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Abstract In order to reduce the use of peat resources and realize the sustainable development of tobacco， the pH of distillers grain substrate was adjusted with humic acid， and the effects of various distillers grain substrates on the growth of tobacco seedlings were studied to find out the method of cultivating highquality tobacco seedlings by using distillers grain substrate. The results showed that when 60% of humic acid was added to the distillers grain substrate made from coarse distillers grains， fine distillers grains， perlite and vermiculite， both the pH and electrical conductivity decreased significantly and were similar to that of the conventional substrate. Moreover， the emergence rate of tobacco seedlings， the rate of strong tobacco seedlings， leaf number， stem height， root growth and quality of tobacco seedlings were good.
Key words Distillers grains； Dubstrate； Fluecured tobacco； Floating breeding of seedlings
Floating breeding of seedlings means after seeds are sown in a seedlingraising disk filled with a substrate， the disk is placed in a nursery pond with a specific nutrient solution[1-2]. The floating breeding of tobacco seedlings has many characteristics， such as high production efficiency， and superior tobacco seedlings， so since the introduction from foreign countries， its research and application in China have developed rapidly， and it has gradually become the main method for the production of fluecured tobacco in China[3-4]. The floating breeding matrix of tobacco seedlings can support and fix the plants in the process of floating breeding， and transport the nutrients and water from nutrient solution to the important parts of tobacco seedlings. It determines the growth environment of roots of tobacco seedlings and is the key to the floating breeding of tobacco seedlings[5-6]. At present， the main materials of the special breeding substrate for commodity tobacco in China include peat， vermiculite， expanded perlite， etc. Among them， peat is nonrenewable， has limited resources， is unevenly distributed in China， and will cause damage to the environment during its mining process， so China has banned peat extraction since 2006. Therefore， it is extremely important to look for a material that can replace peat as a floating breeding substrate and be able to achieve the same effect of peat substrate. To maintain the sound development of tobacco floating breeding technology and ensure the sustainable development of fluecured tobacco production， it is of great significance to finding and developing new environmental matrix materials[7]. At present， there are a large number of studies on floating breeding of seedlings. For instance， Ma Xiao et al.[8]mixed rotten peanut bran with river sand to make a substrate in in different proportions， and the results showed that the comprehensive properties of tobacco seedlings bred in the substrate composed of a suitable proportion of rotten peanut bran and river sand were good， and it was feasible to use the substrate for the floating breeding of seedlings. Xu Fuyin et al.[9]found that the emergence rate and growth of tobacco seedlings growing in the substrates made from sludge compost， perlite， and vermiculite in appropriate proportions could meet the requirements of the substrate standard for the floating breeding of seedlings. Compared with the use of peat， the use of sludge compost could not only reduce the use of fertilizers and production cost， but also decrease the damage of sludge to the environment， and improve the environment. Ren Lantian et al.[10]proposed that decomposed wheat straw could be used to completely replace peat in a substrate， and the quality of seedlings bred by this method met the requirements of breeding seedlings by conventional methods， and the cost of seedling raising was low， which is in line with the need for the sustainable development of breeding of tobacco seedlings. Therefore， finding a substance that can be used to replace the peat in a substrate for the floating breeding of tobacco seedlings is a major problem that needs to be solved in the sustainable development of floating breeding of tobacco seedlings. Distillers grains must be fermented and decomposed before being used as a substrate or organic fertilizer. During the process of fermenting and decomposing， some lime will be added， so that distillers grains have strong alkalinity after being fermented. However， pH of the substrate used for the floating breeding of seedlings should be 5-7， so humic acids are used to adjust the pH of the strong alkaline environment. Humic acids can not only properly increase the bulk density and electrical conductivity of a substrate， but also is weakly acidic and will have no toxicity the substrate. It can also improve the agrochemical properties and structure of soil and increase soil temperature， so it is very suitable to use it to adjust pH[11-13]. Therefore， the pH of distillers grain substrate was adjusted with humid acid， and the effects of different distillers grain substrates on the growth and development of tobacco seedlings were studied to find out suitable pH for the growth of tobacco seedlings and the method for using distillers grains to breed highquality tobacco seedlings by using floating breeding system. It was proved that the properties of distillers grain substrate used in the experiment were similar to that of commodity substrate， and its water absorption was good， while its pH needs to be further regulated. Therefore， after the pH of distillers grain substrate was adjusted with humic acid， the effects of various distillers grain substrates on the growth of tobacco seedlings were studied[14-17].
Materials and Methods
Experimental materials
The test substrates were selfmade distillers grain substrate， which were made from smashed distillers grains， peat， perlite， and vermiculite according to a certain proportion. The variety of fluecured tobacco was Yunyan 85. The floating disks were polystyrene plastic disks， with the size of 55 cm×37.4 cm×6 cm. There were 160 holes on each disk， and the volume of humic acids was 27 cm3. The test fertilizer was "Xinye" special fertilizer for breeding of tobacco seedlings that was produced by Sichuan Jinye Chemical Fertilizer Co.， Ltd. （total nutrient 48%， NP2O5K2O 181020， and nitrate nitrogen content accounted for 55% of total nitrogen content）. The test humic acid was the raw powder of an exportgrade， highcontent， highquality， and weathered brown coal humic acid. In the humic acid， the content of humic acid （dry basis）， fulvic acid （dry basis）， and ash was ≥ 70%， 15%-20%， and 10% respectively. There was a variety of mineral components. The pH was 4-6， and they were black powder that could pass through a sieve with 100-120 meshes. They were from weathered brown coal and slightly soluble in water. Experimental design
The experiment was conducted in Kunming， Yunnan Province in March 2017. The pH of the test substrates was adjusted by the addition the humic acid. The test substrates mainly made from distillers grains were made according to the volume ratio of coarse distillers grains∶fine distillers grains∶perlite∶vermiculite of 3∶3∶2∶2. The total mass of distillers grains prepared in each group was the same. There were four treatments and two control groups， including distillers grain substrate+humic acid accounting for 40% of total mass of distillers grains （A）， distillers grain substrate+50% of humic acid （B）， distillers grain substrate+60% of humic acid （C）， distillers grain substrate+70% of humic acid （D）， distillers grain substrate without humic acid （CK1）， commodity substrate without humic acid （CK2）. Each treatment had four repetitions. The special fertilizer for the floating breeding of seedlings was added to the disks when water was released into the disks， so that the mass concentration of nitrogen was 150 mg/L in each treatment. After the disks were put in water for 30 and 45 d， the special fertilizer for the floating breeding of seedlings （100 and 50 mg/L） was added to the disks. Other managements were conducted according to conventional methods.
Measurement indicators and methods
On the 30th， 40th and 50th day after sowing， five plants with uniform growth were selected from each treatment， and the leaf number （leaves were greater than 2 cm in length） and stem height of tobacco seedlings in each treatment were measured. At the same time， the total number of roots， the number of primary lateral roots， and the number of secondary lateral roots were measured on the 60th day after sowing. At last， the pH and electrical conductivity of the substrates， the emergence rate of tobacco seedlings， the rate of strong tobacco seedlings， seedling time， growth vigor， etc. at the time of emergence of tobacco seedlings were recorded. Around 10：00， five healthy and uniform seedlings were selected， and their functional leaves （removing their veins） were taken to measure the activity of nitrate reductase， chlorophyll， and root vigor. Among them， root vigor was measured by methylene blue method， and the activity of nitrate reductase was measured by living body method.
Data processing and analysis
All experimental data were calculated， compiled and statistically analyzed using Excel and Spss13.0.
Results and Analysis Effects of different distillers grain substrates on pH and electrical conductivity
According to Table 1， the commodity substrate （CK2） was acidic， but the distillers grain substrate without humic acid （CK1） was alkaline. With the increase of humic acid dosage， the pH of the substrates in treatments A， B， C and D reduced， and was close to that of the commodity substrate gradually. The electrical conductivity of the distillers grain substrate without humic acid was up to 560.49 μs/cm， while that of the commodity substrate was only 351.28 μs/cm. Along with the increase of humic acid dosage， the electrical conductivity of the substrates decreased firstly and then increased.
Seen from Table 2， the emergence rate of tobacco seedlings in treatment C exceeded that of tobacco seedlings growing in the commodity substrate （CK2）， while the emergence rate of tobacco seedlings in other treatments was lower. It is seen that there was no relationship between the emergence rate of tobacco seedlings and humic acid dosage. The rate of strong tobacco seedlings in treatment C was slightly lower that of tobacco seedlings growing in the commodity substrate， and the rate of strong tobacco seedlings in treatment CK1 was the lowest. The rate of strong tobacco seedlings in the first three treatments A， B and C increased gradually but decreased in the fourth treatment D， because humic acid might be excessive. The number of seedling days in treatments A， B， C and D increased firstly and then reduced， lower than that of treatment CK1 and higher than that of treatment CK2. In terms of growth vigor， tobacco seedlings in treatments C and CK2 grew more luxuriantly than that in other treatments.
Agricultural Biotechnology2018
Effects of different distillers grain substrates on the leaf number of tobacco seedlings
According to Table 3， the leaf number of tobacco seedlings in treatments C and CK2 was larger than that of other treatments 30 days after sowing. It was the largest in treatment CK2， but the smallest in treatment CK1. 30-40 days after sowing， it increased most slowly in treatment CK1. 40-50 days after sowing， the increase in the leaf number of tobacco seedlings became slow in treatments A and B， while it was fast in treatment CK1. At this time， the leaf number of tobacco seedlings in various treatments is as follows： C>CK2>D>A>B>CK1.
Seen from Table 4， there were small differences between various treatment in the stem height of tobacco seedlings 30 days after sowing. 30-40 days after sowing， there were certain differences between various treatments in the stem height of tobacco seedlings， and the height difference between various treatments was up to about 3 cm， the increase in the stem height of tobacco seedlings was the least in treatment CK1. 40-50 days after sowing， the increase in the stem height of tobacco seedlings was the largest in different treatments. With the increase of humic acid dosage in treatments A， B and C， the stem height of tobacco seedlings increased. It declined obviously in treatment D. To sum up， tobacco seedlings in treatments C and CK2 grew fastest， while the stem height of tobacco seedlings was the lowest in treatment CK1. According to Table 5， the total number of roots of tobacco seedlings in different treatments is as follows： CK2>C>D>B>A>CK1. The number of primary lateral roots was the largest in the commodity substrate （CK2） and the smallest in treatment B. It was larger in treatment C than that of treatment D， and it was smaller in treatment CK1 than that of treatment D but larger than that of treatment A. The number of secondary lateral roots in different treatments is as follows： CK2 >C>D>A>CK1>B. On the whole， the number of roots， primary lateral roots or secondary lateral roots was the largest in treatment CK2， followed by treatments C and D， while it was relatively small in treatment B. There were significant differences between treatments C and CK2 in the number of roots， primary lateral roots and secondary lateral roots.
Effects of various distillers grain substrates on the quality of tobacco seedlings
Seen from Table 6， the activity of nitrate reductase in treatments B， A， D and CK1 was similar， and the activity of nitrate reductase in treatments CK2 and C was also similar. On the whole， there was no obvious difference between the six treatments in terms of chlorophyll content. The chlorophyll content of tobacco seedlings in various treatments is as follows： CK1>A>B>D>C and CK2， and the differences in various treatments were not obvious. The root vigor of tobacco seedlings in various treatments is as follows： CK2>C>D>A>CK1>B. There were significant differences between treatment C or CK2 and other treatments in terms of root vigor and chlorophyll content.
Conclusions and Discussion
Different distillers grain substrates with humic acid had effects on their pH and electrical conductivity. With the increase of humic acid dosage， the pH of the substrates decreased. That is， the larger the humic acid dosage was， the better its adjustment effect was. Along with the increase of humic acid dosage， the electrical conductivity of the substrates also reduced. The possible reason is that humic acid had an adsorption effect on free ions in the substrates， and the specific reason needs to be studied further. Various distillers grain substrates with humic acid also affected the emergence rate of tobacco seedlings， the rate of strong tobacco seedlings， and the growth and development of roots of tobacco seedlings to a certain degree. When pH was too high or low， root vigor was low. Withe the increase of pH from 6.75 to 7.13， root vigor reduced， which is similar to the study of Guo Peiguo et al.[18]. Roots play an important role in the growth and development of fluecured tobacco and directly affect the absorption and transport of mineral nutrition by fluecured tobacco. Therefore， Changes in pH indirectly affected the emergence rate of tobacco seedlings and the rate of strong tobacco seedlings. As a result， with the increase of humic acid dosage， the emergence rate of tobacco seedlings and the rate of strong tobacco seedlings increased firstly and then decreased. At the same time， the number of primary lateral roots and the number of secondary lateral roots had a positive correlation. Various distillers grain substrates with humic acid also had certain effects on the leaf number and stem height of tobacco seedlings. With the increase of humic acid dosage， the leaf number and stem height of tobacco seedlings increased firstly and then decreased. Different distillers grain substrates with humic acid also affected the quality of tobacco seedlings. there were certain differences between various treatments in terms of root vigor， the activity of nitrate reductase， and chlorophyll content. There were significant differences between treatment C and other treatments in terms of root vigor and chlorophyll content. The reason may be that pH had certain influences on root vigor and the absorption of water and mineral nutrients by roots in soil and then indirectly affected the production of chlorophyll and various enzymes in tobacco seedlings， thereby resulting in differences in photosynthesis of tobacco seedlings and thus affecting the agronomic characters and quality of tobacco seedlings.
Among the distillers grain substrates with humic acid， the values of the indicators in the distillers grain substrate with 60% of humic acid were similar to that of the commodity substrate. In the environment with the pH of 6.75， tobacco seedlings could grow in the floating breeding system of the distillers grain substrate， so the distillers grain substrate can be used to replace the commodity substrate in the floating breeding of tobacco seedlings.
References
[1]PENG XQ， WU JZ， LU ZS， et al. Current status of application，research progress and future development in tobacco floatingbed seedling production technology in China[J]. Acta Tabacaria Sinica， 2010， 16（3）：90-94. （in Chinese）.
[2]AN DY， LONG GC， KOU XP. A preliminary study on growing seedling of tobacco by floating plate[J]. Journal of Mountain Agriculture & Biology，2001，20（1）：76-78. （in Chinese）.
[3]DAVIS DL， NIELSEN MT. Tobacco production， chemistry and technology[M]. Beijing： Chemical Industry Press， 2003：68-69. （in Chinese）.
[4]SHI XD， SUN JW， XIE XB. Research advance on medium of tobacco floating system[J]. Chinese Tobacco Science，2008，29（5）：64-68. （in Chinese）.
[5]TIAN JL， WANG YH. Current situations and prospects of researches on soillessculture substrates[J]. Acta Agriculturae Shanghai， 2000，16（4）：87-92. （in Chinese）.
[6]XU WB. Influences of different matrixes for raising seedlings in floating system on quality of tobacco seedling[J]. Hunan Agricultural Sciences， 2013（17）：41-44. （in Chinese）. [7]WU T， JIN Y， YANG YH， et al. The research of substituting the peat in the medium of floating systemI The primary study of using liginite， screw extrusion and other material to substitute peat[J]. Journal of Yunnan Agricultural University，2007，22（2）：234-240.
[8]MA X， SUN JW， XU XH， et al. Application of peanut bran and sand medium to tobacco float seedling production[J]. Chinese Tobacco Science， 2009，30（6）：61-64. （in Chinese）.
[9]XU FY， BAI JY， HU YY， et al. Effect of sewage sludge compost for float seedlings of tobacco[J]. Heilongjiang Agricultural Sciences， 2017（1）：42-44. （in Chinese）.
[10]REN LT， LIU Q， MEI YY， et al. Study on wheat straw substituted medium in the float seedling system[J]. Chinese Tobacco Science，2017，38（3）：26-36. （in Chinese）.
[11]WANG QY， ZHANG C， CHEN B， et al. Formula selection and effects comparison on media for tobacco float system[J]. Journal of Mountain Agriculture & Biology， 2015， 34（6）： 20-24. （in Chinese）.
[12]CHENG G， KANG J， ZHANG YL， et al. Effects of biomaterial carbon application on seedling emergence and seedling growth of tobacco floating seedlings[J]. Shaanxi Journal of Agricultural Sciences，2017，63（3）： 6 -10. （in Chinese）.
[13]YANG ZH， LIU DS， SHI FB， et al. Problems and countermeasures in the floating breeding of tobacco seedlings[J]. Modern Agricultural Science and Technology， 2010（13）：82-83. （in Chinese）.
[14]GAO L. Comprehensive utilization of distillers grains[J]. LiquorMaking Science & Technology， 2004（5）：101-102. （in Chinese）.
[15]HAO ZX， HUANG DP， GU SH， et al. Screening of melon and fruit of vegetables grown substrate from different vinasse and cow dung composts mixtures[J]. Journal of Nanjing Agricultural University，2017， 40（3）： 457-463. （in Chinese）.
[16]ZHAO M. Effects of distillers grain substrate on growth and development of potted peonies[J]. Xiandai Horticulture，2015（6）：12-13. （in Chinese）.
[17]ZHU B， HUANG YD. Effect of the distillers grains on the nutrient composition， property and production performance of Agaricus bisporus[J]. Journal of Anhui Agricultural Sciences，2010（11）：5606-5607. （in Chinese）.
[18]GUO PG， CHEN JJ， LI RH. Effects of pH values on the activity of roots and chemical compositions of the cured leaves in fluecured tobacco[J].Scientia Agricultura Sinica，2000，33（1）：39-45. （in Chinese）.
Editor： Yingying YANG Proofreader： Xinxiu ZHU
Key words Distillers grains； Dubstrate； Fluecured tobacco； Floating breeding of seedlings
Floating breeding of seedlings means after seeds are sown in a seedlingraising disk filled with a substrate， the disk is placed in a nursery pond with a specific nutrient solution[1-2]. The floating breeding of tobacco seedlings has many characteristics， such as high production efficiency， and superior tobacco seedlings， so since the introduction from foreign countries， its research and application in China have developed rapidly， and it has gradually become the main method for the production of fluecured tobacco in China[3-4]. The floating breeding matrix of tobacco seedlings can support and fix the plants in the process of floating breeding， and transport the nutrients and water from nutrient solution to the important parts of tobacco seedlings. It determines the growth environment of roots of tobacco seedlings and is the key to the floating breeding of tobacco seedlings[5-6]. At present， the main materials of the special breeding substrate for commodity tobacco in China include peat， vermiculite， expanded perlite， etc. Among them， peat is nonrenewable， has limited resources， is unevenly distributed in China， and will cause damage to the environment during its mining process， so China has banned peat extraction since 2006. Therefore， it is extremely important to look for a material that can replace peat as a floating breeding substrate and be able to achieve the same effect of peat substrate. To maintain the sound development of tobacco floating breeding technology and ensure the sustainable development of fluecured tobacco production， it is of great significance to finding and developing new environmental matrix materials[7]. At present， there are a large number of studies on floating breeding of seedlings. For instance， Ma Xiao et al.[8]mixed rotten peanut bran with river sand to make a substrate in in different proportions， and the results showed that the comprehensive properties of tobacco seedlings bred in the substrate composed of a suitable proportion of rotten peanut bran and river sand were good， and it was feasible to use the substrate for the floating breeding of seedlings. Xu Fuyin et al.[9]found that the emergence rate and growth of tobacco seedlings growing in the substrates made from sludge compost， perlite， and vermiculite in appropriate proportions could meet the requirements of the substrate standard for the floating breeding of seedlings. Compared with the use of peat， the use of sludge compost could not only reduce the use of fertilizers and production cost， but also decrease the damage of sludge to the environment， and improve the environment. Ren Lantian et al.[10]proposed that decomposed wheat straw could be used to completely replace peat in a substrate， and the quality of seedlings bred by this method met the requirements of breeding seedlings by conventional methods， and the cost of seedling raising was low， which is in line with the need for the sustainable development of breeding of tobacco seedlings. Therefore， finding a substance that can be used to replace the peat in a substrate for the floating breeding of tobacco seedlings is a major problem that needs to be solved in the sustainable development of floating breeding of tobacco seedlings. Distillers grains must be fermented and decomposed before being used as a substrate or organic fertilizer. During the process of fermenting and decomposing， some lime will be added， so that distillers grains have strong alkalinity after being fermented. However， pH of the substrate used for the floating breeding of seedlings should be 5-7， so humic acids are used to adjust the pH of the strong alkaline environment. Humic acids can not only properly increase the bulk density and electrical conductivity of a substrate， but also is weakly acidic and will have no toxicity the substrate. It can also improve the agrochemical properties and structure of soil and increase soil temperature， so it is very suitable to use it to adjust pH[11-13]. Therefore， the pH of distillers grain substrate was adjusted with humid acid， and the effects of different distillers grain substrates on the growth and development of tobacco seedlings were studied to find out suitable pH for the growth of tobacco seedlings and the method for using distillers grains to breed highquality tobacco seedlings by using floating breeding system. It was proved that the properties of distillers grain substrate used in the experiment were similar to that of commodity substrate， and its water absorption was good， while its pH needs to be further regulated. Therefore， after the pH of distillers grain substrate was adjusted with humic acid， the effects of various distillers grain substrates on the growth of tobacco seedlings were studied[14-17].
Materials and Methods
Experimental materials
The test substrates were selfmade distillers grain substrate， which were made from smashed distillers grains， peat， perlite， and vermiculite according to a certain proportion. The variety of fluecured tobacco was Yunyan 85. The floating disks were polystyrene plastic disks， with the size of 55 cm×37.4 cm×6 cm. There were 160 holes on each disk， and the volume of humic acids was 27 cm3. The test fertilizer was "Xinye" special fertilizer for breeding of tobacco seedlings that was produced by Sichuan Jinye Chemical Fertilizer Co.， Ltd. （total nutrient 48%， NP2O5K2O 181020， and nitrate nitrogen content accounted for 55% of total nitrogen content）. The test humic acid was the raw powder of an exportgrade， highcontent， highquality， and weathered brown coal humic acid. In the humic acid， the content of humic acid （dry basis）， fulvic acid （dry basis）， and ash was ≥ 70%， 15%-20%， and 10% respectively. There was a variety of mineral components. The pH was 4-6， and they were black powder that could pass through a sieve with 100-120 meshes. They were from weathered brown coal and slightly soluble in water. Experimental design
The experiment was conducted in Kunming， Yunnan Province in March 2017. The pH of the test substrates was adjusted by the addition the humic acid. The test substrates mainly made from distillers grains were made according to the volume ratio of coarse distillers grains∶fine distillers grains∶perlite∶vermiculite of 3∶3∶2∶2. The total mass of distillers grains prepared in each group was the same. There were four treatments and two control groups， including distillers grain substrate+humic acid accounting for 40% of total mass of distillers grains （A）， distillers grain substrate+50% of humic acid （B）， distillers grain substrate+60% of humic acid （C）， distillers grain substrate+70% of humic acid （D）， distillers grain substrate without humic acid （CK1）， commodity substrate without humic acid （CK2）. Each treatment had four repetitions. The special fertilizer for the floating breeding of seedlings was added to the disks when water was released into the disks， so that the mass concentration of nitrogen was 150 mg/L in each treatment. After the disks were put in water for 30 and 45 d， the special fertilizer for the floating breeding of seedlings （100 and 50 mg/L） was added to the disks. Other managements were conducted according to conventional methods.
Measurement indicators and methods
On the 30th， 40th and 50th day after sowing， five plants with uniform growth were selected from each treatment， and the leaf number （leaves were greater than 2 cm in length） and stem height of tobacco seedlings in each treatment were measured. At the same time， the total number of roots， the number of primary lateral roots， and the number of secondary lateral roots were measured on the 60th day after sowing. At last， the pH and electrical conductivity of the substrates， the emergence rate of tobacco seedlings， the rate of strong tobacco seedlings， seedling time， growth vigor， etc. at the time of emergence of tobacco seedlings were recorded. Around 10：00， five healthy and uniform seedlings were selected， and their functional leaves （removing their veins） were taken to measure the activity of nitrate reductase， chlorophyll， and root vigor. Among them， root vigor was measured by methylene blue method， and the activity of nitrate reductase was measured by living body method.
Data processing and analysis
All experimental data were calculated， compiled and statistically analyzed using Excel and Spss13.0.
Results and Analysis Effects of different distillers grain substrates on pH and electrical conductivity
According to Table 1， the commodity substrate （CK2） was acidic， but the distillers grain substrate without humic acid （CK1） was alkaline. With the increase of humic acid dosage， the pH of the substrates in treatments A， B， C and D reduced， and was close to that of the commodity substrate gradually. The electrical conductivity of the distillers grain substrate without humic acid was up to 560.49 μs/cm， while that of the commodity substrate was only 351.28 μs/cm. Along with the increase of humic acid dosage， the electrical conductivity of the substrates decreased firstly and then increased.
Seen from Table 2， the emergence rate of tobacco seedlings in treatment C exceeded that of tobacco seedlings growing in the commodity substrate （CK2）， while the emergence rate of tobacco seedlings in other treatments was lower. It is seen that there was no relationship between the emergence rate of tobacco seedlings and humic acid dosage. The rate of strong tobacco seedlings in treatment C was slightly lower that of tobacco seedlings growing in the commodity substrate， and the rate of strong tobacco seedlings in treatment CK1 was the lowest. The rate of strong tobacco seedlings in the first three treatments A， B and C increased gradually but decreased in the fourth treatment D， because humic acid might be excessive. The number of seedling days in treatments A， B， C and D increased firstly and then reduced， lower than that of treatment CK1 and higher than that of treatment CK2. In terms of growth vigor， tobacco seedlings in treatments C and CK2 grew more luxuriantly than that in other treatments.
Agricultural Biotechnology2018
Effects of different distillers grain substrates on the leaf number of tobacco seedlings
According to Table 3， the leaf number of tobacco seedlings in treatments C and CK2 was larger than that of other treatments 30 days after sowing. It was the largest in treatment CK2， but the smallest in treatment CK1. 30-40 days after sowing， it increased most slowly in treatment CK1. 40-50 days after sowing， the increase in the leaf number of tobacco seedlings became slow in treatments A and B， while it was fast in treatment CK1. At this time， the leaf number of tobacco seedlings in various treatments is as follows： C>CK2>D>A>B>CK1.
Seen from Table 4， there were small differences between various treatment in the stem height of tobacco seedlings 30 days after sowing. 30-40 days after sowing， there were certain differences between various treatments in the stem height of tobacco seedlings， and the height difference between various treatments was up to about 3 cm， the increase in the stem height of tobacco seedlings was the least in treatment CK1. 40-50 days after sowing， the increase in the stem height of tobacco seedlings was the largest in different treatments. With the increase of humic acid dosage in treatments A， B and C， the stem height of tobacco seedlings increased. It declined obviously in treatment D. To sum up， tobacco seedlings in treatments C and CK2 grew fastest， while the stem height of tobacco seedlings was the lowest in treatment CK1. According to Table 5， the total number of roots of tobacco seedlings in different treatments is as follows： CK2>C>D>B>A>CK1. The number of primary lateral roots was the largest in the commodity substrate （CK2） and the smallest in treatment B. It was larger in treatment C than that of treatment D， and it was smaller in treatment CK1 than that of treatment D but larger than that of treatment A. The number of secondary lateral roots in different treatments is as follows： CK2 >C>D>A>CK1>B. On the whole， the number of roots， primary lateral roots or secondary lateral roots was the largest in treatment CK2， followed by treatments C and D， while it was relatively small in treatment B. There were significant differences between treatments C and CK2 in the number of roots， primary lateral roots and secondary lateral roots.
Effects of various distillers grain substrates on the quality of tobacco seedlings
Seen from Table 6， the activity of nitrate reductase in treatments B， A， D and CK1 was similar， and the activity of nitrate reductase in treatments CK2 and C was also similar. On the whole， there was no obvious difference between the six treatments in terms of chlorophyll content. The chlorophyll content of tobacco seedlings in various treatments is as follows： CK1>A>B>D>C and CK2， and the differences in various treatments were not obvious. The root vigor of tobacco seedlings in various treatments is as follows： CK2>C>D>A>CK1>B. There were significant differences between treatment C or CK2 and other treatments in terms of root vigor and chlorophyll content.
Conclusions and Discussion
Different distillers grain substrates with humic acid had effects on their pH and electrical conductivity. With the increase of humic acid dosage， the pH of the substrates decreased. That is， the larger the humic acid dosage was， the better its adjustment effect was. Along with the increase of humic acid dosage， the electrical conductivity of the substrates also reduced. The possible reason is that humic acid had an adsorption effect on free ions in the substrates， and the specific reason needs to be studied further. Various distillers grain substrates with humic acid also affected the emergence rate of tobacco seedlings， the rate of strong tobacco seedlings， and the growth and development of roots of tobacco seedlings to a certain degree. When pH was too high or low， root vigor was low. Withe the increase of pH from 6.75 to 7.13， root vigor reduced， which is similar to the study of Guo Peiguo et al.[18]. Roots play an important role in the growth and development of fluecured tobacco and directly affect the absorption and transport of mineral nutrition by fluecured tobacco. Therefore， Changes in pH indirectly affected the emergence rate of tobacco seedlings and the rate of strong tobacco seedlings. As a result， with the increase of humic acid dosage， the emergence rate of tobacco seedlings and the rate of strong tobacco seedlings increased firstly and then decreased. At the same time， the number of primary lateral roots and the number of secondary lateral roots had a positive correlation. Various distillers grain substrates with humic acid also had certain effects on the leaf number and stem height of tobacco seedlings. With the increase of humic acid dosage， the leaf number and stem height of tobacco seedlings increased firstly and then decreased. Different distillers grain substrates with humic acid also affected the quality of tobacco seedlings. there were certain differences between various treatments in terms of root vigor， the activity of nitrate reductase， and chlorophyll content. There were significant differences between treatment C and other treatments in terms of root vigor and chlorophyll content. The reason may be that pH had certain influences on root vigor and the absorption of water and mineral nutrients by roots in soil and then indirectly affected the production of chlorophyll and various enzymes in tobacco seedlings， thereby resulting in differences in photosynthesis of tobacco seedlings and thus affecting the agronomic characters and quality of tobacco seedlings.
Among the distillers grain substrates with humic acid， the values of the indicators in the distillers grain substrate with 60% of humic acid were similar to that of the commodity substrate. In the environment with the pH of 6.75， tobacco seedlings could grow in the floating breeding system of the distillers grain substrate， so the distillers grain substrate can be used to replace the commodity substrate in the floating breeding of tobacco seedlings.
References
[1]PENG XQ， WU JZ， LU ZS， et al. Current status of application，research progress and future development in tobacco floatingbed seedling production technology in China[J]. Acta Tabacaria Sinica， 2010， 16（3）：90-94. （in Chinese）.
[2]AN DY， LONG GC， KOU XP. A preliminary study on growing seedling of tobacco by floating plate[J]. Journal of Mountain Agriculture & Biology，2001，20（1）：76-78. （in Chinese）.
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Editor： Yingying YANG Proofreader： Xinxiu ZHU