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 2,400 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.
The tested soil is Phaeozem (IUSS, 2014), and tobacco seedlings were transplanted on May 10, 2015, 2016, and 2017. The basic physico-chemical properties of tobacco-growing soil are as follows: soil pH of 6.9; C, N and S content of 1.371, 0.126, and 0.073%, respectively; organic matter content of 2.31%; AN content of 89%. For AN 55 mg/kg, AP is 21.587 mg/kg, and AK is 165.453 mg/kg. The biochar used in the experiment was provided by Biochar Laboratory of Tobacco College of Henan Agricultural University. Its basic physico-chemical indexes include total carbon (76.50%), moisture (9.0%), pH (8.6), TN (1.90%), TP (1.50%), and TK (0.80%).
1.2.1 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 T1, T2, T3, T4, and T5). After ridging the tobacco field, the fertilizers were applied 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 K₂SO₄ 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.
1.2.2 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, 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 1,000 μmol/(m2·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 CO2 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＋2W)/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 wetted at 115°C for 25 min and then dried at 65°C before it was weighed. The result obtained was the dry matter mass of the tobacco plant.
1.3 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.