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Abstract Pot experiments were conducted to study the effects of different organic fertilizers made from distillers grains on the physiological characteristics and growth of flue-cured tobacco. The results showed that the agronomic traits and biomass of flue-cured tobacco in the three treatments were higher than that of the control group， and the mixed application of different organic fertilizers made from distillers grains and compound fertilizer could promote the growth and development of flue-cured tobacco significantly.
Key words Organic fertilizers made from distillers grains； Agronomic characters； Biomass； Chlorophyll content
Organic fertilizers， carbonaceous materials， mainly derive from plants and （or） animals， and their main function is to provide nutrition for plants after being applied to soil. Organic fertilizers are closely related to flue-cured tobacco. Experimental studies have shown that different types of organic fertilizers can significantly increase the quantity of bacteria and actinomycetes in the rhizosphere of tobacco soil. Different organic fertilizers can significantly increase the yield， output value， and proportion of upper-middle tobacco[1]. The application of biological organic fertilizer can not only greatly increase the number of bacteria， fungi and actinomycetes in the rhizosphere soil of tobacco plants， but also increase the plant height and leaf area coefficient of tobacco and effectively inhibit the incidence of tobacco black shank[2]. The organic fertilizers made from distillers grains are fully fermented and produced by mixing the distillers grains that are left in the process of producing white spirit with oil cake， cow dung and fermentation inocula in a certain proportion. The use of distillers grains to produce organic fertilizers is not only environmentally friendly， but also can provide a conducive environment for the growth of green flue-cured tobacco and improve ecological， economic and social benefits[3]. In distillers grains， the content of organic matter is higher， and the ratio of carbon to nitrogen is suitable. After being applied into soil， they can not only promote the growth of flue-cured tobacco， but also increase the yield of flue-cured tobacco[4]. They not only can provide comprehensive nutrition for crops， but also have long fertilizer efficiency. In addition， they can increase and renew soil organic matter， promote microbial reproduction， and improve physical and chemical properties and biological activity of soil， and they are one of the main nutrients for the production of green crops. At present， there are many studies on the effects of organic fertilizers on the growth of flue-cured tobacco[5-7]. For instance， Wang Lifang et al.[8] pointed out that the organic fertilizers made from distillers grains could improve the the appearance quality， chemical composition and sensory quality of tobacco to a certain degree. Shao Fuwen et al.[9] suggested that the combination of low-dose sulphur and distillers grains could significantly increase tobacco yield and proportion of upper-middle tobacco[10]. However， effects of the organic fertilizers made from distillers grains on the growth and development of flue-cured tobacco have been rarely reported at present. In this study， through pot experiments， the effects of different organic fertilizers made from distillers grains on the growth and development of flue-cured tobacco were studied to provide a basis for the application of organic fertilizers made from distillers grains in the production of flue-cured tobacco. Materials and Methods
Experimental materials
The experiment was conducted at the tobacco testing base of Yunnan Agricultural University in 2017. The variety of flue-cured tobacco was Yunyan 85. The soil used in the pot experiment was yellow loam that was collected from the field behind Yunnan Agricultural University. The chemical properties of the soil are as follows： pH was 7.29， and the content of organic matter was 2.94%； the content of available potassium， available phosphorus， and alkaline nitrogen was 72， 6.0， and 117 mg/kg respectively. Three types of organic fertilizers made from distillers grains were fully fermented and made by mixing the distillers grains with oil cake， cow dung and fermentation inocula in a certain proportion （Table 1）.
Experimental design
There were three treatments， including T1 （18.5 g/kg of organic fertilizer A + 50 g of compound fertilizer）， T2 （18.5 g/kg of organic fertilizer B + 50 g of compound fertilizer）， T3 （18.5 g/kg of organic fertilizer C + 50 g of compound fertilizer）， and CK （130 g of compound fertilizer）. Each treatment had four repetitions， and there were 16 pots that were arranged randomly. Each pot contained 20 kg of the soil. The amount of pure nitrogen applied in the soil was 105 kg/hm2， and the planting density of tobacco was 16 500 plants/hm2. After the even mixture of the organic fertilizers， compound fertilizer and soil was put in a pot， tobacco seedlings were planted in the pot.
Main measurement indicators and methods
Agronomic characters
According to the Investigating and Measuring Methods of Agronomical Characters of Tobacco， the plant height， stem girth， maximum leaf length， and maximum leaf width of flue-cured tobacco in the resettling period， vigorous growth period and maturation period were measured， and the maximum leaf area was calculated （maximum leaf length×maximum leaf width×0.634 5）.
Biomass
After the tobacco plants in each treatment were mature physiologically， the samples were devastatingly collected. The roots， stems and leaves of the plants were separated， washed with tap water， and then wiped with clean paper. After there was no water on them， they were weighed， and their fresh weight was recorded separately. They were dried for 30-60 min at 105 ℃ and then dried at 70 ℃ until their weight was constant. Finally， they were weighed to record their dry weight.
Chlorophyll content Firstly， 2 g of tobacco leaves were taken from the upper， middle and lower part of each tobacco plant respectively， and three copies of the tobacco leaves were collected from each part， so there were 126 treatments. After chlorophyll was extracted with 96% ethanol （or 80% acetone）， the absorbance values at wavelength 645 and 663 nm were measured with a spectrophotometer， and chlorophyll content in each treatment was calculated. The formula is as follows： Ca=12.72A663-2.59A645， Cb=22.88A663-4.67A649， CT=Ca+Cb； chlorophyll content （mg/g）=（chlorophyll concentration×volume of extract×dilution ratio）/fresh weight of samples. Statistical method
Results and Analysis
Effects of application of organic fertilizers made from distillers grains on the agronomic characters of flue-cured tobacco in various periods
Agronomic traits reflect the growth status of tobacco plants at different growth stages and more intuitively illustrate the growth of tobacco plants. According to Table 2， in the resettling period， vigorous growth period and maturation period， the plant height of tobacco in various treatments was as follows： T2>T1>T3>CK， T2>T1>T3>CK， T1>T2>T3>CK. In the three periods， there were obvious differences between CK and T1， T2 or T3. In the three periods， the number of effective leaves was larger in treatments T1， T2 and T3 than CK， but the differences were not obvious. In the three periods， the maximum leaf area was larger in treatments T1， T2 and T3 than CK， and the differences were significant， but there was no obvious difference between treatments T1， T2 and T3. The above results showed that the growth of tobacco plants in treatment T2 was the best， followed by treatment T1. The growth of tobacco plants in treatments T1， T2 and T3 was better than CK. The combination of organic fertilizers made from distillers grains and special compound fertilizer for tobacco could promote the growth of tobacco plants. The agronomic characters of flue-cured tobacco in different treatments 98 d after the tobacco seedlings were transplanted are shown in Table 3.
Biomass referred to the dry and fresh weight of roots， stems and leaves of the harvested tobacco plants， and the quality of the roots， stems and leaves reflected the growth of the tobacco plants. Seen from Table 4， in the maturation period， the dry and fresh weight of the stems and the dry weight of the leaves in treatments T1， T2 and T3 were larger than CK. The fresh weight of the roots in treatments T1 and T2 was higher， while there was no obvious difference between treatment T3 and CK. The results revealed that the combination of organic fertilizers made from distillers grains and special compound fertilizer for tobacco could promote the accumulation of tobacco biomass obviously.
Chlorophyll content reflects the ability of plants to perform photosynthesis. The chlorophyll content of flue-cured tobacco in various treatments was as follows： CK>T3>T1>T2. The results showed that the combination of organic fertilizers made from distillers grains and special compound fertilizer for tobacco could promote the growth and development of tobacco plants. In the same time， the more mature the tobacco leaves were， the lower the chlorophyll content in tobacco leaves was. Therefore， the promoting effect of various treatments is as follows： T2>T1>T3. In the same treatment， the chlorophyll content of leaves in different parts of tobacco plants is as follows： upper leaves>middle leaves>lower leaves. In the upper leaves， chlorophyll content in various treatments is as follows： T1>T2>T3>CK. In the middle leaves， chlorophyll content in different treatments is as follows： CK>T1>T3>T2. In the lower leaves， chlorophyll content in various treatments is as follows： T2>CK>T1>T3. It can be seen that chlorophyll content in tobacco leaves in different parts of tobacco plants had no obvious regularity. Conclusions and Discussion
According to the above results， the promoting effect of application of organic fertilizers made from distillers grains and compound fertilizer is as follows： B>A>C>CK. The growth of tobacco plants in treatments T1， T2 and T3 was better than CK. In the three treatments， the fertilizer efficiency of the organic fertilizers A， B and C is as follows： B>A>C. Seen from the agronomic characters， they were better in the three treatments than CK. In the three periods， the plant height， the number of effective leaves， and the maximum leaf area of tobacco in the three treatments were obviously larger than CK， and the promoting effect of organic fertilizer B （treatment T2） was the best， followed by organic fertilizer A， and the promoting effect of organic fertilizer C was not significant. Seem from biomass， the biomass of tobacco plants （including the fresh and dry weight of the roots， stems and leaves） in the three treatments increased. That is， the fresh and dry weight of the roots， stems and leaves in the three treatments were higher than CK. Seen from chlorophyll content， tobacco plants in CK grew most slowly， and chlorophyll content was the highest. In the three treatments， the growth and development of tobacco plants was promoted， and chlorophyll content was low in the same harvest period， showing as CK>C>A>B. In general， the application of organic fertilizers made from distillers grains and compound fertilizer could promote the growth and development of tobacco plants， showing as B>A>C>CK. Moreover， the growth and development of tobacco plants in the three treatments were better than CK. The combination of organic fertilizers made from distillers grains and a certain amount of chemical fertilizer can replace complete chemical fertilizer， and the effect exceeds that of single application of chemical fertilizer to a certain degree. There were certain differences between different organic fertilizers made from distillers grains in terms of the promoting effect. On the basis of the predecessors， the optimal formula of organic fertilizers made from distillers grains should be studied， which can not only promote the growth and development of tobacco plants， but also promote the harmonious development of ecological and economic benefits.
References
[1] PENG ZL， HUANG YJ， LIU GS， et al. Effects of different organic fertilizer on soil microbe and hence quality and yield of tobacco leaves[J]. Acta Tabacaria Sinica， 2009（2）：41-45. [2] CHAI WY， LI HL， WANG Y， et al. Effect of bio-organic fertilizer on prevention and control of tobacco black shank disease and its influence on tobacco growth[J]. Tianjin Agricultural Sciences，2014（4）：24-27.
[3] FAN GX， CUI TY， LEI YC. Experimental demonstration of organic fertilizers made from Maotai distillers grains in the production of sorghum[J]. Liquor Making， 2007，34（2）：34-35.
[4] DOU S. The fertilizer effect of distillers grains and other organic materials on black clay[J]. Journal of Shenyang University， 1988（4）： 45-52.
[5] LIN GH， YANG B， SHANGGUAN KP， et al. Effect of organic fertilizers on volume and quality of aroma in flue-cured tobacco in Longyan[J]. Chinese Tobacco Science， 2003，24（3）： 9-10.
[6] ZHANG CD. Effect of medium and trace elements， organic fertilizer on qualities of baoshan sun-cured tobacco[J]. Chinese Agricultural Science Bulletin， 2006， 22（8）：320-323.
[7] CHAI JR， HUANG CJ. Effects of organic fertilizer on volatile matter and smoking quality in burley tobacco[J]. Crop Research， 200（2）：127-130.
[8] WANG LF， WANG Y， LUO DL. Effect of distillers grains organic fertilizer on tobacco quality[J]. Journal of Anhui Agricultural Sciences， 2016， 44（6）： 157-158.
[9] SHAO FW， JIANG CQ， ZU CL， et al. Influence of sulfur and stillage fertilizer on the growth， quality of flue-cured tobacco， and pH in alkaline soil[J]. Acta Botanica Boreali-Occidentalia Sinica，2012，32（12）：247.
[10] ZHAO PB， ZHENG PF， LI ST， et al. Effects of different ratios of vermicompost and vinasse on soil physical and chemical properties and quality of flue-cured tobacco[J]. Chinese Journal of Soil Science， 2017， 48（3）： 676-682.
Key words Organic fertilizers made from distillers grains； Agronomic characters； Biomass； Chlorophyll content
Organic fertilizers， carbonaceous materials， mainly derive from plants and （or） animals， and their main function is to provide nutrition for plants after being applied to soil. Organic fertilizers are closely related to flue-cured tobacco. Experimental studies have shown that different types of organic fertilizers can significantly increase the quantity of bacteria and actinomycetes in the rhizosphere of tobacco soil. Different organic fertilizers can significantly increase the yield， output value， and proportion of upper-middle tobacco[1]. The application of biological organic fertilizer can not only greatly increase the number of bacteria， fungi and actinomycetes in the rhizosphere soil of tobacco plants， but also increase the plant height and leaf area coefficient of tobacco and effectively inhibit the incidence of tobacco black shank[2]. The organic fertilizers made from distillers grains are fully fermented and produced by mixing the distillers grains that are left in the process of producing white spirit with oil cake， cow dung and fermentation inocula in a certain proportion. The use of distillers grains to produce organic fertilizers is not only environmentally friendly， but also can provide a conducive environment for the growth of green flue-cured tobacco and improve ecological， economic and social benefits[3]. In distillers grains， the content of organic matter is higher， and the ratio of carbon to nitrogen is suitable. After being applied into soil， they can not only promote the growth of flue-cured tobacco， but also increase the yield of flue-cured tobacco[4]. They not only can provide comprehensive nutrition for crops， but also have long fertilizer efficiency. In addition， they can increase and renew soil organic matter， promote microbial reproduction， and improve physical and chemical properties and biological activity of soil， and they are one of the main nutrients for the production of green crops. At present， there are many studies on the effects of organic fertilizers on the growth of flue-cured tobacco[5-7]. For instance， Wang Lifang et al.[8] pointed out that the organic fertilizers made from distillers grains could improve the the appearance quality， chemical composition and sensory quality of tobacco to a certain degree. Shao Fuwen et al.[9] suggested that the combination of low-dose sulphur and distillers grains could significantly increase tobacco yield and proportion of upper-middle tobacco[10]. However， effects of the organic fertilizers made from distillers grains on the growth and development of flue-cured tobacco have been rarely reported at present. In this study， through pot experiments， the effects of different organic fertilizers made from distillers grains on the growth and development of flue-cured tobacco were studied to provide a basis for the application of organic fertilizers made from distillers grains in the production of flue-cured tobacco. Materials and Methods
Experimental materials
The experiment was conducted at the tobacco testing base of Yunnan Agricultural University in 2017. The variety of flue-cured tobacco was Yunyan 85. The soil used in the pot experiment was yellow loam that was collected from the field behind Yunnan Agricultural University. The chemical properties of the soil are as follows： pH was 7.29， and the content of organic matter was 2.94%； the content of available potassium， available phosphorus， and alkaline nitrogen was 72， 6.0， and 117 mg/kg respectively. Three types of organic fertilizers made from distillers grains were fully fermented and made by mixing the distillers grains with oil cake， cow dung and fermentation inocula in a certain proportion （Table 1）.
Experimental design
There were three treatments， including T1 （18.5 g/kg of organic fertilizer A + 50 g of compound fertilizer）， T2 （18.5 g/kg of organic fertilizer B + 50 g of compound fertilizer）， T3 （18.5 g/kg of organic fertilizer C + 50 g of compound fertilizer）， and CK （130 g of compound fertilizer）. Each treatment had four repetitions， and there were 16 pots that were arranged randomly. Each pot contained 20 kg of the soil. The amount of pure nitrogen applied in the soil was 105 kg/hm2， and the planting density of tobacco was 16 500 plants/hm2. After the even mixture of the organic fertilizers， compound fertilizer and soil was put in a pot， tobacco seedlings were planted in the pot.
Main measurement indicators and methods
Agronomic characters
According to the Investigating and Measuring Methods of Agronomical Characters of Tobacco， the plant height， stem girth， maximum leaf length， and maximum leaf width of flue-cured tobacco in the resettling period， vigorous growth period and maturation period were measured， and the maximum leaf area was calculated （maximum leaf length×maximum leaf width×0.634 5）.
Biomass
After the tobacco plants in each treatment were mature physiologically， the samples were devastatingly collected. The roots， stems and leaves of the plants were separated， washed with tap water， and then wiped with clean paper. After there was no water on them， they were weighed， and their fresh weight was recorded separately. They were dried for 30-60 min at 105 ℃ and then dried at 70 ℃ until their weight was constant. Finally， they were weighed to record their dry weight.
Chlorophyll content Firstly， 2 g of tobacco leaves were taken from the upper， middle and lower part of each tobacco plant respectively， and three copies of the tobacco leaves were collected from each part， so there were 126 treatments. After chlorophyll was extracted with 96% ethanol （or 80% acetone）， the absorbance values at wavelength 645 and 663 nm were measured with a spectrophotometer， and chlorophyll content in each treatment was calculated. The formula is as follows： Ca=12.72A663-2.59A645， Cb=22.88A663-4.67A649， CT=Ca+Cb； chlorophyll content （mg/g）=（chlorophyll concentration×volume of extract×dilution ratio）/fresh weight of samples. Statistical method
Results and Analysis
Effects of application of organic fertilizers made from distillers grains on the agronomic characters of flue-cured tobacco in various periods
Agronomic traits reflect the growth status of tobacco plants at different growth stages and more intuitively illustrate the growth of tobacco plants. According to Table 2， in the resettling period， vigorous growth period and maturation period， the plant height of tobacco in various treatments was as follows： T2>T1>T3>CK， T2>T1>T3>CK， T1>T2>T3>CK. In the three periods， there were obvious differences between CK and T1， T2 or T3. In the three periods， the number of effective leaves was larger in treatments T1， T2 and T3 than CK， but the differences were not obvious. In the three periods， the maximum leaf area was larger in treatments T1， T2 and T3 than CK， and the differences were significant， but there was no obvious difference between treatments T1， T2 and T3. The above results showed that the growth of tobacco plants in treatment T2 was the best， followed by treatment T1. The growth of tobacco plants in treatments T1， T2 and T3 was better than CK. The combination of organic fertilizers made from distillers grains and special compound fertilizer for tobacco could promote the growth of tobacco plants. The agronomic characters of flue-cured tobacco in different treatments 98 d after the tobacco seedlings were transplanted are shown in Table 3.
Biomass referred to the dry and fresh weight of roots， stems and leaves of the harvested tobacco plants， and the quality of the roots， stems and leaves reflected the growth of the tobacco plants. Seen from Table 4， in the maturation period， the dry and fresh weight of the stems and the dry weight of the leaves in treatments T1， T2 and T3 were larger than CK. The fresh weight of the roots in treatments T1 and T2 was higher， while there was no obvious difference between treatment T3 and CK. The results revealed that the combination of organic fertilizers made from distillers grains and special compound fertilizer for tobacco could promote the accumulation of tobacco biomass obviously.
Chlorophyll content reflects the ability of plants to perform photosynthesis. The chlorophyll content of flue-cured tobacco in various treatments was as follows： CK>T3>T1>T2. The results showed that the combination of organic fertilizers made from distillers grains and special compound fertilizer for tobacco could promote the growth and development of tobacco plants. In the same time， the more mature the tobacco leaves were， the lower the chlorophyll content in tobacco leaves was. Therefore， the promoting effect of various treatments is as follows： T2>T1>T3. In the same treatment， the chlorophyll content of leaves in different parts of tobacco plants is as follows： upper leaves>middle leaves>lower leaves. In the upper leaves， chlorophyll content in various treatments is as follows： T1>T2>T3>CK. In the middle leaves， chlorophyll content in different treatments is as follows： CK>T1>T3>T2. In the lower leaves， chlorophyll content in various treatments is as follows： T2>CK>T1>T3. It can be seen that chlorophyll content in tobacco leaves in different parts of tobacco plants had no obvious regularity. Conclusions and Discussion
According to the above results， the promoting effect of application of organic fertilizers made from distillers grains and compound fertilizer is as follows： B>A>C>CK. The growth of tobacco plants in treatments T1， T2 and T3 was better than CK. In the three treatments， the fertilizer efficiency of the organic fertilizers A， B and C is as follows： B>A>C. Seen from the agronomic characters， they were better in the three treatments than CK. In the three periods， the plant height， the number of effective leaves， and the maximum leaf area of tobacco in the three treatments were obviously larger than CK， and the promoting effect of organic fertilizer B （treatment T2） was the best， followed by organic fertilizer A， and the promoting effect of organic fertilizer C was not significant. Seem from biomass， the biomass of tobacco plants （including the fresh and dry weight of the roots， stems and leaves） in the three treatments increased. That is， the fresh and dry weight of the roots， stems and leaves in the three treatments were higher than CK. Seen from chlorophyll content， tobacco plants in CK grew most slowly， and chlorophyll content was the highest. In the three treatments， the growth and development of tobacco plants was promoted， and chlorophyll content was low in the same harvest period， showing as CK>C>A>B. In general， the application of organic fertilizers made from distillers grains and compound fertilizer could promote the growth and development of tobacco plants， showing as B>A>C>CK. Moreover， the growth and development of tobacco plants in the three treatments were better than CK. The combination of organic fertilizers made from distillers grains and a certain amount of chemical fertilizer can replace complete chemical fertilizer， and the effect exceeds that of single application of chemical fertilizer to a certain degree. There were certain differences between different organic fertilizers made from distillers grains in terms of the promoting effect. On the basis of the predecessors， the optimal formula of organic fertilizers made from distillers grains should be studied， which can not only promote the growth and development of tobacco plants， but also promote the harmonious development of ecological and economic benefits.
References
[1] PENG ZL， HUANG YJ， LIU GS， et al. Effects of different organic fertilizer on soil microbe and hence quality and yield of tobacco leaves[J]. Acta Tabacaria Sinica， 2009（2）：41-45. [2] CHAI WY， LI HL， WANG Y， et al. Effect of bio-organic fertilizer on prevention and control of tobacco black shank disease and its influence on tobacco growth[J]. Tianjin Agricultural Sciences，2014（4）：24-27.
[3] FAN GX， CUI TY， LEI YC. Experimental demonstration of organic fertilizers made from Maotai distillers grains in the production of sorghum[J]. Liquor Making， 2007，34（2）：34-35.
[4] DOU S. The fertilizer effect of distillers grains and other organic materials on black clay[J]. Journal of Shenyang University， 1988（4）： 45-52.
[5] LIN GH， YANG B， SHANGGUAN KP， et al. Effect of organic fertilizers on volume and quality of aroma in flue-cured tobacco in Longyan[J]. Chinese Tobacco Science， 2003，24（3）： 9-10.
[6] ZHANG CD. Effect of medium and trace elements， organic fertilizer on qualities of baoshan sun-cured tobacco[J]. Chinese Agricultural Science Bulletin， 2006， 22（8）：320-323.
[7] CHAI JR， HUANG CJ. Effects of organic fertilizer on volatile matter and smoking quality in burley tobacco[J]. Crop Research， 200（2）：127-130.
[8] WANG LF， WANG Y， LUO DL. Effect of distillers grains organic fertilizer on tobacco quality[J]. Journal of Anhui Agricultural Sciences， 2016， 44（6）： 157-158.
[9] SHAO FW， JIANG CQ， ZU CL， et al. Influence of sulfur and stillage fertilizer on the growth， quality of flue-cured tobacco， and pH in alkaline soil[J]. Acta Botanica Boreali-Occidentalia Sinica，2012，32（12）：247.
[10] ZHAO PB， ZHENG PF， LI ST， et al. Effects of different ratios of vermicompost and vinasse on soil physical and chemical properties and quality of flue-cured tobacco[J]. Chinese Journal of Soil Science， 2017， 48（3）： 676-682.