growth stage, and maturity stage, as detailed below: Plant height: During the rosette stage, the LL-8 treatment group showed a significant increase of 5.35% compared to the control group; during the vigorous growth stage, it showed a significant increase of 3.49% compared to the control group; and during the maturity stage, it showed a significant increase of 3.49% compared to the control group. The plant height maintained a stable growth-promoting effect at all growth stages. Stem circumference: During the rosette stage, the LL-8 treatment group showed a significant increase of 15.56% compared to the control group; during the vigorous growth stage, it showed a significant increase of 11.73% compared to the control group; and during the maturity stage, it showed a significant increase of 11.73% compared to the control group, making it the indicator with the most significant increase among all agronomic traits. Leaf length and leaf width: There were no significant differences in leaf length among the treatments; as for leaf width, the LL-8 treatment group was significantly higher than the CK by 3.87% during the vigorous growth stage, significantly higher than the CK by 3.87% during the maturity stage, and higher than the CK during the clustering stage, but the difference was not significant. SPAD values: During the vigorous growth stage, the LL-8 treatment group showed a significant increase of 10.89% compared to the control group; during the maturity stage, the SPAD value was significantly higher than that of the control group by 11.74%; and during the rosette stage, although the SPAD value was higher than that of the control group, the difference was not significant. This indicates that the LL-8 strain can continuously increase the chlorophyll content of flue-cured tobacco and enhance its photosynthetic capacity.

[0057] Table 3. Effects of ammonia-converting bacteria on flue-cured tobacco biomass

[0058] Table 3 (Effects of ammonia-converting bacteria on flue-cured tobacco biomass) shows that the root, stem, and leaf dry matter content of the LL-8 treatment group was significantly higher than that of the control group (CK), indicating a significant synergistic effect. Root biomass: The LL-8 treatment group had 26.97 g / plant, which was significantly higher than the control (19.73 g / plant) by 36.70%. Stem biomass: The LL-8 treatment group had a biomass of 49.55 g / plant, which was significantly higher than the control group (39.68 g / plant) by 24.88%. Leaf biomass: The LL-8 treatment group had 46.68 g / plant, which was significantly higher than the CK (36.44 g / plant) by 28.10%.

[0059] In summary, LL-8 inoculant significantly enhances the accumulation of root, stem, and leaf biomass by continuously optimizing core agronomic traits such as plant height, stem circumference, and SPAD value throughout the entire growth period of flue-cured tobacco, thus laying a solid foundation for high-yield and high-quality flue-cured tobacco through vegetative growth.

[0060] Experimental Example 6: Effect of LL-8 inoculant on nitrogen accumulation and distribution in flue-cured tobacco The experimental materials, group design, and culture management methods in this experiment are completely identical to those in Experiment 1, with the only difference being: S3. Sample Collection and Measurement Whole tobacco plants were collected at the ripening stage. Roots, stems and leaves (leaf parts were divided into upper, middle and lower parts) were separated. The surface impurities were first washed with clean water, and then rinsed twice with sterile water. After blanching at 105℃, the samples were dried at 80℃ to constant weight, crushed and sealed for storage, and used for nitrogen content determination.

[0061] Nitrogen content determination: The nitrogen nutrient content in roots, stems and leaves was determined according to the method in "Soil Agrochemical Analysis" (Bao Shidan, 2000); Leaf nitrogen accumulation calculation: Calculated according to the formula "Leaf nitrogen accumulation (mg / plant) = Total nitrogen content of leaves (%, g / 100g) × Leaf dry weight (g / plant) × 10"; Table 4. Effects of ammonia-converting bacteria on nitrogen accumulation and distribution in flue-cured tobacco.

[0062] Table 4 (Effects of ammonia-converting bacteria on nitrogen accumulation and distribution in flue-cured tobacco) shows that there were significant differences between the LL-8 treatment group and the control group (CK) in nitrogen content, nitrogen accumulation, and nitrogen use efficiency, as detailed below: Nitrogen content distribution: Root nitrogen content: The LL-8 treatment group had a root nitrogen content of 1.96 g / kg, which was significantly higher than the CK (1.44 g / kg) by 36.11%, making it the group with the highest root nitrogen content among all treatments; Stem nitrogen content: The LL-8 treatment group had a nitrogen content of 1.29 g / kg, which was significantly higher than the control (1.13 g / kg) by 14.16%, demonstrating its optimization of nitrogen translocation efficiency in the stem. Leaf nitrogen content: The LL-8 treatment group was 4.91 g / kg, which was not significantly different from the CK (4.93 g / kg), indicating that while it increased nitrogen accumulation in the roots and stems, it did not sacrifice nitrogen supply in the leaves, and the nitrogen distribution was more balanced.

[0063] Leaf nitrogen accumulation: The LL-8 treatment group reached 243.44 mg / plant, which was significantly increased by 24.36% compared with CK (195.8 mg / plant). This was closely related to the efficient absorption by the roots and the nitrogen translocation capacity of the stems, providing sufficient nitrogen guarantee for the formation of flue-cured tobacco quality.

[0064] Nitrogen use efficiency: The LL-8 treatment group had a nitrogen use efficiency of 20.38%, which was not significantly different from the CK (20.29%). This indicates that while significantly increasing nitrogen accumulation, it maintained a stable nitrogen use efficiency and did not result in nitrogen waste.

[0065] In summary, LL-8 inoculant significantly improves nitrogen content in roots and stems and nitrogen accumulation in leaves by optimizing the nitrogen distribution pattern in flue-cured tobacco roots, stems, and leaves, while maintaining stable nitrogen utilization efficiency. This achieves efficient absorption and rational distribution of nitrogen in flue-cured tobacco, providing a nutritional foundation for improving tobacco yield and quality.

[0066] 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 by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial agent based on strain LL-8 that regulates soil nitrogen transformation and promotes efficient nitrogen utilization in flue-cured tobacco, characterized in that, include: This bacterial agent was prepared by inoculating LL-8 strain into a culture medium. The preservation information of the LL-8 strain is as follows: Name of depositary institution: China General Microbiological Culture Collection Center; Address of depositary institution: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Date of deposit: January 28, 2026; Accession number: CGMCCNo.37560.

2. The microbial agent based on strain LL-8 for regulating soil nitrogen transformation and promoting efficient nitrogen utilization in flue-cured tobacco, as described in claim 1, is characterized in that... The inoculation method for the LL-8 strain is as follows: S1. Seed culture: A single colony of strain LL-8 was inoculated into 10 mL of beef extract peptone culture medium and cultured for 24 h to obtain seed culture; S2. Expanded culture: Take 1 mL of the seed culture and add it to 100 mL of beef extract peptone culture medium. Expand the culture by 1% inoculum for 24 h. S3. Centrifugation and washing: Centrifuge the fermentation broth after expansion culture at 4000 rpm for 10 min, discard the supernatant, and wash the precipitate twice with an equal volume of sterile water. After each wash, centrifuge at 4000 rpm for 10 min and retain the precipitate. S4. Preparation of bacterial agent: The washed precipitate is suspended in an equal volume of sterile water to obtain the LL-8 bacterial agent, with a concentration of 10. 8 cfu / mL.

3. A method for using the microbial agent based on strain LL-8 to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco, as described in claim 1, characterized in that: The LL-8 inoculant was applied to flue-cured tobacco at an inoculum rate of 1%, which promoted the efficient utilization of nitrogen in flue-cured tobacco by regulating soil nitrogen transformation and nitrogen metabolism pathways.

4. The method for using the microbial agent based on LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco according to claim 3, characterized in that, The application is a soil-based root irrigation application, which includes the following steps: after transplanting flue-cured tobacco seedlings that are 40-50 days old and have uniform growth, the LL-8 microbial agent is applied to the root zone of each flue-cured tobacco plant at the time of transplanting and 25-35 days after transplanting.

5. A method for using the microbial agent based on LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco according to claim 4, characterized in that, The dosage of LL-8 bacteria per plant per irrigation is 20 mL. Before transplanting, mix the soil with tobacco base fertilizer. Apply tobacco top dressing on days 22 and 37 after transplanting. During the growth period of flue-cured tobacco, maintain the soil field water holding capacity at about 70%.

6. A method for using the microbial agent based on LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco according to claim 5, characterized in that, The N-P2O5-K2O of the tobacco base fertilizer is 10-10-25, and the application rate is 91 grams per 10 kilograms of soil. The N-P2O5-K2O ratio of the tobacco topdressing is 15-0-30, and the application rate is 45.5 grams per 10 kilograms of soil.

7. The method for using the microbial agent based on the LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco according to claim 3, characterized in that, The application method is hydroponic root soaking, which includes the following steps: rinse the roots of flue-cured tobacco seedlings that are 40-50 days old and have uniform growth twice with sterile water, then soak the roots in Hogland nutrient solution containing bacteria for 1 hour, and then fix and cultivate them. The Hogland nutrient solution containing bacteria is prepared by adding 3 mL of LL-8 bacterial agent to Hogland nutrient solution at a 1% inoculation rate and mixing them evenly.

8. A method for using the microbial agent based on the LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco according to claim 7, characterized in that, The ammonium-nitrate ratio of the Hoglandian nutrient solution containing bacteria is 25:

75.

9. A method for using the microbial agent based on LL-8 strain to regulate soil nitrogen transformation and promote efficient nitrogen utilization in flue-cured tobacco according to claim 7, characterized in that, When soaking roots in hydroponics, the initial concentration of the Hogrange nutrient solution containing bacteria should be 1 / 2 of the original concentration. Maintain this concentration for the first week, and then replace it with the full concentration of the Hogrange nutrient solution containing bacteria for the next two weeks. Change the nutrient solution every 3 days, and expose the roots to air for 0.5 hours every day. Adjust the pH of the nutrient solution to 6.0 every 2 days.

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