Method for exploring influence of magnesium application technology on K326 flue-cured tobacco rhizosphere soil metabolome expression characteristics and tobacco chemical component content

Abstract

The invention discloses a method for exploring the influence of a magnesium application technology on K326 flue-cured tobacco rhizosphere soil metabolome expression characteristics and tobacco chemical component content. The method comprises the following steps: (1) selecting a test site; (2) selecting test materials; (3) designing a test treatment scheme; (4) formulating cultivation measures; and (5) selecting observation indexes. The invention aims to scientifically adjust the proportions of effective potassium / magnesium and calcium / magnesium in soil by increasing the application of a magnesium fertilizer, correspondingly reduce the proportions of potassium / magnesium and calcium / magnesium in flue-cured tobacco leaves, deeply explore the specific influence of the series of adjustment on the yield and quality of flue-cured tobacco, and improve the yield and quality of flue-cured tobacco. Powerful practical guidance and theoretical support are provided for optimizing the quality of flue-cured tobacco products and improving the yield and economic benefits.

Description

Technical field

[0001] The present invention belongs to the technical field of research on the influence of magnesium application technology on the expression characteristics of soil metabolome and tobacco leaf performance, and specifically relates to a method for exploring the influence of magnesium application technology on the expression characteristics of K326 flue-cured tobacco rhizosphere soil metabolome and the content of chemical components in tobacco leaves. [Background Technology]

[0002] As an important leaf-producing cash crop, the yield and quality of flue-cured tobacco are significantly influenced by soil ecological conditions and production technology. During the middle and late stages of tobacco cultivation, nutrient deficiencies and nutrient deficiencies are common. This is closely related to the narrow range of medium and trace element requirements and the slow rate of nutrient fixation and release in the soil. In current production, high-input, excessive fertilization not only fails to achieve rapid nutritional replenishment and balance, but also causes tobacco plant toxicity, fertilizer waste, and environmental pollution.

[0003] Currently, common potash fertilizers used in flue-cured tobacco cultivation include potassium sulfate, potassium nitrate, and compound fertilizers containing these ingredients. Potassium sulfate, a physiologically acidic fertilizer, can easily lead to soil acidification when applied. The nitrate ions in potassium nitrate are easily lost from the soil, making it unable to sustainably supply nitrogen nutrients to the tobacco. Furthermore, excessive application of carbon-free chemical fertilizers can deplete tobacco soil microbial life, disrupting the soil's microbial ecosystem and leading to a homogenization of microbial species, a decrease in beneficial microbial populations, and an increase in harmful microbial populations.

[0004] Magnesium is an essential secondary element for tobacco growth, playing important roles in chlorophyll synthesis, promoting photosynthesis, participating in protein synthesis, and activating and regulating enzymatic reactions. However, insufficient magnesium levels in the soil or excessive levels of potassium, calcium, and ammonium nitrogen can easily lead to magnesium deficiency in tobacco. Magnesium deficiency typically occurs in the middle and late stages of tobacco growth. Low-magnesium stress can lead to impaired chlorophyll synthesis, leaf chlorosis, and even necrosis, severely impacting tobacco growth. Potassium and magnesium interact antagonistically during absorption, and tobacco, a potassium-loving and potassium-intensive crop, emphasizes potassium fertilizer application during production. Low-magnesium stress occurs when the effective potassium to magnesium ratio in the rhizosphere soil exceeds a certain value (generally 2-3:1). Furthermore, applying large amounts of magnesium fertilizer during transplanting can severely inhibit potassium absorption, reducing leaf quality.

[0005] In view of the current situation that the ratio of effective potassium to magnesium, and calcium to magnesium in the soil of tobacco-growing areas is relatively high, which makes it difficult for flue-cured tobacco to absorb magnesium, and then causes magnesium deficiency, which seriously affects the yield and quality of tobacco leaves. It is urgent to explore a reasonable magnesium fertilizer application technology to reduce the potassium / magnesium and calcium / magnesium ratios in the soil and flue-cured tobacco leaves, and improve the yield and quality of flue-cured tobacco. [Summary of the invention]

[0006] In view of the problem that the ratios of effective potassium and magnesium, as well as calcium and magnesium, in the tobacco-growing soil in this tobacco-growing area are unbalanced, which makes it difficult for flue-cured tobacco to effectively absorb magnesium, thereby causing magnesium deficiency and seriously affecting the yield and quality of tobacco leaves, the present invention provides a method for exploring the effects of magnesium application technology on the metabolome expression characteristics of the rhizosphere soil of K326 flue-cured tobacco and the content of chemical components in tobacco leaves.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A method for exploring the effects of magnesium application technology on the metabolome expression characteristics of rhizosphere soil of K326 flue-cured tobacco and the chemical component content of tobacco leaves, comprising the following steps:

[0009] (1) Selection of trial sites;

[0010] (2) Selection of test materials;

[0011] (3) Experimental treatment plan design;

[0012] (4) Development of cultivation measures;

[0013] (5) Selection of observation indicators.

[0014] Furthermore, the experimental site in step (1) was selected as Changfu Village, Xinjing Town, Jingxi City.

[0015] Furthermore, the test materials in step (2) include flue-cured tobacco varieties and fertilizers.

[0016] Furthermore, the flue-cured tobacco variety is K326 flue-cured tobacco.

[0017] Furthermore, the fertilizer includes tobacco compound fertilizer, potassium nitrate, potassium sulfate, and potassium magnesium sulfate.

[0018] Furthermore, the potassium oxide content in the potassium magnesium sulfate is 24%, and the magnesium content is 6%.

[0019] Furthermore, the experimental treatment scheme in step (3) is designed as follows: the experiment is designed based on the balance relationship of soil available potassium / magnesium less than 1.4 and calcium / magnesium less than 20, and the magnesium dosage per mu is set to 3kg, 5kg and 7kg. A control treatment without magnesium application is designed, and a randomized block design is repeated 3 times.

[0020] Furthermore, the cultivation measures in step (4) are formulated according to the Jingxi high-quality tobacco production procedures, including: variety layout, seedling cultivation, field cultivation management, and integrated pest and disease control.

[0021] Furthermore, field cultivation management includes:

[0022] 1) Soil improvement and systematic land preparation for tobacco planting;

[0023] 2) Fertilization;

[0024] 3) Transplanting;

[0025] 4) Field management.

[0026] Furthermore, the observation indicator selection in step (5) includes:

[0027] 1) Agronomic traits of tobacco plants during the vigorous growth period and topping period;

[0028] 2) Collect soil and tobacco leaf samples for testing during the vigorous growth period and topping period;

[0029] 3) Tobacco leaf yield per mu, output value and grade indicators;

[0030] 4) Determine the chemical composition of tobacco leaves after flue-curing.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) Significantly increase tobacco yield and economic benefits

[0033] In the prior art, potassium and magnesium antagonism leads to magnesium deficiency in flue-cured tobacco, seriously affecting yield and quality. The present invention significantly increases the dry weight of individual leaves in the upper part of flue-cured tobacco by rationally increasing the application of magnesium fertilizer without inhibiting potassium absorption. The increase in tobacco leaf yield reaches 7.89% to 11.27%, and the increase in output value reaches 7.42% to 14.01%. The JMg3 treatment significantly outperforms the control in increasing yield and income, breaking through the technical bottleneck of traditional excessive magnesium application that inhibits potassium absorption, achieving a dual increase in yield and economic benefits.

[0034] (2) Accurately adjust soil nutrient balance and improve soil fertility

[0035] In the prior art, excessively high effective potassium / magnesium and calcium / magnesium ratios in the soil lead to magnesium absorption disorders. The present invention scientifically sets the magnesium fertilizer application rate (e.g., 5kg or 7kg per mu), effectively reducing the potassium / magnesium and calcium / magnesium ratios while increasing the available potassium and effective magnesium content in the soil, thus breaking the potassium-magnesium antagonism. This not only avoids the inhibition of potassium absorption caused by traditional large-scale magnesium application, but also specifically addresses the problem of soil nutrient imbalance, creating a more optimized rhizosphere soil nutrient environment for flue-cured tobacco growth.

[0036] (3) In view of the problem that the ratio of effective potassium to magnesium, and calcium to magnesium in the tobacco-growing soil in this tobacco-growing area is too high, this situation makes it difficult for flue-cured tobacco to absorb magnesium, which in turn causes magnesium deficiency, seriously affecting the yield and quality of tobacco leaves. In view of this, the present invention is committed to developing a set of experimental schemes for magnesium fertilizer application technology, aiming to scientifically adjust the ratio of effective potassium / magnesium and calcium / magnesium in the soil by increasing the application of magnesium fertilizer, and correspondingly reduce the potassium / magnesium and calcium / magnesium ratios in the flue-cured tobacco leaves, and deeply explore the specific effects of this series of adjustments on the yield and quality of flue-cured tobacco. The present invention explores a set of magnesium fertilizer application strategies that are most suitable for this tobacco-growing area, providing strong practical guidance and theoretical support for optimizing the quality of flue-cured tobacco products, increasing yield and economic benefits.

Brief Description of the Drawings

[0037] Figure 1 This is the principal component analysis diagram of the rhizosphere soil metabolites of flue-cured tobacco under different treatments. The upper frame is the positive ion mode; the lower frame is the negative ion mode. The horizontal axis PC1 and the vertical axis PC2 in the figure represent the scores of the first and second principal components respectively. Scatter points of different colors represent samples from different groups. [Specific implementation method]

[0038] 1. Purpose of the Experimental Study

[0039] In order to address the problem that the effective potassium / magnesium and calcium / magnesium in the tobacco-growing soil in this tobacco-growing area are too high, resulting in magnesium deficiency in flue-cured tobacco due to difficulty in absorbing magnesium, which affects the yield and quality of tobacco leaves, a magnesium application technology experiment was designed to study the effects of increasing magnesium application to reduce the effective potassium / magnesium and calcium / magnesium ratios in the soil and reduce the potassium / magnesium and calcium / magnesium ratios in tobacco leaves on the yield and quality of flue-cured tobacco, and to explore the magnesium application technology for flue-cured tobacco in this tobacco-growing area.

[0040] 2 Experimental materials and methods

[0041] 2.1 Test time

[0042] March 2023 to August 2023.

[0043] 2.2 Test location

[0044] Changfu Village, Xinjing Town, Jingxi City.

[0045] 2.3 Test materials

[0046] Flue-cured tobacco variety: K326;

[0047] Fertilizer: tobacco-specific compound fertilizer (9-9-26), potassium nitrate (13.5-0-44.5), potassium sulfate (0-0-50), potassium magnesium sulfate (24% potassium oxide, 6% magnesium).

[0048] 2.4 Experimental treatment plan

[0049] The experiment was designed based on a balanced relationship between available potassium and magnesium in the soil less than 1.4 and calcium and magnesium less than 20. Treatments with magnesium application rates of 3kg, 5kg, and 7kg per mu were set, and no magnesium application was designed as a control treatment. A randomized block design was used with three replicates. Plot area: 55.3m 2 The specific test plan and fertilization plan are shown in Table 1 and Table 2.

[0050] Table 1 Field test treatment plan kg / mu

[0051]

[0052] Table 2 Field test fertilization table kg / plot

[0053]

[0054]

[0055] 2.5 Other cultivation measures

[0056] According to Jingxi's high-quality tobacco production procedures, including: variety layout, seedling cultivation, field cultivation management, and integrated prevention and control of diseases and pests.

[0057] Furthermore, field cultivation management includes

[0058] 1) Soil improvement and systematic land preparation for tobacco planting;

[0059] 2) Fertilization;

[0060] 3) Transplanting;

[0061] 4) Field management.

[0062] 2.6 Observation indicators

[0063] 1. Agronomic characteristics of tobacco plants during vigorous growth and topping periods

[0064] The agronomic traits of tobacco plants (plant height, stem girth, leaf number, leaf area, etc.) during the vigorous growth period and topping period were measured using YCT 142-2010 “Survey and Measurement Methods for Agronomic Traits of Tobacco”.

[0065] 2. Collect soil and tobacco leaf samples for testing during the vigorous growth period and topping period

[0066] 1) Sample collection and analysis

[0067] 1-1) Collection of rhizosphere soil

[0068] After K326 tobacco plants matured, they were dug out, carefully maintaining their root systems intact. The tobacco was shaken vigorously to remove most of the soil from the roots. The soil adhering to the root surface (rhizosphere soil) was scraped and removed of impurities before being stored in a –80°C refrigerator for testing. Four replicates were set for each treatment.

[0069] 1-2) Determination of rhizosphere soil metabolites

[0070] The sample was processed as follows: After thawing, 1 g of the sample was weighed into a 50 mL centrifuge tube, and 10 mL of 80% methanol / water extract containing 0.1% formic acid was added. After vortexing, ultrasonic extraction was performed for 30 minutes (4°C, 40 kHz), and the sample was allowed to stand for 30 minutes. The sample was centrifuged at 15,000 r / min and 4°C for 10 minutes, and the supernatant was collected and injected into UPLC-Q-Tof for analysis. 20 μL of supernatant was transferred from each sample and mixed as a quality control sample (QC). In this experiment, Waters' ultra-high performance liquid chromatography tandem time-of-flight mass spectrometry UPLC-Q-TOF system was used to analyze the metabolites of the rhizosphere treated with CK and magnesium (see Table 3 for the chromatographic separation gradient elution program). Chromatographic conditions: Column:

[0071] Waters ACQUITY UPLC BEH C18 1.7 μm 2.1*100 mm; column temperature: 40°C; column flow rate: 0.3 ml / min; injection volume: 2 μL; mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol.

[0072] Table 3 Chromatographic separation gradient elution program

[0073]

[0074] During the instrument analysis, a QC sample was inserted into every 6 analytical samples to examine the stability of the entire detection process. Electrospray ionization (ESI) was used to collect mass spectrometry signals in positive and negative ion scanning modes. Mass spectrometry parameters:

[0075] Ion source: ESI

[0076] Mass scan range: 50-1200Da

[0077] Atomizing gas: Nitrogen (N2, PEAKNM32LA nitrogen generator)

[0078] Collision gas: Argon (Ar, purity 99.999%)

[0079] Capillary voltage: 2kV

[0080] Sampling Cone Voltage: 40

[0081] Source Offset Voltage: 80

[0082] Source Temperature: 100°C

[0083] Desolvation temperature: 450°C

[0084] Cone Gas Flow: 50L / h

[0085] Desolvation Gas Flow: 650L / h

[0086] Collision Energy (CE): Low Energy: Off, High Energy: 10-40V

[0087] Calibration solution (LockMass): Leucine enkephalin, positive ion: m / z 556.2771; negative ion: m / z 554.2615

[0088] 1-3) Data processing: The metabolomics software Progenesis QI was used for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Multivariate statistical analysis was performed using SIMCA, and the data were subjected to principal component analysis (PCA) and orthogonal partial least squares analysis (OPLS-DA).

[0089] 3. Tobacco leaf yield, output value and grade indicators per mu

[0090] Yield per mu: The actual weight after picking is converted into yield per mu based on the planting area.

[0091] Output value: Statistics are collected on the output of tobacco leaves of each processing type, the proportion of high-quality tobacco leaves, and the proportion of medium-quality tobacco leaves, and the average price and output value of tobacco leaves are calculated based on the purchase price of the year.

[0092] Grade classification: Implement GB2635-1992 "Flue-cured Tobacco" national standard, and refer to YC / T210.6-2006 Tobacco Leaf Code Part 6: Tobacco Leaf Grade Code.

[0093] 4. Determination of chemical composition of tobacco leaves after flue-curing

[0094] Calcium determination standards

[0095] YC / T 174-2003 Tobacco and tobacco products-Determination of calcium-Atomic absorption method

[0096] Determination of magnesium

[0097] YC / T 175-2003 Determination of magnesium in tobacco and tobacco products-Atomic absorption method

[0098] Determination of water-soluble sugars

[0099] YC / T 159—2019 Tobacco and tobacco products—Determination of water-soluble sugars—Determination of reducing sugars by continuous flow method

[0100] YC / T 251-2008 Tobacco and tobacco products-Determination of glucose, fructose and sucrose by ion chromatography

[0101] Determination of nicotine

[0102] Hydrochloric acid extraction UV spectrophotometry

[0103] Protein determination standards

[0104] YC / T 166-2003 Tobacco and tobacco products - Determination of total protein content 3 Test results and analysis

[0105] 3.1 Analysis of soil test results

[0106] (1) Mass spectra of rhizosphere soil metabolites showed 1878 and 384 significant peaks were identified in the 16 soil samples in positive and negative ion modes, respectively. Based on the HMDB and metlin databases, a total of 134 named metabolites were identified, including 99 in positive ion mode and 35 in negative ion mode.

[0107] (2) Principal component analysis

[0108] The rhizosphere soil samples of three treatments and one control were subjected to principal component analysis (Principal Component Analysis PCA) Figure 1 ).from Figure 1 It can be seen that, regardless of the positive or negative ion mode, the rhizosphere soil samples of flue-cured tobacco were all within their respective 95% confidence intervals, and the treatments and blank controls were significantly distinguished.

[0109] (3) Screening of differential metabolites among rhizosphere soil groups

[0110] The OPLS-DA method combined with VIP value (>1) was used to screen the differential metabolites among treatments based on the HMDB and metlin databases, and a total of 134 metabolites with significant differences (P<0.05) were identified.

[0111] (4) Effects of rhizosphere soil metabolites on tobacco leaf components

[0112] Previous studies have shown that magnesium application technology can improve the yield and quality of K326 flue-cured tobacco. In this study, correlation analysis between the abundance of differential metabolites in rhizosphere soil CK and between treatments and the nutrient content of flue-cured tobacco leaves revealed that differential metabolites were associated with differences in organic matter, total potassium, alkaline-hydrolyzable nitrogen, and available potassium content in tobacco leaves.

[0113] 3.2 Effect of magnesium application on single leaf weight of flue-cured tobacco

[0114] From the results in Table 4, it can be seen that the dry weight of the upper tobacco leaves treated with magnesium application was significantly or extremely significantly higher than that of the control treatment, the dry weight of the lower tobacco leaves treated with magnesium application was not significantly different from that of the control treatment, and the dry weight of the middle tobacco leaves treated with JMg3 was significantly higher than that of the control treatment.

[0115] Table 4 Effects of different treatments on dry weight of single fresh leaves in May and June

[0116] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 12.16±0.36bB 9.54±0.32bA 5.53±0.40aA JMg1 14.91±1.33aAB 10.18±0.45abA 5.05±0.55aA JMg2 15.28±1.11aA 10.65±0.23abA 5.45±0.90aA JMg3 14.45±0.31aAB 11.16±0.83aA 5.25±0.62aA

[0117] 3.3 Effects of magnesium application on agronomic traits of flue-cured tobacco

[0118] As shown in Table 5, magnesium application increased tobacco stem width and chlorophyll content. The stem widths of tobacco leaves treated with JMg2 and JMg3 were significantly greater than those in the control, while the chlorophyll content of tobacco leaves treated with JMg3 was significantly higher than that in the control. Magnesium application also decreased the maximum leaf width of tobacco leaves, with the maximum leaf width of tobacco leaves treated with JMg2 being significantly lower than that in the control. There were no significant differences in stem width or leaf number between the different treatments and the control.

[0119] Table 5 Agronomic traits of flue-cured tobacco under different treatments

[0120]

[0121] 3.4 Effects of magnesium application on nutrient content of fresh flue-cured tobacco leaves

[0122] As shown in Table 6, magnesium fertilization treatments (JMg1, JMg2, and JMg3) had little effect on the nitrogen content of fresh flue-cured tobacco leaves. There were no significant differences in nitrogen content between the leaves of each part and the control (JMgCK) (same letter in the same column). Nitrogen content in the upper leaves ranged from 1.46% to 1.65%, in the middle from 1.16% to 1.33%, and in the lower leaves from 2.24% to 2.36%. Nitrogen content in the lower leaves was significantly higher than in the upper and middle parts, possibly due to leaf maturity or nutrient distribution in the lower leaves. However, magnesium fertilization did not significantly alter nitrogen accumulation patterns.

[0123] Table 6 Nitrogen content of fresh leaves of tobacco plants under different treatments in May and June (dry basis)

[0124] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 1.65±0.0aA 1.22±0.19aA 2.34±0.03aA JMg1 1.55±0.12aA 1.16±0.12aA 2.36±0.05aA JMg2 1.57±0.10aA 1.28±0.10aA 2.24±0.14aA JMg3 1.46±0.21aA 1.33±0.13aA 2.33±0.15aA

[0125] As shown in Table 7, phosphorus content fluctuated slightly among the different treatments. The highest phosphorus content in the upper tobacco leaves was in the JMg3 treatment (0.22%), while the lowest was in the JMg2 treatment (0.16%). Phosphorus content in the middle and lower tobacco leaves ranged from 0.23% to 0.26% and 0.24% to 0.28%, respectively, with no significant differences among the treatments (identical letters in the same column). This suggests that magnesium application had no significant effect on phosphorus absorption in flue-cured tobacco, and that phosphorus content was primarily controlled by the variety or basic nutrient conditions.

[0126] Table 7 Phosphorus content in fresh leaves of tobacco plants under different treatments (dry basis) in May and June

[0127] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 0.19±0.01aA 0.23±aA 0.28±0.04aA JMg1 0.20±0.07aA 0.26±aA 0.27±0.02aA JMg2 0.16±0.00aA 0.25±aA 0.27±0.01aA JMg3 0.22±0.02aA 0.24±aA 0.24±0.02aA

[0128] As shown in Table 8, the potassium content in the upper tobacco leaves increased with the increase of magnesium application (JMgCK to JMg3: 2.68%→3.06%), and JMg3 was significantly higher than the control (P<0.05, letters avsb), indicating that magnesium fertilizer promoted the accumulation of potassium in the upper leaves.

[0129] There was no significant difference in potassium content in the middle tobacco leaves (all in group aA), but the potassium content in the JMg2 treatment was slightly higher (3.71%). The potassium content in the lower tobacco leaves gradually decreased with increasing magnesium application (JMgCK to JMg3: 7.26% → 6.14%), and JMg3 was significantly lower than the control (P < 0.05, letters avsb). This may be due to the competitive effect of magnesium and potassium ions in the root absorption or transportation process, resulting in the transfer of potassium from the lower part to the middle and upper part.

[0130] Table 8 Potassium content of fresh leaves of tobacco plants with different treatments in May and June (dry basis)

[0131] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 2.68±0.10bA 3.50±0.43aA 7.26±0.58aA JMg1 2.79±0.05abA 3.55±0.34aA 7.29±0.78aA JMg2 2.87±0.08abA 3.71±0.07aA 6.90±0.18aA JMg3 3.06±0.26aA 3.61±0.22aA 6.14±0.06aA

[0132] As shown in Table 9, there was no significant difference in calcium content in the upper tobacco leaves (2.17% to 2.38%); the calcium content in the middle tobacco leaves was highest in the JMg2 treatment (2.86%) and lowest in the JMg3 treatment (2.42%), but the overall difference was not significant (same letters in the same column).

[0133] The calcium content in the lower tobacco leaves first increased and then decreased with the amount of magnesium applied, with the highest content in the JMg2 treatment (4.91%) and significantly lower in JMg3 than in the control and JMg1 and JMg2 (P<0.05, letters bvsab), which may be related to the antagonism between magnesium and calcium ions. High magnesium inhibited the accumulation of calcium in the lower leaves.

[0134] Table 9 Calcium content of fresh leaves of tobacco plants with different treatments in May and June (dry basis)

[0135] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 2.38±0.26aA 2.75±0.24aA 4.45±0.13abA JMg1 2.17±0.19aA 2.77±0.17aA 4.63±0.58abA JMg2 2.29±0.17aA 2.86±0.45aA 4.91±0.33aA JMg3 2.32±0.22aA 2.42±0.29aA 4.05±0.37bA

[0136] As shown in Table 10, magnesium application significantly increased the magnesium content in tobacco leaves, and the content increased with the increase in magnesium application (especially in the middle and lower parts):

[0137] The magnesium content in the middle of tobacco leaves was: JMgCK (0.14%) → JMg2 (0.17%), and JMg1 and JMg2 were significantly higher than the control (P<0.05, the difference within the letter aA group was small, but the magnesium content in the middle of JMg1 and JMg2 was higher than that of JMgCK).

[0138] The magnesium content in the lower tobacco leaves was as follows: JMgCK (0.22%) → JMg2 (0.27%). JMg1, JMg2 and JMg3 were significantly higher than the control (P<0.05), indicating that magnesium fertilizer directly promoted magnesium accumulation in leaves and that the lower leaves were more sensitive to magnesium.

[0139] Table 10 Magnesium content in fresh leaves of tobacco plants with different treatments in May and June (dry basis)

[0140] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 0.20±0.02aA 0.14±0.00aA 0.22±0.01aA JMg1 0.20±0.02aA 0.16±0.02aA 0.26±0.06aA JMg2 0.21±0.01aA 0.17±0.03aA 0.27±0.01aA JMg3 0.22±0.02aA 0.15±0.00aA 0.25±0.02aA

[0141] As shown in Table 11, there was no significant difference in the potassium-to-magnesium ratio in the upper tobacco leaves (13.25-13.89), while the middle and lower leaves showed a downward trend with the increase of magnesium application:

[0142] The potassium-magnesium ratio of the lower tobacco leaves was: JMgCK (33.07) → JMg3 (24.49). JMg1, JMg2, and JMg3 were significantly lower than the control (P < 0.05, letters b / cBvsaA), indicating that magnesium application reduced the potassium-magnesium ratio of the lower leaves, which may be related to the fact that the increase in magnesium content was greater than that of potassium.

[0143] The potassium-magnesium ratio affects the combustibility and stress resistance of tobacco leaves. A decrease in the potassium-magnesium ratio of lower leaves may improve their combustion characteristics, but this needs to be comprehensively evaluated in combination with quality indicators.

[0144] Table 11 Potassium-magnesium ratio of fresh leaves of tobacco plants with different treatments in May and June (dry basis)

[0145] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 13.25±0.87aA 25.07±2.98aA 33.07±0.81aA JMg1 13.89±1.37aA 22.33±4.41aA 28.84±3.12bAB JMg2 13.68±1.07aA 22.48±3.74aA 25.70±1.58bcB JMg3 13.78±0.44aA 23.67±0.69aA 24.49±2.47cB

[0146] As shown in Table 12, the calcium-magnesium ratio of each part decreased significantly with the increase of magnesium application (P<0.05):

[0147] Upper tobacco leaves: JMgCK (11.69) → JMg3 (10.44); middle: JMgCK (19.71) → JMg3 (15.87); lower: JMgCK (20.32) → JMg3 (16.11).

[0148] The calcium-magnesium ratio reflects the intracellular ion balance. A decrease in the ratio indicates an increase in the relative magnesium content, which may alleviate the damage of excessive calcium to cell membranes and improve the physiological function of leaves.

[0149] Table 12 Calcium-magnesium ratio of fresh leaves of tobacco plants with different treatments in May and June (dry basis)

[0150] deal with Upper tobacco leaves Central tobacco leaves Lower tobacco leaves JMgCK 11.69±0.24aB 19.71±1.40aA 20.32±0.86aA JMg1 10.74±0.14bB 17.25±1.07abA 18.30±2.13abA JMg2 10.91±0.39bB 16.99±0.52abA 18.27±0.82abA JMg3 10.44±0.27bB 15.87±1.64bA 16.11±1.53bA

[0151] 3.5 Effects of magnesium application on the chemical composition content of flue-cured tobacco leaves

[0152] 3.5.1 Effects of magnesium application on nutrient content in the middle leaves of flue-cured tobacco

[0153] From Table 13 we can see that:

[0154] Nitrogen content: It showed an upward trend with the increase of magnesium application (JMgCK to JMg3: 1.67%→1.79%), but there was no significant difference among the treatments (letters aA in the same column), indicating that magnesium fertilizer had little effect on nitrogen accumulation in the middle tobacco leaves.

[0155] Phosphorus content: JMg1 (0.26%) and JMg2 (0.23%) were slightly higher than the control (0.19%), and JMg3 was the same as the control (0.19%). The overall fluctuation was small and there was no significant difference, indicating that magnesium application did not significantly change the phosphorus absorption pattern.

[0156] Potassium content: JMg3 treatment had significantly lower potassium levels than the other treatments (2.05%, bB), while JMg1 and JMg2 showed no significant differences from the control (3.75% to 4.05%, aA). The sudden drop in potassium content under the high-magnesium treatment (JMg3) may be due to competition between magnesium and potassium ions, which inhibits potassium absorption or promotes its transfer to other parts of the body (consistent with the downward trend in potassium content in the lower part of Table 8).

[0157] Table 13 Nitrogen, phosphorus and potassium content of middle tobacco leaves under different treatments (dry basis)

[0158]

[0159]

[0160] From Table 14 we can see that:

[0161] Calcium content: JMg1 treatment was significantly higher than the other treatments (3.65%, aA), while JMg2 and JMg3 were close to the control (2.76%-3.01%, bB). The medium magnesium treatment (JMg1) promoted calcium accumulation, while the high magnesium treatment (JMg3) did not significantly increase calcium content, possibly indicating a threshold effect in the magnesium-calcium interaction.

[0162] Magnesium content: It showed a significant increasing trend with the increase of magnesium application amount (JMgCK to JMg3: 0.12%→0.18%), and JMg3 was significantly higher than the control and JMg1 (aAvsbA), confirming that magnesium fertilizer directly and efficiently increased the magnesium nutritional level of the middle tobacco leaves, and the high magnesium treatment had the most significant effect.

[0163] Table 14 Calcium and magnesium content of middle tobacco leaves of different treatments (dry basis)

[0164] deal with calcium magnesium JMgCK 2.80±0.13bB 0.12±0.02bA JMg1 3.65±0.14aA 0.13±0.02bA JMg2 2.76±0.26bB 0.16±0.01abA JMg3 3.01±0.16bB 0.18±0.03aA

[0165] From Table 15 we can see that:

[0166] Potassium to magnesium ratio (K / Mg):

[0167] The K / Mg ratio decreased significantly with increasing magnesium application (JMgCK to JMg3: 31.50 → 11.64), reaching only 37% of the control in the JMg3 treatment and significantly lower than in the other treatments (cBvsaA). The decrease in the K / Mg ratio is directly related to the increase in magnesium content, which may affect tobacco leaf burning characteristics (a low ratio is generally associated with good combustibility). However, this assessment should be combined with the absolute potassium content (excessively low potassium content in JMg3 may be detrimental).

[0168] Calcium to magnesium ratio (Ca / Mg):

[0169] The JMg1 treatment showed a significantly higher calcium-to-magnesium ratio than the other treatments (28.46, aA), while JMg2 and JMg3 showed significantly lower calcium-to-magnesium ratios than the control (17.59-17.11, bB). High-magnesium treatment significantly reduced the calcium-to-magnesium ratio by increasing magnesium content, potentially alleviating potential cellular damage from calcium excess and improving ion balance. In contrast, the medium-magnesium treatment (JMg1) exhibited an elevated calcium-to-magnesium ratio due to a sudden increase in calcium content, potentially related to a dose-dependent synergistic or antagonistic effect between magnesium and calcium.

[0170] Table 15 Potassium, calcium and magnesium ratios of the middle leaves of flue-cured tobacco with different treatments (dry basis)

[0171] deal with Potassium / Magnesium Calcium / Magnesium JMgCK 31.50±3.16aA 23.64±2.97abAB JMg1 28.85±1.21abA 28.46±4.21aA JMg2 25.89±2.31bA 17.59±1.06bB JMg3 11.64±1.35cB 17.11±2.87bB

[0172] 3.5.2 Effects of magnesium application on the chemical composition content of middle flue-cured tobacco leaves

[0173] From Table 16 and Table 17, we can see that:

[0174] Main chemical components:

[0175] Nicotine: JMg3 treatment had the highest content (2.50%), while JMg1 had the lowest content (1.83%), but there was no significant difference among the treatments, indicating that magnesium fertilizer had little effect on nicotine synthesis.

[0176] Total water-soluble sugar and reducing sugar: The reducing sugar of JMg3 was significantly lower than that of other treatments (20.00%, bA), and the total water-soluble sugar showed a downward trend (23.10%), which may be due to the influence of high magnesium treatment on carbon metabolism or sugar transport.

[0177] Protein: It increased slightly with the increase of magnesium application (7.94%→8.57%), but there was no significant difference, indicating that magnesium fertilizer has limited effect on protein accumulation.

[0178] Coordination indicators:

[0179] Schmuck value (sugar / protein): There was no significant difference among the treatments (2.72-3.13, aA), indicating that the ratio of sugar to protein was stable and magnesium fertilizer did not significantly affect the balance between the two.

[0180] Sugar-nitrogen ratio and sugar-alkali ratio: The JMg1 treatment had the highest sugar-nitrogen ratio (15.34), while the JMg3 treatment had the lowest (12.99). The sugar-alkali ratio was higher in JMg1 (14.15) and JMg2 (12.26) than in the control (10.63), while it dropped to 9.43 in JMg3. The sugar-alkali ratio is a key indicator of tobacco leaf taste harmony. The medium magnesium treatments (JMg1 and JMg2) improved the sugar-alkali ratio by increasing sugar content or reducing nicotine content, while the high magnesium treatment (JMg3) exhibited slightly poorer harmony due to a decrease in reducing sugars.

[0181] Table 16 Chemical component content of middle tobacco leaves under different treatments (%)

[0182] deal with Nicotine Total water-soluble sugars reducing sugars protein JMgCK 2.37±0.49aA 24.60±2.10aA 23.50±1.60aA 7.94±0.67aA JMg1 1.83±0.40aA 25.20±0.10aA 23.10±1.00aA 8.37±0.22aA JMg2 2.13±0.45aA 25.30±1.20aA 22.90±0.30aA 8.45±1.07aA JMg3 2.50±0.36aA 23.10±1.99aA 20.00±1.71bA 8.57±0.78aA

[0183] Table 17 Chemical composition harmony of middle leaves of flue-cured tobacco with different treatments

[0184] deal with Schmuck value Sugar-nitrogen ratio Sugar-alkali ratio JMgCK 3.13±0.51aA 14.80±1.63aA 10.63±1.72 JMg1 3.01±0.07aA 15.34±0.70aA 14.15±2.72 JMg2 3.02±0.36aA 14.86±1.24aA 12.26±2.82 JMg3 2.72±0.41aA 12.99±1.78aA 9.43±2.06

[0185] 3.6 Effects of magnesium application on tobacco yield and output value

[0186] As shown in Table 18, when converted to per-acre tobacco leaf yield, the magnesium-treated flue-cured tobacco yield reached 118.03-1121.73 kg per acre, and the tobacco leaf output value reached 3891.45-4130.02 kg per acre. The plot experiment showed that magnesium application increased tobacco leaf yield and output value by 7.89%-11.27% and 7.42%-14.01%, respectively. The increases in yield and income from the JMg3-treated flue-cured tobacco were significantly higher than those from the control.

[0187] Table 18 Tobacco yield and output value under different treatments

[0188] deal with Yield per mu (kg) Increase in production (%) Output value per mu (yuan) Value-added (%) JMgCK 109.40±2.73bA — 3622.54±64.66bA — JMg1 118.03±10.10abA 7.89 <h2 style=";text-align:left;direction:ltr">3891.45±213.70abA 7.42 <h2 style=";text-align:left;direction:ltr"> JMg2 <h2 style=";text-align:left;direction:ltr"> 118.55±5.72abA 8.36 <h2 style=";text-align:left;direction:ltr"> 3928.86±263.94abA 8.46 <h2 style=";text-align:left;direction:ltr"> JMg3 <h2 style=";text-align:left;direction:ltr"> 121.73±7.68 aA 11.27 <h2 style=";text-align:left;direction:ltr"> 4130.02±392.67aA 14.01

[0189] 4 Research Conclusions

[0190] 1) Excessive levels of available potassium / magnesium and calcium / magnesium in tobacco-growing soils in the Jingxi tobacco-growing region lead to magnesium deficiency due to difficulty absorbing magnesium, which in turn affects tobacco leaf yield and quality. Therefore, a magnesium application experiment was designed to investigate its effects on rhizosphere soil metabolites, which may be key to regulating tobacco leaf quality and yield. This study, based on the K326 flue-cured tobacco variety, employed different magnesium application protocols. Rhizosphere soil samples were collected during the harvest period of the K326 tobacco plant. Ultra-performance liquid chromatography–time-of-flight mass spectrometry (UPLC-Q-Tof-MS) was used to characterize the composition and function of rhizosphere soil metabolites during the harvest period of the K326 tobacco plant, and their relationship to yield development was investigated. The results showed that: ① OPLS-DA modeling was used to screen for differentially expressed metabolites (HMDB and MetlinMSMS databases). 387 significantly different (VIP > 1) rhizosphere soil metabolites were identified, of which 134 were specifically identified.

[0191] 2) Direct effect of magnesium fertilizer: Magnesium application significantly increased the magnesium content of tobacco leaves (especially in the middle and lower parts), and the content increased with the increase of magnesium application rate, indicating that magnesium fertilizer effectively supplemented the magnesium nutrition of tobacco plants.

[0192] 3) Indirect effects on other elements: promoting potassium accumulation in upper leaves but inhibiting potassium content in lower leaves, which may be related to ion competition or transport distribution.

[0193] 4) There was no significant effect on nitrogen and phosphorus content, indicating that the interaction between magnesium and nitrogen and phosphorus was weak.

[0194] 5) The calcium content of the lower leaves increased slightly in the medium and low magnesium treatments (JMg1 and JMg2), but decreased significantly in the high magnesium treatment (JMg3), which may indicate the existence of a magnesium-calcium antagonism.

[0195] 6) Changes in ion ratios: Magnesium application reduced the calcium-magnesium ratio in each part and the potassium-magnesium ratio in the lower part, which was beneficial to improving the ion balance and combustion characteristics of the leaves.

[0196] 7) Direct effects of magnesium fertilizer on middle tobacco leaves:

[0197] The significant increase in magnesium content (especially JMg3) verified the effectiveness of magnesium fertilizer supplementation; the medium magnesium treatment (JMg1) unexpectedly promoted calcium accumulation, which may be related to the root ion absorption dynamics.

[0198] 8) Interaction with other elements:

[0199] High magnesium treatment (JMg3) significantly inhibited potassium absorption, aggravated magnesium-potassium antagonism, and led to a sharp drop in the potassium-magnesium ratio; medium magnesium treatment (JMg1) may increase the calcium-magnesium ratio by promoting calcium absorption, showing the complexity of the magnesium-calcium relationship.

[0200] 9) Impact on chemical composition:

[0201] Medium magnesium treatments (JMg1 and JMg2) improved the harmony of tobacco leaves by optimizing the sugar-alkali ratio, while high magnesium treatments may have a potential negative impact on quality due to decreased reducing sugar and low potassium content.

[0202] 10) Magnesium application significantly or extremely significantly increased the dry weight of single leaf of the upper tobacco leaf, and significantly increased the yield and output value of tobacco leaves, with yield increases of 7.89%-11.27% and income increases of 7.42%-14.01%, respectively. The yield and income increases of tobacco leaves treated with JMg3 were significantly higher than those of the control treatment.

[0203] 11) Magnesium application had no significant effect on agronomic traits of flue-cured tobacco.

[0204] 12) The treatments with magnesium application rates of 5 and 7 kg per mu significantly increased the content of available potassium and effective magnesium in the flue-cured tobacco soil in May.

[0205] 13) Production inspiration:

[0206] Moderate magnesium application (such as JMg1 and JMg2) can improve the coordination of chemical components while enhancing magnesium nutrition, and avoid abnormal potassium and sugar content caused by high magnesium treatment.

[0207] The above content cannot be used to determine that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the concept of the present invention, which should be regarded as falling within the scope of patent protection of the present invention determined by the submitted claims.

Claims

1. A method for exploring the effects of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the content of chemical components in tobacco leaves, characterized in that: The following steps are involved: (1) Selection of trial sites; (2) Selection of test materials; (3) Experimental treatment plan design; (4) Development of cultivation measures; (5) Selection of observation indicators.

2. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 1, characterized in that: The experimental site in step (1) was selected as Changfu Village, Xinjing Town, Jingxi City.

3. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 1, characterized in that: The test materials in step (2) include flue-cured tobacco varieties and fertilizers.

4. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 3, characterized in that: The flue-cured tobacco variety is K326 flue-cured tobacco.

5. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 3, characterized in that: The fertilizer includes tobacco compound fertilizer, potassium nitrate, potassium sulfate, and potassium magnesium sulfate.

6. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 5, characterized in that: The potassium magnesium sulfate has a potassium oxide content of 24% and a magnesium content of 6%.

7. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 1, characterized in that: The experimental treatment scheme in step (3) is designed as follows: the experiment is designed based on the balance relationship of soil available potassium / magnesium less than 1.4 and calcium / magnesium less than 20, and the magnesium dosage per mu is set to 3kg, 5kg and 7kg. A control treatment without magnesium application is designed, and a randomized block design is repeated 3 times.

8. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of rhizosphere soil of K326 flue-cured tobacco and the content of chemical components in tobacco leaves according to claim 1, characterized in that: The cultivation measures in step (4) are formulated according to the Jingxi high-quality tobacco production procedures, including: variety layout, seedling cultivation, field cultivation management, and integrated pest and disease control.

9. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of rhizosphere soil of K326 flue-cured tobacco and the content of chemical components in tobacco leaves according to claim 8, characterized in that: Field cultivation management includes: 1) Soil improvement and systematic land preparation for tobacco planting; 2) Fertilization; 3) Transplanting; 4) Field management.

10. The method for exploring the effect of magnesium application technology on the metabolome expression characteristics of K326 flue-cured tobacco rhizosphere soil and the chemical component content of tobacco leaves according to claim 1, characterized in that: The observation indicators selected in step (5) include: 1) Agronomic traits of tobacco plants during the vigorous growth period and topping period; 2) Collect soil and tobacco leaf samples for testing during the vigorous growth period and topping period; 3) Tobacco leaf yield per mu, output value and grade indicators; 4) Determine the chemical composition of tobacco leaves after flue-curing.

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