A method for predicting and regulating the quality of flue-cured tobacco based on evaluating soil microorganisms

Abstract

This invention discloses a method for predicting and regulating the quality of flue-cured tobacco based on the assessment of soil microorganisms, belonging to the field of tobacco planting technology. The method includes: collecting topsoil samples from the planting area; determining the relative abundance of Acidobacteria and Vicinamibacterales in the soil, calculating the index F = relative abundance of Acidobacteria / relative abundance of Vicinamibacterales; comparing the F value with a preset threshold to predict tobacco quality: F < 0.1 indicates poor prediction, 0.1 ≤ F ≤ 50 indicates moderate prediction, and F > 50 indicates good prediction; for plots predicted as Class III or Class II soil, applying a microbial agent before transplanting, wherein the agent contains Acidobacteria as an active functional bacterial group, capable of promoting the proliferation of Acidobacteria and / or inhibiting the growth of Vicinamibacterales. This invention is the first to determine the quantitative relationship between Acidobacteria and Vicinamibacterales and tobacco quality, establish an F-value prediction model, and provide a microbial agent for targeted regulation of F-value, achieving a technological leap from passive prediction to active regulation, and providing an effective means to steadily improve tobacco quality.

Description

Technical Field

[0001] This invention belongs to the field of tobacco planting technology, specifically relating to a method for predicting and regulating the quality of flue-cured tobacco based on the assessment of soil microorganisms. Background Technology

[0002] Flue-cured tobacco is an important economic crop in my country, and the quality of tobacco leaves directly determines its industrial value, economic benefits, and the sensory quality of cigarette products. Therefore, how to stabilize and improve the quality of tobacco leaves has always been a core research topic in the fields of tobacco cultivation, breeding, and processing, and is also the key to the sustainable development of the tobacco industry.

[0003] For a long time, those skilled in the art have conducted extensive and in-depth research on improving tobacco leaf quality, resulting in various technical approaches. In genetic breeding, through systematic selection, hybridization breeding, and molecular marker-assisted breeding, a series of new flue-cured tobacco varieties with excellent quality traits and adaptability to different ecological zones have been successfully cultivated, laying the foundation for improved tobacco leaf quality from a genetic perspective. In cultivation management, soil testing and formula fertilization techniques are used to precisely control the application rates and proportions of nitrogen, phosphorus, potassium, and micronutrients; scientific irrigation, reasonable planting density, and timely topping and pruning optimize the population structure and individual development of tobacco plants. In processing, by improving tobacco leaf harvesting standards, promoting mature harvesting, and combining this with the application of intensive curing barns and intelligent curing equipment, temperature and humidity parameters during the curing process are precisely controlled, promoting the degradation and transformation of macromolecules such as starch and protein within the tobacco leaves, as well as the formation and accumulation of aroma compounds. These traditional technologies have played an irreplaceable and crucial role in promoting the large-scale and standardized production of tobacco leaves and ensuring a stable supply of raw materials.

[0004] However, despite continuous progress and improvement in the aforementioned technological systems, the tobacco industry still faces a long-standing and fundamentally unresolved problem: the poor stability of tobacco leaf quality and significant differences between plots. Specifically, even within the same ecological zone, using the same flue-cured tobacco varieties and implementing highly consistent cultivation management measures and curing processes, tobacco leaves from different plots may still exhibit significant differences in chemical composition and sensory quality. Furthermore, in some tobacco-growing areas with long-term continuous cropping, soil degradation, or newly reclaimed land, even with increased management efforts, the quality of the produced tobacco leaves often fails to meet the standards for premium tobacco. This phenomenon indicates that existing technological means are still insufficient to address certain soil-related issues affecting tobacco leaf quality.

[0005] In-depth analysis reveals that soil is not only the carrier for tobacco plant growth but also a complex micro-ecosystem. Soil microorganisms profoundly influence tobacco plant growth, development, and secondary metabolism through their participation in processes such as nutrient transformation, organic matter decomposition, and plant hormone synthesis, ultimately determining the quality characteristics of tobacco leaves. However, current cultivation and management techniques primarily focus on regulating soil physicochemical properties, such as adjusting nutrient content through fertilization, improving organic matter through the application of organic fertilizers, and adjusting pH through the application of lime. These techniques fail to delve into the fundamental factor of soil microecology that affects tobacco leaf quality. Therefore, identifying key microbial groups closely related to tobacco leaf quality from the perspective of soil microecology and establishing effective control measures has become a pressing technical problem to be solved in this field.

[0006] To address the above problems, this invention is proposed. Summary of the Invention

[0007] In view of the problems of poor stability of tobacco quality, significant differences between plots, and the fact that existing cultivation and management techniques mainly focus on the regulation of soil physicochemical properties without delving into the fundamental factor of soil microecology, this invention provides a method for predicting and regulating flue-cured tobacco quality based on the assessment of soil microorganisms.

[0008] The purpose of this invention is to establish a quantitative prediction model based on specific microbial markers closely related to the quality of flue-cured tobacco leaves by screening such markers, and to provide microbial agents that can target and regulate the soil microbial flora for plots with unsatisfactory prediction results. This will enable a technological leap from passive prediction to active regulation and provide an effective means to steadily improve the quality of tobacco leaves.

[0009] The present invention solves the technical problem by adopting the following technical solution:

[0010] The first aspect of this invention is a method for predicting and regulating the quality of flue-cured tobacco based on the assessment of soil microorganisms, comprising the following steps:

[0011] (1) Collect soil samples from the topsoil of the planting area before transplanting flue-cured tobacco;

[0012] (2) Microbial community analysis was performed on soil samples to determine the relative abundance of Acidobacteria and Vicinamibacterales, and an index F that reflects the soil microecological balance was calculated. The index F is the ratio of the relative abundance of Acidobacteria to the relative abundance of Vicinamibacterales.

[0013] (3) Compare the index F obtained in step (2) with the preset threshold to predict the quality of tobacco leaves:

[0014] If F is less than 0.1, the tobacco quality of this plot is predicted to be Class III soil.

[0015] If F is between 0.1 and 50, the tobacco quality of this plot is predicted to be Class II soil.

[0016] If F is greater than 50, the tobacco quality of the plot is predicted to be Class I soil.

[0017] (4) For plots predicted as Class III or Class II soil in step (3), apply microbial agents to the soil before transplanting flue-cured tobacco. The microbial agents contain effective components that can directionally regulate the soil microbial community. The directional regulation refers to increasing the proliferation of Acidobacterales microorganisms and / or reducing the growth of Vicinamibacterales microorganisms, thereby increasing the F value.

[0018] It should be noted that, for ease of distinction, this invention refers to soil that produces Class I quality tobacco leaves as Class I soil, soil that produces Class II quality tobacco leaves as Class II soil, and soil that produces Class III quality tobacco leaves as Class III soil.

[0019] Preferably, the effective components of the microbial agent in step (4) include microorganisms of the phylum Acidobacteria or order Acidobacteriales as active functional microbial groups.

[0020] Preferably, the microbial agent further comprises an organic carrier selected from vermiculite.

[0021] Preferably, the microbial agent further comprises a synergistic functional component, which is selected from at least one of humic acid, amino acid chelated calcium, and trace elements, wherein the trace elements are selected from one or more of iron, zinc, boron, manganese, molybdenum, and copper.

[0022] Preferably, the microbial agent further comprises a special adjuvant selected from one or more biosurfactants selected from rhamnolipin, sophorolipid, and trehalolipid, and one or more trace elements selected from EDTA chelated iron, EDTA chelated zinc, and EDTA chelated manganese.

[0023] Preferably, the formulation of the microbial agent includes: active functional bacteria, organic carrier, synergistic functional ingredients, and special adjuvants.

[0024] Preferably, the formulation of the microbial agent is as follows:

[0025] Active functional bacteria: Acidobacteria, with a viable count of not less than 1×10⁻⁶. 8 CFU / g;

[0026] Solid matrix: Composed of organic carrier, synergistic functional components, and special additives, including:

[0027] The organic carrier contains vermiculite, and is supplemented with 5% by weight of precipitated silica and 1% by weight of sodium carboxymethyl cellulose from the vermiculite.

[0028] The amount of the synergistic functional ingredient added is 5%-10% of the vermiculite mass, and it is selected from at least one of humic acid, amino acid chelated calcium and trace elements, wherein the trace elements are selected from one or more of iron, zinc, boron, manganese, molybdenum and copper.

[0029] The special additives include biosurfactants and EDTA-chelated trace elements, wherein the amount of biosurfactants added is 0.5%-2% of the mass of vermiculite, the amount of EDTA-chelated trace elements added is 0.5%-1% of the mass of vermiculite, and the total amount of biosurfactants and EDTA-chelated trace elements added is 1%-3% of the mass of vermiculite; the biosurfactants are selected from one or more of rhamnolipids, sophorolipids, and trehalolipids, and the EDTA-chelated trace elements are selected from one or more of EDTA-chelated iron, EDTA-chelated zinc, and EDTA-chelated manganese;

[0030] The bacterial solution and the solid matrix are mixed in a volume-to-mass ratio of 2:1.

[0031] Preferably, the application rate of the microbial agent in step (4) is 5-15 kg per mu.

[0032] Preferably, the microbial agent in step (4) is applied by spreading the agent evenly on the soil surface, then tilling it to a depth of 15-20cm to fully mix it with the topsoil, and maintaining the soil moisture at a suitable range of 60%-70% for 5-7 days after application.

[0033] A second aspect of the present invention provides a microbial agent for use in the method described in the first aspect, comprising Acidobacterium as an active functional bacterial group, and the formulation thereof is as defined above.

[0034] The present invention has the following beneficial effects:

[0035] 1. This invention, through systematic analysis of 240 soil samples from three major tobacco-growing areas in Yunnan, employs a three-tiered quality comparison strategy ("Class I soil (good), "Class II soil (medium), and Class III soil (poor)") and a three-level progressive screening strategy to construct a tobacco leaf quality prediction index F based on the abundance ratio of the orders Acidobacteria and Vicinamibacterales. This index shows a highly consistent correlation with tobacco leaf quality: good quality corresponds to F > 50, medium quality corresponds to 0.1 ≤ F ≤ 50, and poor quality corresponds to F < 0.1. The F value ranges for the three categories of samples do not overlap and the boundaries are clear. Validated in 10 independent plots, the prediction accuracy reached 90%, indicating that the index has good accuracy and reliability. This quantitative index transforms tobacco leaf quality from empirical judgment into a quantifiable microbial indicator, providing an objective basis for the scientific selection of tobacco planting sites.

[0036] 2. For plots with a predicted F-value of poor or moderate (F < 50), this invention provides a microbial agent with *Acidobacterium* as the active functional flora. The agent's formulation is scientifically complete, containing an organic carrier (vermiculite), synergistic functional components (humic acid, amino acid chelated calcium, and trace elements), and special adjuvants (biosurfactants and EDTA chelated trace elements). Field trials have demonstrated that after application of this agent, the F-value of low-F plots (F < 0.1) significantly increased from 0.06-0.09 to 35.6-42.3, and the F-value of medium-F plots (0.1 ≤ F ≤ 50) significantly increased from 8.3-25.6 to 40.6-48.3, both reaching above-average levels, while the control group showed no significant change. Simultaneously, the sensory scores and chemical composition coordination of the tobacco leaves in the experimental group were significantly better than those in the control group. The organic carriers in the formula (vermiculite, silica, sodium carboxymethyl cellulose) provide attachment sites and a protective environment for acid bacteria, extending the survival time of the bacterial community in the soil. The synergistic functional components (humic acid, amino acid chelated calcium, and trace elements) provide nutritional support for the bacterial community and tobacco plants, promoting bacterial metabolism and tobacco plant growth. Special adjuvants (biosurfactants) help the bacterial community rapidly spread and colonize in the soil, improving colonization efficiency and duration of effectiveness. The synergistic effect of all components ensures a stable increase in the F-value of the inoculant.

[0037] 3. The Acidobacteria order microorganisms used in this invention can achieve targeted regulation of the F value. The mechanism of action is as follows: After colonization, Acidobacteria order microorganisms secrete metabolic products such as organic acids, amino acids, and extracellular polysaccharides. Because Acidobacteria order microorganisms share homologous metabolic pathways with closely related species in the same order, they can preferentially recognize and utilize these products as nutrient sources, thus rapidly proliferating. This process follows the basic principles of metabolic interactions between closely related species in microbial ecology. Acidobacteria order and Vicinamibacterales order overlap in carbon source utilization and ecological niches in the soil, leading to resource competition. As Acidobacteria order gains numerical dominance, its ability to seize nutrients and living space increases. According to the principle of microbial competitive exclusion, Vicinamibacterales order growth is inhibited due to nutrient limitations. Ultimately, this dual effect of promoting similar species and inhibiting dissimilar species leads to an increase in the abundance of Acidobacteria order and a decrease in the abundance of Vicinamibacterales order, resulting in a significant increase in the F value. Detailed Implementation

[0038] The present invention will be further described below through embodiments, but is not limited to these embodiments. Experimental methods not specifically described in the embodiments generally use conventional conditions and conditions described in manuals, or conditions recommended by the manufacturer. The general equipment, materials, reagents, etc., used are all commercially available unless otherwise specified.

[0039] Example 1

[0040] This embodiment aims to screen key microbial communities in soil microbial communities that have a stable quantitative correlation with the quality of flue-cured tobacco leaves, and to establish a predictive model based on these microbial communities. The specific steps are as follows:

[0041] 1. Soil and tobacco leaf sample collection

[0042] From 2023 to 2024, in three major tobacco-growing areas in Yunnan Province—Shilin County in Kunming City, Mile County in Honghe Prefecture, and Nanjian County in Dali Prefecture—16 townships with flue-cured tobacco cultivation were selected. Five sampling points were randomly chosen from each township, resulting in a total of 240 topsoil samples (0-20cm). The selection criteria for sampling points were: continuous tobacco cultivation for more than 3 years, red soil type, and previous crop being rapeseed. Each sample was collected using the S-shaped sampling method. After removing impurities on-site, 100g of soil was placed in a sterile sampling bag, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0043] All sampling points correspond to the planting of flue-cured tobacco (variety Honghua Dajinyuan), and are managed uniformly according to the local high-quality tobacco production technical regulations. After the tobacco leaves are harvested and cured, 2 kg of C3F grade primary cured tobacco leaves are taken from each point.

[0044] 2. Tobacco Leaf Quality Evaluation and Classification

[0045] Two kg of C3F grade uncured tobacco leaves collected at each point were placed in a constant temperature and humidity chamber for 48 hours to equilibrate the moisture content. After equilibration, the tobacco leaves were shredded and rolled into single-material cigarettes according to industry standards for subsequent sensory quality evaluation.

[0046] To objectively evaluate the impact of this invention on the core sensory quality of tobacco leaves, seven qualified professional tasters were organized to conduct a systematic taste evaluation of the prepared samples in accordance with the industry standard "Sensory Evaluation Methods for Tobacco and Tobacco Products" (YC / T 138-1998).

[0047] The evaluation adopted a 9-point scoring system. Judges independently scored nine indicators: aroma quality (8 points), aroma quantity (8 points), off-flavors (6 points), concentration (6 points), strength (6 points), irritation (4 points), aftertaste (6 points), combustibility (9 points), and ash color (8 points). The final result was the average score of the seven judges. The total score for the nine indicators was 61 points. To ensure consistent evaluation standards, each judge's original total score was converted to a percentage score using the following formula: Percentage Score = (Original Score / 61) × 100. The final result was the average of the seven judges' percentage scores. Based on industry-standard single-material tobacco evaluation criteria, and considering both sensory evaluation and chemical composition compatibility, sensory grades were divided as follows: Sensory score ≥ 80 points was good; Sensory score 70-80 points was average; Sensory score < 70 points was poor.

[0048] According to the tobacco industry standards "Determination of Water-Soluble Sugars in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T159-2002), "Determination of Total Nitrogen in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T161-2002), "Determination of Total Alkaloids in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T160-2002), "Determination of Chlorine in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T162-2011), and "Determination of Potassium in Tobacco and Tobacco Products - Flame Photometry" (YC / T173-2003), the total sugar and reducing sugar, total nitrogen, nicotine, chlorine, and potassium of freshly flue-cured tobacco leaves were determined. The sugar-to-alkaloid ratio (reducing sugar / nicotine), nitrogen-to-alkaloid ratio (total nitrogen / nicotine), and potassium-to-chlorine ratio (potassium / chlorine) were calculated. According to the generally accepted standards in the tobacco industry, the suitable range for high-quality flue-cured tobacco is generally: sugar-to-alkaloid ratio 8-12, nitrogen-to-alkaloid ratio 0.8-1.1, and potassium-to-chlorine ratio ≥4.

[0049] Based on the aforementioned national and industry standards, the 240 tobacco leaf samples were divided into three categories:

[0050] Good quality (78 samples): Sensory score ≥80 points, and sugar-alkali ratio (total sugar / nicotine) in the range of 8-15, nitrogen-alkali ratio (total nitrogen / nicotine) in the range of 0.8-1.1, and potassium-chlorine ratio (potassium / chlorine) ≥4, all three indicators are met;

[0051] Poor quality (80 samples): Sensory score <70 points, and at least two of the three indicators of sugar-alkali ratio, nitrogen-alkali ratio, and potassium-chloride ratio meet the following deviation standards: sugar-alkali ratio <6 or >14, nitrogen-alkali ratio <0.6 or >1.3, potassium-chloride ratio <3;

[0052] Medium quality (82 samples): Sensory scores are between 70 and 80 points, and do not meet the criteria for good quality and poor quality. That is, among the three indicators of sugar-alkali ratio, nitrogen-alkali ratio and potassium-chlorine ratio, neither the three requirements of good quality are met at the same time, nor the condition of "at least two deviations" of poor quality is met.

[0053] 3. Soil microbial community analysis

[0054] Total DNA was extracted from soil samples using the YH-soil FastPure Soil DNA Isolation Kit (Magnetic bead) (MJYH, Shanghai, China), and its purity and concentration were determined using NanoDrop 2000. PCR amplification was performed targeting the V3-V4 region of the 16S rRNA gene using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and...

[0055] The reaction system, 806R (5'-GGACTACHVGGGTWTCTAAT-3'), contained FastPfu Buffer, dNTPs, FastPfu DNA Polymerase, and template DNA. Amplification conditions: 95℃ pre-denaturation for 3 min, 27 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s), followed by a stable extension at 72℃ for 10 min, and finally storage at 4℃ (PCR instrument: ABI GeneAmp® 9700). The product was purified using a DNA gel extraction and purification kit, quantified using Qubit 4.0, and a library was constructed using the NEXTFLEX Rapid DNA-Seq Kit. Paired-end sequencing was performed on the Illumina Nextseq2000 platform. The original sequences were controlled by FASTP and assembled by FLASH. UPARSE v11.0.667[4,5] software (http: / / drive5.com / uparse / ) was used to perform OTU (Operational Taxonomic Unit) clustering and remove chimeras based on 97% similarity. RDP classifier[6] (http: / / rdp.cme.msu.edu / , version 2.11) was used to compare the OTU species taxonomic annotation with the Silva 16S rRNA gene database (v138), with a confidence threshold of 70%. The community composition of each sample was calculated at different species taxonomic levels. The relative abundance of each sample at the "order" level was obtained.

[0056] 4. Screening of key microbial communities

[0057] Based on the sensory quality evaluation results of the tobacco leaves, the corresponding soil samples were divided into three categories:

[0058] Class I soil (good): Soil samples that produce high-quality tobacco leaves (sensory score ≥80 points, chemical composition in harmony);

[0059] Class II soil (medium): Soil samples that produce medium-quality tobacco leaves (sensory score 70-80 points);

[0060] Class III (Poor): Soil samples that produce low-quality tobacco leaves (sensory score <70 points, chemical composition is not harmonious).

[0061] Independent samples t-tests were used to analyze the significance of differences (p < 0.05). Following a progressive screening method, microbial groups meeting the following three criteria were selected:

[0062] Condition 1: Microbial groups with a significantly higher relative abundance in Class I soil than in Class III soil are denoted as potentially positively correlated microbial groups;

[0063] Condition 2: Microbial groups whose relative abundance is significantly higher in the three types of soil than in the first type of soil are denoted as potentially negatively associated microbial groups;

[0064] Condition 3: For the two potential microbial communities obtained from the initial screening, they must exhibit a continuous gradient distribution in good, medium, and poor soil types.

[0065] For potentially positively associated microbial communities, the soil type must be: Class I soil > Class II soil > Class III soil.

[0066] For potentially negatively associated microbial communities, the soil type must be: Class III soil > Class II soil > Class I soil;

[0067] In soils that produce medium-quality tobacco leaves, the relative abundance of the two types of microbial communities should meet the following requirements: they should be on the same order of magnitude and have no significant difference.

[0068] After these three layers of screening:

[0069] Only the Acidobacteria order satisfies the "Class I soil > Class II soil > Class III soil" gradient of conditions one and three, and is identified as the core positively correlated bacterial group (beneficial bacteria).

[0070] Only Vicinamibacterales met the "Class III soil > Class II soil > Class I soil" gradient of conditions two and three, and was identified as the core negatively associated microbial community (harmful bacteria).

[0071] 5. F-value calculation and prediction threshold determination

[0072] For 240 samples, the relative abundance (%) of the Acidobacteria order and the Vicinamibacterales order were counted, and the index F was calculated as F = relative abundance of Acidobacteria order / relative abundance of Vicinamibacterales order.

[0073] The correlation analysis between the F-values ​​of each sample and the quality grade of tobacco leaves was performed, and the results are shown in Table 1.

[0074] Table 1. Range of F values ​​corresponding to different tobacco leaf qualities

[0075]

[0076] To further determine the optimal prediction threshold, receiver operating characteristic (ROC) curve analysis was performed using SPSS software. ROC curves were plotted with good-quality samples as positive and poor-quality samples as negative. The results showed an area under the curve (AUC) of 0.96, indicating that the F-value has excellent discriminatory power for good and poor-quality samples. Based on the Youden index maximization principle and combined with the ROC analysis results, the classification thresholds for good and poor quality were determined to be F=50 and F=0.1, respectively.

[0077] Meanwhile, based on the F-value distribution characteristics of the three types of samples shown in Table 1 (good quality F > 52.3, poor quality F < 0.095, and medium quality F between 0.12 and 48.9), the F-values ​​of the three types of samples have significant distinguishability, which can effectively achieve three-level classification. To ensure classification accuracy, reserve a safety buffer for measurement errors and future new samples, and avoid misjudgment of boundary samples, the final quality grading prediction threshold is determined based on the statistical distribution of F-values ​​for different quality tobacco leaves shown in Table 1 as follows:

[0078] F > 50: Predicted as good quality

[0079] F < 0.1: Predicted as poor quality

[0080] 0.1 ≤ F ≤ 50: Predicted as medium quality

[0081] Verification showed that the threshold system achieved a prediction accuracy of over 95% for all three categories of samples (good, medium, and poor), indicating that the prediction model has good reliability.

[0082] Example 2

[0083] To verify the reliability of the above prediction model, 10 tobacco-growing plots (non-modeling samples) independent of Example 1 were randomly selected in Dali and Honghe prefectures of Yunnan Province. Soil samples were collected from the topsoil before tobacco transplanting, and 16S rRNA amplicon sequencing and bioinformatics analysis were performed according to the method in Example 1. The F-value of each sample was calculated. Simultaneously, flue-cured tobacco (variety: Honghua Dajinyuan) was planted in each plot and managed according to local technical specifications. After harvesting and curing, C3F grade samples were taken for independent sensory quality evaluation. The verification results are shown in Table 2.

[0084] Table 2. Validation results of the F-value prediction model

[0085]

[0086] Of the 10 validation points, 9 were correctly predicted, achieving an accuracy rate of 90%, indicating that the prediction model based on the abundance ratio F between Acidobacteriaceae and Vicinamibacterales has good accuracy and reliability. Sample number 3 was predicted to be of medium quality (F=1.07) but actually was of poor quality, which may have stemmed from random errors in sample processing, sequencing, or field management. However, overall, the model still demonstrates stable and reliable predictive ability.

[0087] Example 3

[0088] This embodiment aims to verify the regulatory effect of the microbial agent of the present invention on the F value, and its effect on improving the quality of tobacco leaves after regulation. Specifically:

[0089] 1. Preparation of microbial inoculants:

[0090] (1) Active functional bacterial flora: In this example, Acidobacterium capsulatum DSM 11244 (commercially available) is used as an example. The freeze-dried strain was inoculated into modified R2A liquid medium (formulation: yeast extract 0.5 g / L, peptone 0.5 g / L, acid hydrolyzed casein 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, sodium pyruvate 0.3 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate 0.05 g / L, pH 6.5), and activated by shaking culture at 28℃ and 180 r / min for 24 h. After activation, it was transferred to the same medium at an inoculation rate of 5% (v / v) and cultured at 28℃ and 180 r / min for 7 days to obtain the Acidobacterium capsulatum bacterial suspension. The viable cell count was determined using the plate count method (using modified R2A agar plates as the counting medium, incubated at 28°C for 7 days). The average value of three replicate experiments was 5.2 × 10⁻⁶. 9 CFU / mL (standard deviation ±0.4×10) 9 (CFU / mL).

[0091] (2) Preparation of solid matrix:

[0092] Using vermiculite as a base, 5% silica and 1% sodium carboxymethyl cellulose were added by weight of vermiculite and mixed evenly.

[0093] Add 8% of the synergistic functional ingredients based on the mass of vermiculite (in this embodiment, humic acid and amino acid chelated calcium are mixed at a mass ratio of 1:1).

[0094] Add 2% of a special additive by weight of vermiculite (in this example, rhamnolipin and EDTA chelated iron are mixed at a mass ratio of 1:1).

[0095] The above materials are thoroughly mixed to obtain a solid matrix.

[0096] (3) Preparation of the microbial agent: The *Acidobacterium* bacterial solution was mixed with the above-mentioned solid matrix at a volume ratio of 2:1 to obtain the microbial agent of the present invention. Testing showed that the viable *Acidobacterium* count in the finished agent was 2.1 × 10⁻⁶. 8 CFU / g.

[0097] 2. Selection of test sites:

[0098] In 2025, in a tobacco-growing area of ​​Yuxi City, Yunnan Province, the F-value pre-detection was conducted on 30 plots of land using the prediction model of Example 1, and 9 representative test plots were selected, among which:

[0099] Three plots with low F values ​​(numbered D1-D3): all with F values ​​less than 0.1;

[0100] Three plots with medium F values ​​(numbered M1-M3): F values ​​range from 0.1 to 50;

[0101] Three plots with good F values ​​(numbered G1-G3): all with F values ​​greater than 50.

[0102] The tested soils were all red soils, with the following basic physicochemical properties: pH 6.1-6.5, organic matter content 16.5-22.8 g / kg, available nitrogen 88-115 mg / kg, available phosphorus 13-19 mg / kg, and available potassium 98-142 mg / kg. There were no significant differences in any of these indicators among the groups.

[0103] 3. Application of biological agents:

[0104] Each plot of land was divided into a control group and an experimental group, with each group replicated three times, for a total of 36 experimental plots, each plot measuring 64m². 2 The randomized block arrangement is used, with three protection rows between each block.

[0105] Experimental group treatment:

[0106] Plots D1-D3 (F < 0.1): Apply the microbial agent of this invention at a rate of 10 kg per mu 10 days before tobacco transplanting;

[0107] M1-M3 plots (0.1≤F≤50): Apply the microbial agent of this invention at a rate of 10 kg per mu 10 days before tobacco transplanting;

[0108] For plots G1-G3 (F>50): Apply the microbial agent of this invention at a rate of 10 kg per mu 10 days before transplanting flue-cured tobacco.

[0109] Application method: Spread the microbial agent evenly on the soil surface, then use a rotary tiller to till to a depth of 15-20cm to fully mix the microbial agent with the topsoil, and then create ridges for planting. If drought occurs after application, use sprinkler irrigation to maintain soil moisture at a suitable range of 60%-70% for 5-7 days.

[0110] The control group did not receive any microbial inoculants and only underwent routine land preparation and ridging. Apart from the application of inoculants, all other cultivation and management practices remained consistent throughout the entire growth period of the flue-cured tobacco in all treatment groups: the variety was Honghua Dajinyuan, and fertilization, irrigation, topping, pruning, and pest and disease control were carried out in accordance with local high-quality tobacco production technical regulations.

[0111] 4. Sample collection and testing

[0112] Soil samples from the 0-20cm topsoil layer were collected in each area using a five-point sampling method before inoculum application (10 days before tobacco transplanting) and after tobacco harvest (5 days after full maturity). Impurities were removed and the samples were mixed thoroughly. 16S rRNA amplicon sequencing and bioinformatics analysis were performed according to the method in Example 1, and the F-value for each sample was calculated.

[0113] 5. Evaluation of tobacco leaf quality

[0114] After the tobacco leaves mature, each region harvests, binds, and loads them into the curing ovens separately, using the same intensive curing barns and curing process (three-stage curing process). C3F grade samples of the initially cured tobacco leaves are taken and subjected to sensory quality evaluation and chemical composition analysis according to the method in Example 1.

[0115] The final measurement results are shown in Table 3 below:

[0116] Table 3. Changes in F-values ​​before and after application of microbial inoculants.

[0117]

[0118] Note: Data is presented as "mean ± standard deviation", n=3.

[0119] This embodiment demonstrates that the microbial agent of the present invention can significantly increase the F value of plots with low and medium initial F values, thereby regulating the soil microbial community structure in a direction conducive to improving tobacco quality. For plots with good F values ​​that are already in a high-quality state, there is no significant change in F value after applying the agent, indicating that the agent has good application safety and will not disrupt the already established good soil micro-ecological balance.

[0120] 6. The effect of microbial agents on improving tobacco quality

[0121] After the tobacco leaves matured, each plot was harvested and cured separately. Samples of C3F grade first-cured tobacco leaves were taken and subjected to sensory quality evaluation and chemical composition testing according to the method described in Example 1 for tobacco quality evaluation and classification. The results are shown in Table 4: sugar-alkali ratio 8-12, nitrogen-alkali ratio 0.8-1.1, and potassium-chlorine ratio ≥4.

[0122] Table 4. Evaluation results of tobacco leaf quality under different treatments

[0123]

[0124] This embodiment demonstrates that the microbial agent of the present invention can effectively and directionally regulate the abundance ratio F of Acidobacteria and Vicinamibacterales in soil, significantly increasing the F value. Regarding tobacco quality:

[0125] (1) For plots with low initial F values ​​(D1-D3), the quality of tobacco leaves in the experimental group improved from "poor" to "medium", the sensory score increased significantly from 62.4-63.8 to 75.8-77.2, and the coordination of chemical components such as sugar-alkali ratio, nitrogen-alkali ratio, and potassium-chlorine ratio improved from "uncoordinated" to "relatively coordinated".

[0126] (2) For plots with medium initial F value (M1-M3), although the sensory grade of tobacco leaves in the experimental group did not improve significantly, the score increased from 72.4-73.2 points to 77.5-78.8 points, an increase of about 5-6 points; at the same time, in terms of chemical compatibility, the experimental groups of M2 and M3 improved from relatively compatible to compatible.

[0127] (3) For plots with good initial F value (G1-G3), there were no significant differences in sensory scores and chemical composition indicators between the experimental group and the control group. The tobacco quality remained at the "good" level, indicating that the fungicide would not destroy the good quality that had been formed.

[0128] In summary, the microbial agent of the present invention has significant effects on soil microecological regulation and tobacco quality improvement, and has good application safety.

[0129] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A method for predicting and regulating the quality of flue-cured tobacco based on the assessment of soil microorganisms, characterized in that, Includes the following steps: (1) Collect soil samples from the topsoil of the area to be planted before transplanting flue-cured tobacco; (2) Microbial community analysis was performed on soil samples to determine the relative abundance of Acidobacteria and Vicinamibacterales, and an index F that reflects the soil microecological balance was calculated. The index F is the ratio of the relative abundance of Acidobacteria to the relative abundance of Vicinamibacterales. (3) Compare the index F obtained in step (2) with the preset threshold to predict the quality of tobacco leaves: If F is less than 0.1, the tobacco quality of this plot is predicted to be Class III soil. If F is between 0.1 and 50, the tobacco quality of this plot is predicted to be Class II soil. If F is greater than 50, the tobacco quality of this plot is predicted to be Class I soil. (4) For plots predicted as Class III or Class II soil in step (3), apply microbial agents to the soil before transplanting flue-cured tobacco. The microbial agents contain effective components that can directionally regulate the soil microbial community. The directional regulation refers to increasing the proliferation of Acidobacterales microorganisms and / or reducing the growth of Vicinamibacterales microorganisms, thereby increasing the F value.

2. The method according to claim 1, characterized in that, The effective components of the microbial agent in step (4) include microorganisms of the phylum Acidobacteria or Acidobacteria as active functional microbial groups.

3. The method according to claim 2, characterized in that, The microbial agent also includes an organic carrier, which is selected from vermiculite.

4. The method according to claim 3, characterized in that, The microbial agent also contains a synergistic functional component, which is selected from at least one of humic acid, amino acid chelated calcium, and trace elements, wherein the trace elements are selected from one or more of iron, zinc, boron, manganese, molybdenum, and copper.

5. The method according to claim 2, characterized in that, The microbial agent also includes special adjuvants, which are selected from one or more biosurfactants selected from rhamnolipin, sophorolipid, and trehalolipid, and one or more trace elements selected from EDTA chelated iron, EDTA chelated zinc, and EDTA chelated manganese.

6. The method according to any one of claims 2-5, characterized in that, The formulation of the microbial agent includes: active functional bacteria, organic carrier, synergistic functional ingredients, and special adjuvants.

7. The method according to claim 6, characterized in that, The formula for the microbial agent is as follows: Active functional bacteria: Acidobacteria, with a viable count of not less than 1×10⁻⁶. 8 CFU / g; Solid matrix: Composed of organic carrier, synergistic functional components, and special additives, including: The organic carrier contains vermiculite, and is supplemented with 5% by weight of precipitated silica and 1% by weight of sodium carboxymethyl cellulose from the vermiculite. The amount of the synergistic functional ingredient added is 5%-10% of the vermiculite mass, and it is selected from at least one of humic acid, amino acid chelated calcium and trace elements, wherein the trace elements are selected from one or more of iron, zinc, boron, manganese, molybdenum and copper. The special additives include biosurfactants and EDTA-chelated trace elements, wherein the amount of biosurfactants added is 0.5%-2% of the mass of vermiculite, the amount of EDTA-chelated trace elements added is 0.5%-1% of the mass of vermiculite, and the total amount of biosurfactants and EDTA-chelated trace elements added is 1%-3% of the mass of vermiculite; the biosurfactants are selected from one or more of rhamnolipids, sophorolipids, and trehalolipids, and the EDTA-chelated trace elements are selected from one or more of EDTA-chelated iron, EDTA-chelated zinc, and EDTA-chelated manganese; The bacterial solution and the solid matrix are mixed in a volume-to-mass ratio of 2:

1.

8. The method according to claim 1, characterized in that, The application rate of the microbial agent mentioned in step (4) is 5-15 kg per mu.

9. The method according to claim 8, characterized in that, The application method of the microbial agent in step (4) is as follows: spread the agent evenly on the soil surface, then plow to a depth of 15-20cm to mix it thoroughly with the topsoil. After application, keep the soil moisture at a suitable range of 60%-70% for 5-7 days.

10. A microbial inoculant for use in the method according to any one of claims 1-9, characterized in that, It contains Acidobacterium as an active functional bacterial group, and its formulation is as defined in claim 7.

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