Biochar increases tobacco yield by promoting root growth based on a three-year field application

In order to explore the effects of biochar on root system and growth characteristics of flue-tobacco, three years of field experiments were conducted to study the influence of different biochar application levels [600 (T1), 1200 (T2), 1800(T3), 2400 (T4), 3000 (T5) kg/ha] and no fertilizer (CK) on the root physiological indexes and growth index of tobacco. Compared with local conventional fertilization, the application rate of N fertilizer in each treatment (except for control) was reduced by 40% to analyze the effects of different amount of biochar on the physiological indexes of tobacco roots and leaf photosynthesis during flourishing. The results showed that tobacco plants' root development status in the flourishing period was consistent with the photosynthetic physiological indexes, chlorophyll content, and leaf-area coefficient. Compared with the control, the application of biochar could increase the root vigor by 177.8%. Biochar improved the roots, increasing the total root area by 91.35% and the number of root tips by 100.9%. Meanwhile, biochar increased the net photosynthetic rate of tobacco leaves by 77.3% and the total tobacco biomass by 72.5%. Studies have shown that biochar can promote the development of tobacco roots, and then enhance the photosynthesis of leaves, so that tobacco plants can grow healthily, which is conducive to the tobacco production and the cultivation of soil.

www.nature.com/scientificreports/ At present, many problems exist in tobacco agriculture in Mudanjiang, such as too much chemical fertilizer and weak awareness of land maintenance, which has led to a significant decline in the soil fertility and unsound tobacco plant development. With low leaf maturity, insufficient oil and aroma 20,21 , a suitable soil amendment is needed to repair the local soil, solve the local tobacco-growing soil's problems, and improve the quality and efficiency of tobacco production.
The experiment (biochar localization experiment) was performed from 2015 to 2017 to improve tobaccogrowing soil and plant growth, which could explore the effects of different application rates of biochar on tobacco root development and leaf photosynthetic characteristics, as well as the relationship between the coordinated growth of the overground and underground parts of tobacco. It provides a theoretical reference for further exploring the effects of long-term application of biochar or N fertilizers on crop root growth and photosynthetic characteristics, as well as the rational utilization of biomass resources and soil fertility.

Materials and methods
Materials. The field experiment was performed at the Tobacco Research Institute of Mudanjiang City, Heilongjiang Province, China, from April 27 to October 4, 2015, April 15 to October 8, 2016, and April 20 to October 5, 2017. The experimental site is located in the cold northeast of China (128°02′-131°18′E 43°24′-45°59'N), and has a high-latitude continental monsoon climate featuring four seasons with rain and heat in the same period. Its annual sunshine time is about 2400 h, it has an annual average temperature of 6.1 ℃ and a frost-free period of 140 d, and the annual precipitation averages about 580 mm.
Methods. Experimental design. Six treatments were designed in this experiment, one of which was a control (CK). 600, 1,200, 1,800, 2,400 and 3,000 kg/ha biochar was applied to tobacco-growing soils, respectively (named T 1 , T 2 , T 3 , T 4 , and T 5 ). After ridging the tobacco field, the fertilizer is applied between the two tobacco plants on the ridge platform. The fertilization method was hole application, and the fertilization points were 15 cm from the tobacco plant. 0.06 ha plot was used for each treatment and it was repeated three times. The row spacing was 120 cm and the plant spacing was 55 cm. Base fertilizer (conventional fertilizer) was applied in the strips of 4.5 kg/ha (60% of the normal fertilization amount), and no fertilizer was applied to the blank control. 150 kg/ha K 2 SO 4 was applied in the topdressing stage of each biochar treatment group, and hole application was used for fertilization. After applying the base fertilizer, the field's water-holding capacity was maintained for 15 days before transplanting. The water and fertilizer management of all treatments was consistent with field management.
Research content and methods. Since most of the physiological indexes of tobacco plants reached their peaks on the 60th day after transplantation of tobacco plants 22 , the middle leaf (the 9th leaf from bottom to top, growing well in good light conditions), was selected to measure the photosynthetic characteristics of tobacco leaves during this period. At 9:30-11:30 on a sunny day with the light intensity greater than 1000 μmol/(m 2 ·s), the experiment was repeated three times, with five plants (fixed tobacco plants) for each treatment. LI-6400 (Beijing Ligaotai Technology Co., Ltd.) portable photosynthesis instrument was used to measure the leaf net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, and transpiration rate. After selecting the same leaf of the tobacco plant with the same photosynthetic characteristics, the length, width, and relative chlorophyll value (SPAD value) of the central leaf were determined.
The leaf area is calculated by d = (L + 2 W)/3, where d, L, and W represent the leaf area, maximum leaf length, and maximum leaf width, respectively. The relative value of chlorophyll (SPAD value) was measured by SPAD-502PLUS produced by Shandong Hengmei Electronic Technology Co., Ltd. Root vigor was measured by the TTC method and root sampling. The method was as follows: take three tobacco plants for each replicate of each treatment, as well as the full roots, and store them at low temperature, and measure them in time after washing with clean water.
The root structure was measured by CI-600 produced by Shanghai Zequan Technology Co., Ltd.; the leaf area coefficient was measured by the HJ03-LAI-2200 plant canopy analyzer of Beixin Instrument Company. The obtained whole tobacco plant sample was placed at 115 °C for 25 min to inactivate the cells, then dried at 65 °C, and then weighed. The result obtained was the dry matter mass of the tobacco plant. Data processing. Origin 2017 was used to make histograms and line graphs, and SPSS 20.0 was used to perform the single-factor analysis of variances for different treatments. Besides, the LSD method was used for multiple comparisons.
Ethics approval. The experimental research and field studies on plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. The appropriate permissions and/or licenses for collection of plant or seed specimens were obtained for the study. www.nature.com/scientificreports/

Results and discussions
Effect of biochar on the physiological indexes of tobacco roots. Effect on root vigor. As shown in Fig. 1, the changing trend of root vigor after biochar application was the same from 2015 to 2017. With the increased biochar application, the root vigor first increased and then decreased, in 2015, the root vitality reached the maximum at the T3 treatment, and in 2016-207, the root vitality reached the maximum at the T4 treatment.. There was no significant difference in root vigor among the treatments in 2015, and the difference between the biochar treatment and the control group treatment was not statistically significant. Among them, the root vigor in T 3 and T 4 was 110.01% and 120.03% higher than in the control, respectively. The root vigor in T 3 was higher than in other treatments with a significant difference, while the difference among T 2 , T 4 , and T 5 was not significant. Each treatment was significantly different from the control. T 4 treatment had the highest root vigor, 178.02% higher than in the control. The changing trend of root vigor among treatments in 2017 was similar to that in the previous two years. However, the root vigor in biochar treatment in 2017 was higher than in the previous two years. The vitality of each line in T 3 treatment was the highest, 164.12% higher than in the control group. The treatment of 1800 kg/ha biochar was more conducive to increasing root vigor.
Effect on the total surface area of the root system. As shown in Fig. 2, the total root area in each treatment increased with increased biochar, and the total root area first increased and then decreased, consistent with the increasing trend of root vigor. In 2015, the differences between T 2 , T 3 , T 4 , T 5 , and the control reached a significant level, and the total root area in T 3 and T 4 was 94.00% and 91.04% larger than in the control, respectively. The differences between the treatments in 2016 were similar to those in 2015. T 4 stood out in 2016, with the total root area 73.02% more than in the control; T 4 had the largest total root area in 2017, 96.01% more than in the control. The changing trend of the root system's total variable area that year was similar to that in the previous two years. On the whole, with the increased biochar application, the total surface area of the root system in each treatment showed an increasing trend. The difference among T 3 , T 4 , and T 5 treatments was not significant, but the total surface area in these three treatments were larger than in other sources in three years. The difference between T 1 and T 2 was not significant, but the total surface area in T 1 and T 2 was larger than in the control. It showed that biochar could increase the root system's total area and help the root system absorb nutrient elements in the tobacco-growing soil. The effect of T 4 was the most prominent.
Effect on the total number of root tips. Figure 3 shows the overall change of the total root tip number in each treatment. With the increased biochar, it first increased and then decreased, which was similar to the changing trend of root vigor. The difference between T 2 , T 3 , T 4 , T 5 , and the control in 2015 reached a significant level. The total number of root tips in T3 was 100.00% more than in the control. The difference between the treatments in 2016 was similar to that in 2015, and the total number of root tips in T 4 was 69.57% more than in the control. In 2017, it was the largest in T 4 , 115.00% more than in the control. With the increased biochar application, the total number of root tips of tobacco plants showed an increasing trend. T 3 and T 4 treatments were more conducive to the increase of root tips.
Effect of biochar treatment on the tobacco leaf area. As can be seen from Table 1, the area of middle leaf in each treatment was larger than in the contro l in 2015. Moreover, the difference was significant compared with the control. The changing trend of treatments was as follows: T 4 > T 5 > T 2 > T 3 > T 1 . The maximum leaf area in T 4 was 73% more than in the control. Besides, the changing pattern of the middle leaf area in each treatment    www.nature.com/scientificreports/ Effect of biochar treatment on tobacco chlorophyll. As can be seen from Table 2, the SPAD value (relative value of chlorophyll) of the middle leaf in the peak period of 2015 was higher than that in the control, with a significant difference. The changes among the treatments were as follows: T 4 > T 5 > T 3 > T 1 > T 2 . The SPAD value was 57.68% higher than that in the control; the SPAD change pattern of the treatments in 2016 was similar to that in 2015, and the SPAD value in T 4 was 50.91% higher than in the control. In 2017, the SPAD value decreased after increasing, with the increase of biochar. The SPAD value in T 4 reached its peak, 50.47% higher than in the control. The SPAD value could directly reflect the chlorophyll content, and T 4 could promote the accumulation of chlorophyll in tobacco. Note: Different lowercase letters indicate significant differences between treatments in the same year at the 0.05 level. "SPAD" represents the relative value of chlorophyll content.  Table 1 shows that the leaf opening and SPAD value in T 3 and T 4 were relatively large, which increased the net photosynthetic rate. The two values were higher in biochar treatment than in the control. The changing trend of leaf transpiration rate was consistent with that of the net photosynthetic rate in each treatment, and it was higher than in the control. The three-year data peaked in T 4 .

Effect of biochar on leaf photosynthetic characteristics and leaf area coefficients of flue-cured tobacco. Effect on the photosynthetic characteristics of leaves.
In 2015, 2016, and 2017, the changes of leaf stomatal conductance were also consistent with those of the net photosynthetic rate. Each treatment's intercellular CO 2 concentration decreased with the increased biochar application during the three years and then increased. The changes in photosynthetic characteristics of the treated leaves were the same, which indicated that the various indexes of photosynthetic characteristics were complementary to each other in the changes, and the changes in the data of light and leaf characteristics in the two years were the same, which showed that biochar had a positive effect on flue-cured tobacco leaves.  www.nature.com/scientificreports/ Effect on leaf area coefficient. Figure 5 shows that in 2015, 2016, and 2017, the leaf area coefficients' change remained the same. With the increase of biochar, leaf area coefficient first increased and then decreased, and reached the peak value in T4. The leaf area index in each biochar treatment was higher than in the control. The change of leaf area coefficients in each treatment was T 4 > T 3 > T 5 > T 1 > T 2 > the control (2015); T 4 > T 3 > T 5 > T 2 > T 1 > the control (2016); T 4 > T 2 > T 5 > T 3 > T 1 > the control (2017). It showed that biochar could promote leaf opening and ensure the balanced nutrition of the overground part of the flue-cured tobacco plant. The effect of T 4 was obvious.
Effect on the total biomass of tobacco plants. Figure 6 shows that with the increase of biochar application, the total biomass in each treatment also increased. In 2015, the total biomass of tobacco plants increased with the increase of biochar application. The change was T 4 > T 5 > T 2 > T 3 > T 1 > the control, and each biochar treatment was significantly different from the control. The changing trend of this index in 2016 was similar to that in 2015, and the index in T 4 was 94.56% higher than in the control. The changing trend in 2017 was similar to that in the previous two years. The difference between T 3 and T 4 was not significant, but the total biomass of tobacco plants was higher than that in other treatments. The index in T 4 was 117% higher than in the control. Biochar had a promoting effect on tobacco yield, and the effect of T 4 was prominent.  www.nature.com/scientificreports/

Discussions
Effect of the amount of biochar on the root physiological indexes of flue-cured tobacco. The root system is an active absorber and synthetic organ 23 . Its growth and development status and vitality level affect the overground parts' nutritional status and yield level 24 . The vitality of the root system is an important indicator that responds to developing the root system 25,26 .With the increased biochar application rate, root vigor, total root surface area, and total root-tip number increase because biochar increases soil permeability and porosity and reduces soil bulk density. Finally, the physiological indexes of the root system are improved. When the application rate of biochar was 2400 kg/ha (T 4 ), biochar had the best effect on root physiological indexes, higher than in the control. Biochar has a strong adsorption capacity, reduces the loss of nutrients in the soil, and improves the availability of nutrients in the soil, thereby improving the soil structure and utilizing nutrients by the root system. Biochar can also improve soil's water-holding capacity and soil porosity, and soil CEC can increase by 20% 26 . Meanwhile, biochar can increase the diversity of soil microorganisms 27 . Microorganisms in soil are essential participants in material transformation 28 . When the number of microorganisms involved in the decomposition of organic carbon and nitrogen in the soil increases, the mineralization rate of carbon and nitrogen and the utilization of nutrients will increase 29 . A well-developd tobacco root system helps the growth of the overground parts, and the enhancement of leaf photosynthesis provides sufficient nutrients for root growth 30 .
With the improved soil physico-chemical properties, the growth and development of roots can obtain sufficient nutrients in a suitable growth environment 31 . There are differences in the response of crops to biochar application rates. It may be affected at low application rate, but high application rate inhibits crop growth and development. This result requires further exploration of the reasons or mechanisms 32 , and this conclusion is similar to the experimental results of the work.

Effect of biochar application on flue-cured tobacco leaf area and chlorophyll.
With the increased biochar application rate, the leaf area and SPAD value of the middle leaf increased. The above two indexes in biochar treatment were higher than in the control. Among them, 2400 kg/ha of biochar treatment had apparent advantages in the above indexes because biochar could increase the respiratory metabolism rate of microorganisms 33 . The porous structure and strong adsorption properties of biochar provide a suitable habitat for soil microorganisms and have strong nutrient retention capacity 34 . Biochar can avoid the leaching of nutrient elements by reducing the dissolution and migration of water-soluble nutrient ions. It is slowly and continuously released in the soil, thereby achieving soil fertility 15,35 . It also improves the utilization of microorganisms on the substrate and improves soil fertility 36 , and the root growth. When the roots of flue-cured tobacco grow well, they can deliver sufficient water and mineral nutrients to the leaf 37 . The chlorophyll activity in the leaves is guaranteed, and the flue-cured tobacco leaves are opened in time. Previous studies show that the root system's physiological activity and the chlorophyll content of rice during the grain filling stage are significantly positively correlated 38 . This conclusion is similar to the experimental results of the work.
Effect of biochar application on the photosynthetic characteristics, leaf area coefficients and total biomass of flue-cured tobacco in the flourishing period. Among the biochar treatments, 2400 kg/ha of biochar treatment has the most significant impact on the photosynthetic physiological indexes of flue-cured tobacco leaves because the biochar application in this treatment is more conducive to the development of leaves and the optimization of the physiological indexes of photosynthesis 39 . The change of photosynthetic characteristics is similar to the change of the root physiological index. Furthermore, the tobacco plants in 2400 kg/ha of biochar treatment have greater root vigor, total root tip number, and total root absorption area in the flourishing period, promoting the tobacco plants to maintain good photosynthetic physiological performance and chlorophyll synthesis. Biochar has advantages such as reducing the concentration of carbon dioxide between cells, and increasing net photosynthesis rate, transpiration rate, stomatal conductance, and leaf area coefficients, which may be related to the physiological characteristics of roots and chlorophyll. When the roots and leaves of flue-cured tobacco are well developed, the supply of mineral nutrients, water, and enzymes required for photosynthesis is sufficient to optimize the relevant indexes of photosynthetic characteristics and make the leaf development healthier. Moreover, the leaf area coefficients increase. The growth status of the root system of flue-cured tobacco is closely related to the leaf photosynthetic physiological status, and the physiological indexes of photosynthesis and the development of the overground parts are in connection with the yield and quality of flue-cured tobacco 40 . The addition of biochar increases the net photosynthetic rate of rice and plays a significant role in promoting rice growth during the vegetative and maturing stages 41 . This conclusion is consistent with the results of our work. The increase in leaf area index of the flue-cured tobacco lays the foundation for improving the maturity and yield quality of the flue-cured tobacco at the later stage. Biochar can increase the total biomass of tobacco, which is an important economic crop 42 . Therefore, the increased total biomass is of great significance to farmers.

Conclusions
The application of biochar can improve the environment of tobacco root growth, root vigor, primary root morphology, flue-cured tobacco leaves, and physiological indexes. The effects of biochar on leaf photosynthetic physiological indexes, leaf area, leaf area coefficient, and chlorophyll prove that biochar's role in promoting leaf photosynthetic characteristics results from the coordination, complementation, and synergistic development of flue-cured tobacco roots and overground parts. By changing the characteristics of the soil, biochar promotes tobacco growth and coordinates the relationship between roots and leaves, which is of great significance to the improvement of the quality of raw materials in the tobacco production process.