Xia NI, Shungang SHEN, Xianyi ZENG, Dewen LI, Yeming FU, Min CHEN, Chaokai YU, Libing QI, Yingguo RUAN, Changchong ZI, Haohan GONG, Guangjun HU, Mengs
Abstract ［Objectives］ This study was conducted to improve acidic soil and enhance the quality of tobacco leaves.
［Methods］ The effects of different microbial fertilizers on improving acidic tobacco-planting soil and tobacco leaf quality were investigated through plot experiments.
［Results］ The application of microbial fertilizers could improve the pH value of acidic soil， and composite microbial agent A showed the best application effect. The application of bio-organic fertilizer was beneficial to improving the contents of available phosphorus and available boron in acidic soil. The application of composite microbial agent A was beneficial to improving the contents of available phosphorus and available potassium in acidic soil， and could promote the growth of tobacco plants and improve the economic traits of flue-cured tobacco and the coordination of chemical components in tobacco leaves. The application of composite microbial agent B led to a downward trend in the content of available boron in acidic soil. The application of composite microbial agent B could promote the absorption of nutrients by tobacco plants， and improve their disease resistance and the quality of tobacco leaves. Due to the differences in functional microorganisms contained， the application effects of different microbial fertilizers in improving acidic tobacco-planting soil and improving tobacco leaf quality varied. Overall， the application of microbial fertilizers could increase soil pH， activate soil nutrients， promote tobacco growth， enhance disease resistance， increase tobacco output value， and improve tobacco quality. Microbial fertilizers have good application prospects in improving acidic soil and improving tobacco quality.
［Conclusions］ The application of microbial fertilizers to improve acidic tobacco-planting soil can ensure the normal growth and development of tobacco plants and the improvement of tobacco leaf quality， achieving high-quality and sustainable development of Zhaotong tobacco.
Key words Microbial fertilizer; Flue-cured tobacco; Acidic soil; Tobacco quality
Received：
December 6， 2022 Accepted：
February 7， 2023
Supported by Science and Technology Program Major Project of Yunnan Branch， China National Tobacco Corporation （2020530000241022）.
Xia NI （1968-）， female， P. R. China， senior agronomist， devoted to research about tobacco cultivation and nutritional regulation， physiology and biochemistry.
*Corresponding author. Min CHEN （1970-）， female， P. R. China， senior agronomist， devoted to research about crop diseases and pests and nutritional regulation. E-mail：
928218887@qq.com.
The tobacco industry is one of the leading industries in Yunnan Province， occupying a pivotal position in the national economic development of Yunnan Province［1］. In recent years， with the continuous adjustment of rural industrial structure， the agriculture in Yunnan Province has shown a trend of diversified development， and the contradiction between cash crop such as vegetables and flowers and flue-cured tobacco is increasingly prominent. In order to alleviate this contradiction， many areas in Yunnan have adopted the method of developing new tobacco-growing areas and shifting tobacco planting layout from dam areas to mountainous and semi-mountainous areas. However， severe soil acidification in some mountainous and semi-mountainous areas poses great challenges to the production of high-quality tobacco leaves. To achieve high-quality and sustainable development of Yunnan tobacco， effective measures must be taken to improve acidic soil.
In agricultural production， applying lime to improve acidic soil is the most traditional measure. The experiment carried out by Wang et al.［2］ on red soil in Jiangxi showed that applying 3 000 kg/hm2 of limestone powder could effectively reduce the acidity value of the soil surface by 2-3 units. Duan et al.［3］ increased the pH value of soil by applying lime to acidic yellow soil， and the results showed that applying lime could promote the growth and development of flue-cured tobacco， thereby significantly increasing its yield and quality. Minerals such as dolomite and fly ash， as well as industrial waste， can also improve acidic soil. In tobacco cultivation， an appropriate amount of dolomite powder can promote tobacco plant growth［4］. Organic materials such as crop stems， livestock manure， green manure and plant ash are also used in the improvement of acidic soil. These organic materials can not only increase soil pH， but also effectively reduce soil heavy metal content， and affect the comprehensive fertility level of the soil by enhancing soil enzyme activity［5-9］. Biochar is composed of tightly and irregularly stacked aromatic ring layers， which endow its surface with oxygen-containing functional groups， and it thus exhibits strong alkalinity. Such structure and properties make biochar show great application prospects in improving acidic soils［10-11］. Studies have shown that the application of biochar can effectively increase soil pH， soil carbon pool， soil base saturation， soil cation exchange capacity， as well as the contents of available phosphorus， available potassium， and available nitrogen in the soil. Meanwhile， it effectively reduces the toxic effect of aluminum on plants in acidic soil， thereby changing soil texture and ultimately improving the environment of plant growth ［12-15］.
Microbial agents contain active microorganisms that can promote the transformation of substances in the soil， enhance plant nutrient absorption， improve crop nutrition， and regulate crop growth， thereby achieving the goal of increasing crop yield［16］. In recent years， in order to enhance the disease resistance of tobacco plants and improve the yield and quality of tobacco leaves， microbial fertilizers have been widely used in tobacco production［17-21］， but there have been few reports on their use in the improvement of acidic tobacco-growing soil. In this study， in the context of migration in the Ludian tobacco-growing area of Zhaotong City， microbial fertilizers were applied to improve acidic tobacco-planting soil to ensure the normal growth and development of tobacco plants and the improvement of tobacco leaf quality， so as to achieve high-quality and sustainable development of Zhaotong tobacco.
Materials and Methods
Experimental location
The experiment was carried out in Xinle Village， Longshu Town， Ludian County， Zhaotong City， with an altitude of 2 149.8 m， N：
27.362645 °， E：
103.439865°. The experimental field had a pH of 5.02， and contained organic matter 42.67 g/kg， hydrolyzable nitrogen 194.48 mg/kg， available phosphorus 48.94 mg/kg， available potassium 245.24 mg/kg， available zinc 3.53 mg/kg， available boron 5.63 mg/kg， and chloride ion 12.68 mg/kg， and the soil belonged to acidic soil.
Experimental materials
Test variety：
Yunyan 99.
Test fertilizers：
Compound fertilizer special for tobacco （N-P2O5-K2O=11-15-22） and potassium sulfate （K2O≥50%） were provided by local tobacco companies. Bio-organic fertilizer （containing phosphorus-solubilizing， potassium-dissolving and growth-promoting microorganisms， effective viable count≥20 million/g） was provided by Yunnan Yunye Fertilizer Co.， Ltd. Compound microbial agent A （a combination of Bacillus subtilis and Bacillus amylogenes with phosphorus-solubilizing， potassium-dissolving and growth promoting functions， with an effective viable count≥1 billion/ml） was provided by Yunnan Microbial Fermentation Engineering Research Center Co.， Ltd. Compound microbial agent B （a mixture of Bacillus megaterium， Bacillus licheniformis， Brevibacillus laterosporu and Trichoderma harzianum with growth promoting function， effective viable count≥1 billion/g） was provided by Hunan Zhongxiang Zhenfeng Biological Engineering Co.， Ltd.
Experimental design
A randomized block experimental design was adopted， with 4 treatments in 3 replicates， each of which formed a single plot， totaling 12 plots. Various treatment：
T1 （conventional fertilization）：
75 kg/hm2 of compound fertilizer special for tobacco was applied during transplantation， and 450 kg/hm2 of potassium sulfate was top-applied in a dry manner on a rainy day or in a mixture with water on a sunny day in the period from the rosette stage to the vigorous growth stage at the positions which the leaf tips of the largest leaves were perpendicular to by drilling a hole; T2：
750 kg/hm2 of bio-organic fertilizer was applied during transplantation on the basis of conventional fertilization; T3：
30 L/hm2 of composite microbial agent A was applied on the basis of conventional fertilization; and T4：
30 L/hm2 of composite microbial agent B was applied in a manner of water dilution with a dilution ratio of 300 times on the basis of conventional fertilization in the period from the rosette stage to the vigorous growth stage. All treatments had consistent management measures except for application of different fertilizers.
Investigation items and methods
Soil nutrient investigation
Before fertilization， a basic soil sample was taken before fertilization， and one sample was taken from each treatment after the tobacco leaves were harvested and cured， totaling 5 soil samples （2 kg per sample）. The soil pH value， organic matter， hydrolyzable nitrogen， available phosphorus， available potassium， available zinc， available boron， and chloride ions were detected and analyzed.
Investigation of agronomic traits
After topping， 15 representative tobacco plants were randomly selected from each treatment for the observation of plant height， stem girth， number of leaves， node distance， and leaf length and width of each part of tobacco leaves.
Investigation of disease occurrence
Throughout the entire growth period of flue-cured tobacco， a systematic investigation of major diseases and pests was conducted in accordance with the grading and investigation methods of tobacco diseases and pests in GB/T 23222-2008.
Investigation of economic traits
Tobacco leaves in various treatments were harvested and cured separately in the normal maturation stage， and graded and sold according to the same standard. The economic traits of flue-cured tobacco in various treatments were investigated， including yield， output value， average price， proportion of high-class tobacco， and proportion of mid- and high-class tobacco.
Analysis of tobacco leaf quality
Representative tobacco plants were selected and labeled from each treatment to collect the middle leaves， and after harvesting and curing， a total of 2 kg of tobacco leaves was left for each treatment for routine chemical composition analysis and sensory quality evaluation.
Data analysis
Excel 2016 software was used to collate relevant data. SPSS 22.0 was used for one-way ANOVA， and Duncans new multiple range method was used for significance analysis between different treatments （P<0.05）.
Result and Analysis
Effects of microbial fertilizers on pH and organic matter content of acidic soil
From Table 1， it can be seen that compared with the control T1， the pH values of treatments T2-T4 applied with microbial fertilizers all showed an upward trend， with treatment T3 showing the most obvious trend， indicating that the application of microbial fertilizers could improve the pH value of acidic soil， and composite microbial agent A had the best application effect. The organic matter contents of various treatments after curing were irregular， indicating that different microbial fertilizers had varying effects on the organic matter content of acidic soil. Among them， the application of composite microbial agent A was beneficial to improving the organic matter content of acidic soil.
Effects of microbial fertilizers on hydrolyzable nitrogen， available phosphorus and available potassium contents in acidic soil
The test results of soil hydrolyzable nitrogen， available phosphorus， and available potassium content in each treatment after harvesting and curing of tobacco leaves are shown in Table 2. From the table， it can be seen that there were small differences in the content of hydrolyzable nitrogen among different treatments， indicating that the application of microbial fertilizers had little effect on the nitrogen content of acidic soil. Compared with the control T1， the available phosphorus content of treatment T3 and the available potassium contents of treatments T2 and T3 showed a significant upward trend， indicating that the application of bio-organic fertilizer was beneficial to improving the available phosphorus content of acidic soil， and the application of composite microbial agent A was beneficial to improving available phosphorus and available potassium contents of acidic soil.
Effects of microbial fertilizers on the contents of available zinc， available boron and chloride ions in acidic soil
The detection results of available zinc， available boron， and chloride ion contents in the soil of each treatment after harvesting and curing of tobacco leaves are shown in Table 3. From the table， it can be seen that there were small differences in available zinc content among different treatments. Compared with the control T1， the available boron and chloride ion contents of treatment T2 showed an upward trend， while in treatment T3， the available boron content showed no significant change and the chloride ion content showed a downward trend. The available boron content of treatment T4 showed a significant downward trend， while the chloride ion content did not show a significant change. Overall， the application of microbial fertilizers had little effect on available zinc content of acidic soil. The application of bio-organic fertilizer was beneficial to improving available boron content of acidic soil， while the application of composite microbial agent B could lead to a decrease in available boron content of acidic soil.
Effects of microbial fertilizers on agronomic traits of flue-cured tobacco
The results of the investigation on the agronomic traits of flue-cured tobacco after topping are shown in Table 4. From the table， it can be seen that there were no significant differences in the main agronomic traits of various treatments after topping. The plant height was slightly lower in treatment T2， and the differences among other treatments were not significant. The stem girth was the largest in T3， the smallest in T2， and the remaining two treatments were between the two. Node distance was slightly higher in T1 and T4， and the number of leaves per plant was slightly higher in T3. The length and width of tobacco leaves in various parts were the largest in T3. Overall， T3 performed the best in agronomic traits among various treatments after topping， while treatments showed little difference， indicating that the application of composite microbial agent A could improve the agronomic traits of flue-cured tobacco， while bio-organic fertilizer and composite microbial agent B had little effect on the agronomic traits of flue-cured tobacco.
Xia NI et al. Effects of Microbial Fertilizers in Improving Acidic Tobacco-planting Soil and Tobacco Leaf Quality
Effects of microbial fertilizers on the occurrence of tobacco diseases
It can be seen from Table 5 that the main diseases observed in the test site include potato virus Y disease， common tobacco mosaic disease， climate scab， brown spot disease and spotted wilt virus， of which potato virus Y disease and climate scab were more serious. The incidence rates of potato virus Y disease ranked from high to low as T3>T2>T1>T4. The incidence rates of tobacco mosaic disease were in order of T1>T3>T4>T2 from high to low. The incidence rate of climate scab showed an order of T2>T4>T1>T3 from high to low. The incidence rate of brown spot disease was slightly higher in T1 and T2， and that of spotted wilt virus was slightly higher in T1 and T3. Overall， the occurrence of diseases was relatively mild in T4 among various treatments， while other three treatments showed small differences， indicating that the application of bio-organic fertilizer and microbial inoculant A had little impact on the occurrence of tobacco diseases， while microbial inoculant B could improve the disease resistance of tobacco plants and reduce the occurrence of diseases.
Effects of microbial fertilizers on economic traits of flue-cured tobacco
The statistical results of economic traits of flue-cured tobacco are shown in Table 6. From the table， it can be seen that the yield and output value were highest in T3 among various treatments， followed by T4， and slightly lower in T1 and T2. The average price was the highest in T4， followed by T2 and T3， and slightly lower in T1. The proportions of high-class tobacco and mid- and high-class tobacco ranked from high to low as T4>T3>T2>T1. Overall， the economic traits of treatments T2-T4 were better than those of control T1， and treatment T3 performed the best. It indicated that the application of microbial fertilizers could improve the economic traits of flue-cured tobacco， and composite microbial agent A had the best effect.
Effects of microbial fertilizers on chemical composition of tobacco leaves
The detection results of chemical composition in cutters are shown in Table 7. From the table， it can be seen that the total sugar contents of T1 and T2 were relatively high， while the contents of T3 and T4 were moderate. The reducing sugar content was moderate in various treatments， with T1 and T2 slightly higher. The total nitrogen content was higher in T3 and T4， followed by T1， and lowest in T2. The nicotine content was highest in T2， followed by T3 and T4， and lowest in T1. The potassium ion content was highest in T4， and there were no significant differences among other treatments. The differences in chloride ion content among different treatments were not significant. The differences between the two sugars were relatively high among various treatments， and T2 showed the highest value. Except for T1， which was slightly higher in the ratio of total sugar to nicotine， all other treatments were suitable. The differences in the ratio of total nitrogen to nicotine were not significant among different treatments. Overall， the chemical components in cutters were most coordinated among various treatments in T3， followed by T4， and slightly worse in T1 and T2. It indicated that the application of composite microbial agent A and composite microbial agent B could improve the chemical components of tobacco leaves， while the application of bio-organic fertilizer had no significant impact on the chemical components of tobacco leaves.
Effects of microbial fertilizers on sensory quality of tobacco leaves
The sensory quality evaluation results of cutters are shown in Table 8. From the table， it can be seen that there were small differences in the strength， concentration， aftertaste， and irritancy among various treatments. The aroma quality was best in T4， followed by T1 and T3， and slightly worse in T2. T4 had the most abundant aroma， and there were no significant differences among other treatments. The offensive odor was the lightest in T4， followed by T2 and T3， and slightly heavier in T1. Overall， T4 had the best sensory quality in cutters， and other treatments differed little. It indicated that composite microbial agent B could improve the sensory quality of tobacco， while the application of composite microbial agent A and bio-organic fertilizer had no significant impact on the sensory quality of tobacco.
Conclusions and Discussion
In the process of tobacco production， the yield and quality of tobacco are influenced by various factors， among which good soil quality is the prerequisite for producing high-quality tobacco leaves［22］. Soil pH is one of the important indexes of soil nutrients， as well as a comprehensive reflection of soil physical and chemical properties and fertility characteristics. Soil acidification will reduce soil fertility， significantly affect the activities of some microorganisms in the soil， and then affect the synthesis and decomposition of organic matter， the transformation and release of nutrient elements， and the ability of soil to maintain nutrients， thus affecting the improvement of soil production performance and the growth and development of tobacco［23-26］. At present， inorganic or organic modifiers are mostly used for acid soil improvement， such as lime， dolomite， phosphogypsum， crop straw， livestock manure， green manure and grass ash［27-29］.
In this study， the results of the application of different microbial fertilizers on typical acidic soil in Ludian， Zhaotong showed that the application of microbial fertilizers could improve the pH value of acidic soil， and compound microbial agent A showed the best application effect. Different microbial fertilizers had varying effects on the organic matter content of acidic soil， and the application of composite microbial agent A was beneficial to improving the organic matter content of acidic soil. Microbial fertilizers had little effect on the contents of available nitrogen and available zinc in acidic soil， but the application of bio-organic fertilizer was beneficial to improving the contents of available phosphorus and available boron in acidic soil. The application of composite microbial agent A was beneficial to improving the contents of available phosphorus and available potassium in acidic soil. The application of composite microbial agent B led to a downward trend in the content of available boron in acidic soil. The effects of different microbial fertilizers on the nutrients of acidic soil varied， which was mainly related to the different functional microorganisms they contained. The bio-organic fertilizer and composite microbial agent A both contained functional microorganisms that dissolve phosphorus and potassium， which could increase the content of available phosphorus and available potassium in the soil after application， while the main function of the functional microorganisms contained in composite microbial agent B was to promote growth and the absorption of nutrients by tobacco plants， and the available nutrients in the soil were generally lower than the control after application.
The improvement effects of microbial fertilizers on acidic soil are also reflected in promoting tobacco growth， enhancing disease resistance， and improving economic traits and leaf quality of flue-cured tobacco. In this study， the application of compound microbial agent A was the best in promoting the growth and development of tobacco plants and improving the agronomic traits of flue-cured tobacco. The application of compound microbial agent B was the best in improving the disease resistance of tobacco plants and reducing disease occurrence. In terms of increasing the output value of tobacco leaves and improving the economic traits of flue-cured tobacco， the application effect of composite microbial agent A was the best. In terms of improving the coordination of chemical components in tobacco leaves， the application effects of composite microbial agent A and composite microbial agent B were better. The application of compound microbial agent B was the best in improving the aroma quality， enhancing the aroma content， and enhancing the sensory quality of tobacco leaves.
In summary， due to the differences in functional microorganisms contained， the application effects of different microbial fertilizers in improving acidic tobacco-planting soil and improving tobacco leaf quality varied. Overall， the application of microbial fertilizers could increase soil pH， activate soil nutrients， promote tobacco growth， enhance disease resistance， increase tobacco output value， and improve tobacco quality. Microbial fertilizers have good application prospects in improving acidic soil and improving tobacco quality.
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