Wheat Straw Biochar Amendment Increases Salinity Stress Tolerance in Alfalfa Seedlings by Modulating Physiological and Biochemical Responses

Abstract

Salinity stress is a major environmental challenge that adversely impacts the physiological and biochemical processes of pasture, consequently resulting in reduced yields and compromised quality. Biochar amendment has recently emerged as a promising strategy to alleviate the deleterious effects of salinity stress. However, the interactive influences of salinity stress and wheat straw biochar on the physiological, biochemical, and growth characteristics of alfalfa (Medicago sativa L.) remain underexplored. A factorial experiment was conducted using a randomized complete design with five salinity levels (0, 25, 50, 75, and 100 mM NaCl) and three application rates of biochar (0, 25, and 50 g kg^-1) to evaluate wheat straw biochar's potential in alleviating salinity stress in alfalfa. Results showed that salinity stress increased oxidative stress (hydrogen peroxide and malondialdehyde) and reduced chlorophyll fluorescence (maximum quantum efficiency of photosystem II by 1-27%), leading to decreasing photosynthetic parameters, thereby constraining biomass accumulation by 9-77%. Wheat straw biochar amendment under the highest salinity stress, particularly at 25 g kg^-1, mitigated oxidative stress by reducing H₂O₂ and MDA levels by 35% and 33%, respectively, while decreasing the antioxidant enzymes activities of CAT, POD, and SOD by 47%, 42%, and 39%, respectively, compared to the control (non-biochar addition). Concurrently, biochar restored the osmoregulatory substance concentrations of proline and soluble sugar by 59% and 33%, respectively, compared to the control. Furthermore, wheat straw biochar amendment increased the net CO₂ assimilation rate by 98%, thereby increasing biomass by 63%. Our study demonstrates that wheat straw biochar can contribute to protecting alfalfa against salinity stress by modulating physiological and biochemical responses. These findings demonstrate that the 25 g kg^-1 wheat straw biochar application had the best performance, suggesting this amendment could be a viable strategy for improving alfalfa productivity in salt-affected soils. Future research should explore long-term field applications and the underlying mechanisms of biochar-plant-soil-plant interactions under diverse saline-alkali environments.

Keywords: Medicago sativa; antioxidant enzyme; gas exchange; hormonal regulation; osmotic adjustment.

Figure 1

Shoot and root biomass of alfalfa seedlings under salinity stress and wheat straw biochar amendment treatments. Different capital letters indicate significant differences between measurement dates within the same wheat straw biochar treatment at the 0.05 level (p ≤ 0.05). Different lowercase letters indicate significant differences between salinity stress and wheat straw biochar amendment treatment combinations at the 0.05 level (p ≤ 0.05). S0, S1, S2, S3, and S4 refer to 0 mM, 25 mM, 50 mM, 75 mM, and 100 mM NaCl dose levels, respectively. B0, B1, and B2 refer to 0 g kg⁻¹, 25 g kg⁻¹, and 50 g kg⁻¹ wheat straw biochar amendment levels, respectively. Data are presented as arithmetic mean ± standard error (n = 5).

Figure 2

(a) Leaf net CO₂ assimilation rate (A), (b) stomatal conductance (g_s), (c) transpiration rate (E), (d) intrinsic water-use efficiency (W_g), (e) instantaneous water-use efficiency (W_i), and (f) maximum quantum efficiency of photosystem II (F_V/F_M) in alfalfa seedlings under salinity stress and wheat straw biochar amendment treatments. Different capital letters indicate significant differences (p ≤ 0.05) between measurement dates within the same wheat straw biochar treatment. Different lowercase letters indicate significant differences (p ≤ 0.05) between salinity stress and wheat straw biochar amendment treatment combinations. S0, S1, S2, S3, and S4 refer to 0 mM, 25 mM, 50 mM, 75 mM, and 100 mM NaCl dose levels, respectively. B0, B1, and B2 refer to 0 g kg⁻¹, 25 g kg⁻¹, and 50 g kg⁻¹ wheat straw biochar amendment levels, respectively. Data are presented as arithmetic mean ± standard error (n = 5).

Figure 3

The contents of (a) chlorophyll a (Chl a), (b) chlorophyll b (Chl b), (c) total chlorophyll (total Chl), and (d) the ratio of chlorophyll a to chlorophyll b (Chl a:b ratio) in alfalfa leaves under salinity stress and wheat straw biochar amendment treatments. Different capital letters indicate significant differences (p ≤ 0.05) between measurement dates within the same wheat straw biochar treatment. Different lowercase letters indicate significant differences (p ≤ 0.05) between salinity stress and wheat straw biochar amendment treatment combinations. S0, S1, S2, S3, and S4 refer to 0 mM, 25 mM, 50 mM, 75 mM, and 100 mM NaCl dose levels, respectively. B0, B1, and B2 refer to 0 g kg⁻¹, 25 g kg⁻¹, and 50 g kg⁻¹ wheat straw biochar amendment levels, respectively. Data are presented as arithmetic mean ± standard error (n = 5).

Figure 4

(a) Hydrogen peroxide (H₂O₂) and (b) malondialdehyde (MDA) contents in alfalfa leaves under salinity stress and wheat straw biochar amendment treatments. Different capital letters indicate significant differences (p ≤ 0.05) between measurement dates within the same wheat straw biochar treatment. Different lowercase letters indicate significant differences (p ≤ 0.05) between salinity stress and wheat straw biochar amendment treatment combinations. S0, S1, S2, S3, and S4 refer to 0 mM, 25 mM, 50 mM, 75 mM, and 100 mM NaCl dose levels, respectively. B0, B1, and B2 refer to 0 g kg⁻¹, 25 g kg⁻¹, and 50 g kg⁻¹ wheat straw biochar amendment levels, respectively. Data are presented as arithmetic mean ± standard error (n = 5).

Figure 5

Leaf (a) catalase (CAT) activity, (b) peroxidase (POD) activity, (c) superoxide dismutase (SOD) activity, (d) proline content, and (e) soluble sugar content in alfalfa seedlings under salinity stress and wheat straw biochar amendment treatments. Different capital letters indicate significant differences between measurement dates within the same wheat straw biochar treatment at the 0.05 level (p < 0.05). Different lowercase letters indicate significant differences between salinity stress and wheat straw biochar amendment treatment combinations at the 0.05 level (p ≤ 0.05). S0, S1, S2, S3, and S4 refer to 0 mM, 25 mM, 50 mM, 75 mM, and 100 mM NaCl dose levels, respectively. B0, B1, and B2 refer to 0 g kg⁻¹, 25 g kg⁻¹, and 50 g kg⁻¹ wheat straw biochar amendment levels, respectively. Data are presented as arithmetic mean ± standard error (n = 5).

Figure 6

The contents of (a) indoleacetic acid (IAA_leaf), (b) gibberellic acid (GA_leaf), and (c) abscisic acid (ABA_leaf) in alfalfa leaves under salinity stress and wheat straw biochar amendment treatments. Different capital letters indicate significant differences (p < 0.05) between measurement dates within the same wheat straw biochar treatment. Different lowercase letters indicate significant differences (p ≤ 0.05) between salinity stress and wheat straw biochar amendment treatment combinations. S0, S1, S2, S3, and S4 refer to 0 mM, 25 mM, 50 mM, 75 mM, and 100 mM NaCl dose levels, respectively. B0, B1, and B2 refer to 0 g kg⁻¹, 25 g kg⁻¹, and 50 g kg⁻¹ wheat straw biochar amendment levels, respectively. Data are presented as arithmetic mean ± standard error (n = 5).

Figure 7

Correlation plots between all measured variables in the experiment. Color intensity and circle size represent the absolute value of the Pearson correlation coefficient.

Figure 8

Schematic representation of wheat straw biochar amendment on the growth and physiological adaptations of alfalfa (Medicago sativa L.) under salinity stress, based on the findings of this study.