Experimental design and Plant materials
The study was designed as a two factors factorial experiment following randomized complete block design with the split-plot arrangement. The first factor included two varieties CFSH30 and Siyong 3180 abbreviated as V1 and V2 and the second factor was N rates of 0, 150, and 300 kg N ha− 1, abbreviated as N1, N2, and N3 respectively.
Ethical Statement
Forage Sorghum variety CFSH30 from Canada was used in this study. The variety was improved by Dr OM Dangi in 2012 and provided by Canada Environmental Renew Co. Ltd. Siyong 3180 variety from China was collected in 2018 by Mengma Agriculture Company plz. The plant materials were provided by Chinese Academy of Agricultural Sciences.
The seed was stored in brown bags in fridge (4 oC) to maintain good germination ability. Before sowing, seeds of each variety were selected for uniform color and size. Germination percentage was recorded 90.5% and 90.7% for CFSH30 and Siyong 3180. On May 26, both in 2017 and 2018, the seed was hand broadcast at the rate of 4.5 kg ha− 1 in 18 plots of 10.5 m2 (3.5 m × 3. 0 m) sizes with plot to plot distance of 40 cm.
Phosphorus (P) was broadcasted twice at equal rate of 60 kg ha− 1 per time at seeding and plant elongation stages30. The N was applied as urea to all treatments in solid form. The N and P fertilizer were applied during sowing at the same time to each plot by hand-broadcasting. Others field practices, like, mowing between lines, weeding, and pest and disease control were carried out in accordance with local recommendations.
Observation and measurement
Plant growth parameters
On the 90th day of seeding (DAS) ten uniform plants were randomly collected in each plot and leaf length (cm) and width (cm) were measured31.
Plant height (cm) was measured using a survey rod from the bottom of soil to the top of the panicles32.
To determine specific leaf weight, the second top leaves were selected and weighted through electronic balance.
To determine biomass production, whole plants were harvested at 90th day after sowing from the center of plots and hand-separated into leaf and stem. The fresh weight of leaves and stem were examined33.
Sorghum was cut repeatedly: into two cut-systems. Raw materials were accumulated and determination were made on the numbers of plant m2 and weight kg− 1 through electrical scale34.
Determination of physiological parameter
0.5 g leaves sample were used to determine protein soluble activity. The sample was homogenized at 4 oC in 5 mL Na-phosphate buffer (pH 7.2) and directly centrifuged at 4oC at 10,000 rpm for 15 minutes. The supernatants were kept on ice for further examination. Soluble protein content was measured by using the Coomassie blue dye-binding assay following the method of35. Absorbance readings were converted into protein contents using bovine serum albumin (BSA) as the standard curve36. Supernatants and dye were pipetted in spectrophotometer cuvettes and absorbance was recorded using a spectrophotometer (Model 721, Shanghai Mapada Instruments Co. Ltd, Shanghai, China) at 595 nm.
To determine antioxidant activities, 0.5 g fresh leaves were crushed in a mortar containing 5 ml extraction buffer (50 mM Tris-HCl [pH 7.0], 0.1 mM EDTA, 1 mM AsA, 1 mM dithiothreitol and 5 mM MgCl2). The resultant homogenates were centrifuged at 10,000 g for 15 minutes at 40C, and the obtained supernatants were used to determine the activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)6–7−37. SOD activity was measured by computing the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT)38. The structure of the reaction combination (3 ml) was 50 mM of Tris-HCl (pH 7.8), 13.37 of mM methionine, 0.1 mM of NBT, 0.1 mM of riboflavin, 0.1 mM of EDTA and 0.1 ml of the enzyme extract. Peroxidase (POD) and catalase (CAT) activities were assessed following the methods of Tariq et al. (2018)37 with minor changes. Due to guaiacol oxidation, POD activity was evaluated by calculating the increase in absorbance at 470 nm. The reaction combination was composed of 50 mM Tris-HCl (pH 7.0), 10 mM of guaiacol, 5 mM of H2O2 and 0.1 ml of the enzyme extract. One unit of POD activity was determined as an absorbance variation of 0.1 units per min. In addition, CAT activity was measured by determining the decrease in the absorbance of H2O2 at 240 nm, respectively. The structure of the reaction combination (3 ml) was 50 mM of Tris-HCl (pH 7.0), 0.1 mM of EDTA, 12.5 mM of H2O2 and 0.1 ml of the enzyme extract. One unit of enzyme activity was distinct as a 0.01-change in the absorbance at 240 nm per min.
Statistical analysis
Compound data analysis of two years (2017 and 2018) was conducted based on randomized complete block design with split-plot arrangement (two cultivars and three N rates). Analysis of variance was carried out using Mstate-C sofware39. Least significant difference test (LSD) was used when the F values were significant (P ≤ 0.05).
Conclusions
The study examined the effects of various levels of N on morpho-physiological activities of two sorghum varieties. We concluded that sorghum growth and physiology responded more to higher rates of N as compared with N1. The application of N2 and N3 enhanced all the morpho-physiological activities such as leaf length, leaf width, plant height, Specific leaf weight, stem weight, leaf weight, weight kg− 1, protein contents, and antioxidant enzymes activities. Moreover, higher application of N reduced the number of plants m2 during two cuts as compared to N1. Both sorghum varieties exhibited potential increase but V1 showed higher morpho-physiological activity as compared with V2. All these indices sorted out the best practices for achieving higher sorghum biomass yield but N3 was much appropriated during the two growing seasons particularly for V1 variety. Therefore, it can be concluded from the results that higher rate of N3 and V1 cultivar is promising alternative for good economic revenues at Yangtze River China. However, additional investigation is required to examine the impact of nitrogen from different sources in more sorghum varieties. Consequently, nitrogen fertilizer management is needed to provide growth and yield of crops.