Study site and experimental soil properties
A field experiment was conducted in Sheli Town, Da’an Country, Jilin Province, Northeast China (45°35′N, 123°50′E). This area has a typical dry-cold monsoon climate, with an average annual air temperature of 4.7 °C, average precipitation of approximately 413.7 mm, and average evaporation of approximately 1696.9 mm. The soil texture in this experiment is 23.23% sand, 38.14% silt and 37.60% clay according to the international Society of Soil Science classification. The basic physicochemical characteristics of the soil in this experiment are shown in Table 1. The average value of EC, SAR, pH in saturated paste and ESP is 24.08 μs m-1, 368.11 (mmolc L-1)1/2, 10.10, and 71.11%. Based on the texture classification system of USDA (1954), the soil in this experimental aera is characterized as saline-alkali and had a three years history of rice planting.
Experimental design
The field experiment was conducted in 2021. The trial was established in a split-plot design with nitrogen as the main plot and biochar as the subplot, with three replicates. There are include a total of 24 plots (each 5 m by 6 m) with separated by buffer rows (60 cm in width). Each plot has an independent irrigation and drainage. There are two nitrogen treatments and four biochar applied rate treatments. The two nitrogen treatments include no nitrogen applied (N0) and conventional nitrogen applied level (N225). Four biochar rates were applied into soil layer (0-20cm), refereed as T0 (zero biochar), T1 (1.5% biochar, w/w), T2 (3.0% biochar, w/w) and T3 (4.5% biochar, w/w), respectively.
The rice variety planted in this field study was japonica rice Changbai 9, one of the elite cultivars used in saline-alkali paddy soil in Northeast China. On 12 April, 2021, rice seeds were sown in a greenhouse. Rice seedlings were transplanted to the field plots on May 21, 2021. The transplanting density (per hill) was 30 cm × 16.5 cm, and each hill contained three seedlings. The rice was harvested on September 30, 2021. In the treatment of N225, the application rates of chemical (NPK) fertilizer were as follows: 225 kg N per hectare, 75 kg P per hectare, and 100 kg K per hectare, respectively. In the treatment of N0, the application rates of chemical (NPK) fertilizer were as follows:0 kg N per hectare, 75 kg P per hectare, and 100 kg K per hectare, respectively. Field management was the same as that used in local production fields to minimize yield loss.
Preparation of biochar
The biochar was produced from peanut shells using a vertical kiln, manufactured by Jinhefu Agricultural Development Company, Liaoning Province, China, and the pyrolysis temperature was 350 to 550 °C for 4 h. The peanut shells were obtained from Jinhefu Agricultural Development Company, AnShan city, Liaoning Province, China. The physiochemical properties of the biochar and peanut shells presented in Table 2. Biochar was applied in the spring of 2021 and uniformly spread on the surface of saline-alkali paddy soils before rice transplanting, and then thoroughly ploughed into the topsoil (0 to 20 cm) using a wooden rake.
Leaf concentration of Na+, K+, Na+ / K+ ratio
All leaves of five rice plants were collected in each treatment at maturity stage. These leaves are dried in an oven to constant weight at 60℃ and then ground to fine power. The leaf samples were digested with 1% nitric-perchloric acid (Bastías et al. 2004). The concentration of Na+ and K+ was measured using the fame meter method (M410, Sherwood Scientific Ltd., Cambridge, England), and then calculated the Na+/K+ ratio.
Leaf malondialdehyde content (MDA) and abscisic acid (ABA)
For each treatment, flag leaves from 3 rice plants were analyzed at heading stage (August 12) and filling stage (September 1). MDA was quantified using the method of Stewart and Bewley (1980). Fresh leaves (1 g) were homogenized with quartz and 10 ml of 10% trichloroacetic acid, and then centrifuged at 4000 rpm for 10 min. The supernatant of 2 ml was extracted, add 2 ml of 0.6% thiobarbituric acid and shake well. After react in boiling water for 15 min, and then cooled on ice and centrifuged at 4000 rpm for 10 min. The absorbance of supernatant was read at 532, 600 and 450 nm. MDA content was calculated using the extinction coefficient of 155 mM-1 cm-1 and the content was expressed as nmol g-1 FW.
Leaf abscisic acid content (ABA) was quantified using the method described by Liquid chromatography-Mass Spectrometry (Liu et al., 2021). Fresh leaves (0.5 g) were ground to a powder with liquid nitrogen. The powder was then pooled (100-200 mg) and placed into a 1.5 mL centrifuge tube, and 750 µL of freeze solution A [methanol/water/acetic acid (89/10/1 v/v/v)] containing 30 ng of 2H-ABA [(-)-5,80 ,80 ,80 -d4 ABA] was added. After thorough vortexing, each sample was centrifuged at 13000 rpm for 10 min. The supernatant was placed into a new 1.5 mL centrifuge tube, and 450 µL of solution B [methanol/water/acetic acid (89/10/1 v/v/v)] was added to the precipitate, after which each sample was vortexed thoroughly for 4 h. The samples were then centrifuged at 13000 rpm for 10 min, after which the supernatant was combined with the previous supernatant. The mixed supernatant was used to quantify the ABA contents by an LC-MS system (Ultimate TSQ Quantia, Thermo Fisher Scientific).
Leaf water status, relative electrical leakage
At heading stage (August 12) and filling stage (September 1), based on the method of Dionisio-Sese and Tobita (1998), the relative electrical leakage of leaf was measured. The HR-33T DEW Point Mikrovolt-meter (Wescor Inc., Logan, UT, USA) was used to determine the leaf water potential (Ψw) of rice at 9:00 - 11:00 am in both heading and filling stage.
Leaf chlorophyll content and leaf N content
At heading stage (August 12) and filling stage (September 1), the chlorophyll content of upper canopy three fully expanded leaves from main stem of rice was determined using a portable CCM-200 (Opti-Science, Tyngsboro, MA, USA). The content of total N (% DW) was measured from finely check leaf samples using CHNS/O analyzer (Flash 2000, Thermo Fisher Scientific, Cambridge, UK). The operating procedure was according to the dynamic flash combustion method.
Leaf area index (LAI), Photosynthetic potential, Leaf area decreasing rate (LAD), and Net assimilation rate (NAR)
At heading stage (August 12) and filling stage (September 1), five hills were selected from each treatment for measuring dry matter weight. The leaf area was measured by length × width ×0.75. The leaf area index (LAI) was calculated after the determination of determined.
Leaf area index (LAI) = total leaf area / land area.
Photosynthetic potential = 1/2 (L1+L2) (t2-t1), where L1 and L2 are the leaf areas (m2) measured before and after, t1 and t2 are the time (d) measured before and after.
Leaf area decreasing rate (LAD) = (LAI2-LAI1)/(T2-t1), where LAI1 and LAI2 are the Leaf area index measured before and after, t1 and t2 are the time (d) measured before and after.
Net assimilation rate (NAR) = [ln (LAI2)-ln (LAI1)]/(LAI2-LAI1) × (W2-W1)/(t2-t1), where LAI1 and LAI2 are the Leaf area index measured before and after, W1 and W2 are the dry matter weight (g •m-2) measured before and after, and t1 and t2 are the time (d) measured before and after.
Leaf photosynthesis rates (Pn), stomatal conductance (GS), transpiration rates (Tr) and intercellular CO2 concentrations (Ci)
Photosynthesis-related parameters were measured at heading stage (August 12) and filling stage (September 1). The net photosynthesis rates, stomatal conductance (GS), transpiration rates (Tr) and intercellular CO2 concentrations (Ci) of flag leaves from main stem of rice were measured using a portable photosynthesis system Li-6400 (Li-COR 6400, Li-COR Inc., Nebraska, USA) at 9:00-11:00 am. Measurement conditions in leaf chamber were kept consistent: photosynthetically active radiation (PAR) of 1200 mmol m-2 s-1, CO2 concentration of 370 ppm and LED light source. Nine leaves were measured for each treatment.
Rice Biomass yield (BY), grain yield (GY) and harvest index (HI)
At the mature stage, 15 rice plants were randomly harvested in each treatment. These plants were oven-dried at 105 °C for 30 min and then at 60 °C to a constant weight. The biomass was recorded. The rice plants were selected from 5 m2 for each experimental plot, and then the rice grain yield was calculated. The harvest index (HI) is the ratio of grain yield to biomass.
Statistical analysis
The data were analyzed using SPSS 18.0 software (IBM Corp., Armonk, NY, USA) based on the trial design. One-way ANOVA and Tukey tests were employed to analyze the effect of biochar on the relevant test indicators. The mean value was determined with the least significant difference at the p < 0.05 level.