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Article

Beneficial Effects of Biochar-Based Organic Fertilizer on Nitrogen Assimilation, Antioxidant Capacities, and Photosynthesis of Sugar Beet (Beta vulgaris L.) under Saline-Alkaline Stress

by
Pengfei Zhang
1,
Fangfang Yang
1,
He Zhang
1,
Lei Liu
1,2,
Xinyu Liu
1,
Jingting Chen
1,
Xin Wang
1,
Yubo Wang
1 and
Caifeng Li
1,*
1
College of Agriculture, Northeast Agricultural University, Harbin 150030, China
2
Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Science, Changchun 130102, China
*
Author to whom correspondence should be addressed.
Agronomy 2020, 10(10), 1562; https://doi.org/10.3390/agronomy10101562
Submission received: 8 September 2020 / Revised: 8 October 2020 / Accepted: 9 October 2020 / Published: 14 October 2020
(This article belongs to the Special Issue Applications of Biochar and Other Organic Amendments within a Sustainable Agriculture and Circular Economy)
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Abstract

:
The Songnen Plain, whose climatic conditions are perfectly suited to sugar beet growth, is located in northeastern China. Unfortunately, this region has a lot of saline-alkaline land, which is the most important factor limiting sugar beet production. This study was undertaken to determine whether biochar-based organic fertilizer could alleviate the negative effect of saline-alkaline soil on sugar beet yield and whether such an effect correlated with changes in nitrogen assimilation, antioxidant system, root activity, and photosynthesis. Three treatments were established: Chemical fertilizers were applied to neutral soil (CK), chemical fertilizers were applied to saline-alkaline soil (SA), and biochar-based organic fertilizer was applied to saline-alkaline soil (SA + B). Our results showed that saline-alkaline stress significantly inhibited the nitrogen assimilation and antioxidant enzymes activities in root, root activity, and photosynthesis, thus significantly reducing the yield and sugar content of sugar beet. Under saline-alkaline conditions, the application of biochar-based organic fertilizer improved the activities of nitrogen assimilation enzymes in the root; at the same time, the antioxidant enzymes activities of the root were significantly increased for improving root activity in this treatment. Moreover, the application of biochar-based organic fertilizer could improve the synthesis of photosynthetic pigments, PSII (Photosystem II) activity, stomatal opening, and photosynthesis of sugar beet under saline-alkaline conditions. Hence, the growth and yield of sugar beet were improved by applying biochar-based organic fertilizer to saline-alkaline soil. These results proved the significance of biochar-based organic fertilizer in alleviating the negative effect of saline-alkaline stress on sugar beet. The results obtained in the pot experiment may not be viable in field conditions. Therefore, in the future, we will verify whether biochar-based organic fertilizer could alleviate the adverse effects of saline-alkaline stress on sugar beets yield under field conditions.

1. Introduction

Soil salinization is a major environmental and ecological problem that restricts agricultural production of the world [1]. Changes in enzymes’ activity, reduced root activity (the ability of root to absorb nutrients and water), and photosynthetic capacity are some of main saline-alkaline injury symptoms in plants. Nitrogen assimilation plays an important role in plants, with nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthetase (GOGAT) being key enzymes for nitrogen assimilation [2]. Under saline-alkaline conditions, undernutrition and poor growth of plants is mainly due to the decrease in these enzymes’ activity in the roots [3]. Unconscionable generation of reactive oxygen species (ROS) is one of the main biochemistry changes in plants under salt-alkaline stress [4]. ROS increases the malondialdehyde (MDA) content through oxidative damage to lipids [5]. The antioxidant enzyme system, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), can reduce the damage of ROS to roots, which improves root activity [6]. Photosynthetic pigments (chlorophyll and carotenoids) and gas-exchange parameters (net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration) are regarded as the main photosynthesis parameters [7]. In addition, chlorophyll fluorescence parameters, such as the maximum quantum yield of PSII (Fv/Fm), photochemical quenching (qp), electron transport rate (ETR), PSII actual photochemical efficiency (ΦPSII), and PSII potential photochemical activity (Fv/Fo), can also be used to evaluate photosynthetic performance [8]. Inhibition of photosynthetic pigments, gas exchange, and PSII activity under saline-alkaline stress limit photosynthesis, leading to a decrease in dry matter accumulation, which limits crop yield [9]. In order to overcome the adverse effects of saline-alkaline stress on plants, various strategies are being implemented, such as deep plowing, drip irrigation, and the application of amendments and organic fertilizers.
Biochar-based organic fertilizer is a new type of organic fertilizer, which has been used for agricultural production in recent years [10]. It is composed of biochar and animal manure, which can improve soil properties, such as the pH and nutrient content, and enhance the tolerance of plants to abiotic stresses, such as drought, heavy metal, salt, and acid stress [11,12,13,14]. Under these stresses, inhibition of nitrogen assimilation, root activity, and photosynthesis limits the growth and yield of crops [15]. Recent studies have shown that the maintenance of soil inorganic nitrogen provided by biochar-based organic fertilizer improves root nitrogen assimilation [16]. It can also enhance the activity of antioxidant enzymes to remove excess ROS, which greatly improves root activity [17]. Moreover, application of biochar-based organic fertilizer greatly promotes the accumulation of dry matter because it can significantly improve photosynthesis, thereby enhancing crop yields [18].
Although sugar beet has better saline-alkaline tolerance, the growth and yield of sugar beet will be significantly inhibited when the salt content and pH of the soil are too high [19]. The Songnen Plain, whose climatic conditions are perfectly suited to sugar beet growth, is located in northeast China. Unfortunately, this region has a lot of saline-alkaline land, which is the most important factor limiting sugar beet production [20]. Previous studies have indicated that saline-alkaline stress adversely affects mineral absorption, photosynthesis, ion balance, antioxidant system, growth and development, and physiological metabolism in sugar beet, but few studies have been devoted to alleviating the negative impacts of saline-alkaline soil on sugar beet [21,22,23,24,25,26]. Therefore, effective strategies should be developed to alleviate the adverse effects of saline-alkaline soil on sugar beet throughout the growth period, which is essential for saline-alkaline regions to safeguard the sugar supply and agriculture economic development.
All of the above have stimulated our interest and prompted us to explore the effect of biochar-based organic fertilizer on sugar beet in saline-alkaline soil. We hypothesized that the application of biochar-based organic fertilizer may alleviate the adverse effects of saline-alkaline soil on the growth of sugar beet. This study was undertaken to determine whether biochar-based organic fertilizer could alleviate the negative effects of saline-alkaline soil on sugar beet yield and whether such effects correlated with changes in nitrogen assimilation, antioxidant system, root activity, and photosynthesis. The results could provide a promising strategy for sugar beet production in saline-alkaline areas in the Songnen Plain, and other areas with similar soil conditions.

2. Materials and Methods

2.1. Experimental Design, Materials, and Growing Conditions

The experiment was carried out according to a randomized complete block design in Northeast Agricultural University located in northeast China (126°72′ E, 45°74′ N) from May to September in 2017 and 2018. Four replicates were conducted for each treatment as follows: (1) CK: chemical fertilizers (urea (46% N), diammonium phosphate (18% N and 46% P2O5), and potassium sulfate (50% K2O)) were applied to neutral soil (meadow soil); (2) SA: chemical fertilizers were applied to saline-alkaline soil; and (3) SA + B: biochar-based organic fertilizer was applied to saline-alkaline soil.
Biochar-based organic fertilizer (3.65% N, 3.38% P, and 2.29% K) consisted of the same quality of biochar and matured animal manure, which was provided by RunNong Technology Company (Harbin, China). Biochar (63.2% C, 1.02% N, 0.26% P, and 0.62% K) was made from maize stalk at 450 °C for 3 h in a thermal cracking device. The pH, cation exchange capacity, and specific surface area of biochar were 9.76, 62.32 cmol kg −1, and 89.7 m2 g−1, respectively. Neutral soil was taken from Northeast Agricultural University experimental station, and saline-alkaline soil was procured from Daqing (125°11′ E, 46°37′ N), which is a typical saline-alkaline area of Songnen Plain. The chemical properties of the soils are shown in Table 1. Each pot (20 cm wide and 25 cm height) was filled with 10 kg of soil. The soils were passed through a 4-mesh screen to remove impurities. On April 25 in 2017 and 2018, each pot from CK and SA treatments was fertilized with 4.6 g of urea, 58.8 g of diammonium phosphate, and 19.3 g of potassium sulfate, and well-mixed with the soil. At the same time, 377.9 g of biochar-based organic fertilizer were applied per pot in the SA + B treatment to equal the nutrient contents of each treatment. The pelletized seeds of sugar beet cultivar ‘KWS0143’ were provided by KWS Company (Einbaek, Germany), which were sown in pots on 1 May in 2017 and 2018. In order to avoid diseases and pests, each pot was sprayed with 5 mL of 0.1% carbendazim and 3 mL of 0.5% cypermethrin when the third and sixth pairs of true leaves of sugar beet were fully expanded. Weeds were manually controlled during the growing season.

2.2. Determination of the Properties of Soil and Biochar

Soil pH and electrical conductivity were measured using a pH meter (pH–3110, WTW, Munich, Germany) and conductivity meter (DDSJ–308A, INESA Inc., Shanghai, China), respectively. The available nitrogen of soil was extracted with 2 M KCl and analyzed by using a continuous flow analytical system (FIAstar–5000, Foss, Hilleroed, Denmark) [27]. Soil-available phosphorus was extracted with 0.5 M NaHCO3, adjusted pH to 8.5, and determined at 700 nm [28]. The available potassium of the soil was extracted with 1 M NH4OAc and measured by a flame photometer (FP–6400, Yuefeng Inc., Beijing, China) [29]. Soil C content was determined by an elemental analyzer (EA–3000, Euro Vector, Redavalle, Italy). Organic matter = C content × 1.724 [30].
The methods of measuring the pH and C content of biochar were the same as that of soil. The nitrogen content of biochar was determined by an elemental analyzer. To determine the content of P and K in biochar, the biochar was digested by the mixture of HClO4 and H2SO4. The P content was determined by adding ammonium molybdate, and the K content assessment was made by monitoring the absorbance of the digested liquid at 880 nm [31]. After the biochar was mixed with NaOAc, the cation exchange capacity of the biochar was measured by a flame photometer [32]. The specific surface area of biochar was measured by using an auto fast specific surface area analyzer (BSD–Bet400, Beishide Inc., Beijing, China).

2.3. Sampling and Measurements of Sugar Beet

Gas-exchange parameters and chlorophyll fluorescence parameters of fully expanded top leaves in each treatment were measured at 50, 70, 90, 110, and 130 days after sowing (DAS), which coincided approximately with the seedling, root differentiating and forming, foliage luxuriating, sugar increasing, and sugar accumulating stages. At the same time, eight sugar beets were randomly selected from each treatment. After washing with distilled water, four of them were divided into overground tissues and roots, then frozen in liquid nitrogen, and stored at −80 °C until physiological assays were done, and the others were dried to determine the dry matter accumulation and root/shoot ratio.

2.3.1. Activities of Nitrogen Assimilation Enzymes

The activity of nitrate reductase (NR) in roots was determined by the method of Jaworski [33]. Sugar beet roots (0.5 g), 50 μL of 5% ethyl acetate, and 9 mL of 35 mM phosphate buffer (pH 7.5) were sealed in a vial, and then were placed in the dark at 25 °C for 30 min. The reaction was terminated by adding 1 mL of 3% trichloroacetic acid. The reaction solution was treated by adding 2 mL of N–1–Naphthyl-ethylenediamine hydrochloride and 2 mL of 2% sulfanilamide. After 25 min, the absorbance of the solution was measured using a spectrophotometer (UV–2500, Shimadzu, Kyoto, Japan) at 540 nm.
Nitrite reductase (NiR) activity was determined by testing the reduction of nitrite at 540 nm according to the method of Datta and Sharma [34]. In total, 1 g of root tissue was homogenized in 3 mL of 50 mM Tris–HCl buffer (pH 7.9) containing 5 mM cysteine and 2 mM EDTA. The homogenate was centrifuged at 10,000× g for 20 min at 4 °C. The reaction mixture contained 50 mM Tris–HCl (pH 7.5), 0.5 mM NaNO2, 1 mM methyl viologen, 50 μL of enzyme extract, and distilled water. Then, 0.12 M Na2S2O4 was added to start reaction and incubated at 30 °C for 30 min. After incubation, the reaction mixture was violently shaken until the methyl color completely faded to terminate the reaction. Then, 1 M Zn(CH3COO)2 was added and centrifuged at 10,000× g for 10 min. The supernatant was treated by 0.02% N–1–Naphthyl–ethylenediamine hydrochloride and 1% sulfanilamide containing 1.5 M HCl, and the absorbance was determined at 540 nm.
Glutamine synthetase (GS) activity was measured by the method of Nagy et al. with modification [35]. The sugar beet root (1 g) and 3 mL of 100 mM Tris–HCl (pH 8.0) were homogenized and centrifuged at 12,000× g for 20 min at 4 °C. Enzyme extract was mixed with reaction solution consisting of 100 mM Tris–HCl (pH 8.0), 50 mM glutamate, 5 mM hydroxylamine hydrochloride, 50 mM MgSO4, and 20 mM adenosine triphosphate, and then incubated at 35 °C for 15 min. The reaction was terminated by stop solution consisting of 370 mM FCl3, 670 mM TCA, and 200 mM HCl. After centrifugation (10 min, 5000× g), supernatant absorbance was measured to monitor the formation of γ-glutamyl monohydroxamate at 540 nm.
The activity of glutamate synthase (GOGAT) was determined by testing the formation of glutamate at 340 nm according to the method of Yasuhiro [36]. Enzyme extract was collected by the procedures of GS enzyme extract. The reaction mixture included 100 mM α-ketoglutarate, 10 mM KCl, 25 mM Tris–HCl (pH 8.0), 3 mM nicotinamide adenine dinucleotide, and enzyme extract. After incubation (15 min, 35 °C), the absorbance of the reaction mixture was measured using a spectrophotometer (UV–2500, Shimadzu, Kyoto, Japan).

2.3.2. Activities of Antioxidant Enzymes and Content of MDA

Fresh beetroots (0.5 g) with 1 mL of 0.05 M phosphate buffer (pH 7.0) were homogenized in a pre-cooled mortar and centrifuged at 12,000× g for 20 min at 4 °C. Supernatant was used to determine the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and MDA content by a spectrophotometer.
SOD activity was measured according to the method of Giannopolitis and Ries [37]. The reaction mixture consisted of 1.5 mL of 0.05 M phosphate buffer (pH 7.8), 0.3 mL of 130 mM Met, 0.3 mL of 750 μM NBT, 0.3 mL of 100 μM EDTA–Na2, 0.3 mL of 20 μM FD, 0.3 mL of distilled water, and 20 μL of enzyme extract. After 30 min of illumination by white light at 4000 lx, the absorbance of the reaction mixture at 560 nm was recorded. One unit (U) of SOD activity was defined as the amount of enzyme required for 50% inhibition of the photochemical reduction of NBT.
POD activity assessment was made by monitoring the rate of guaiacol oxidation at 470 nm according to the method of Hernández et al. [38]. In total, 20 μL of enzyme extract were mixed with 3 mL of reaction solution, which consisted of 50 mL of 0.1 M phosphate buffer (pH 6.0), 28 μL of guaiacol, and 19 μL of 30% H2O2.
The activity of CAT was determined by monitoring the decomposition of H2O2 at 240 nm according to the method of Zhang and Kirkham [39]. The reaction mixture included 0.5 mL of 0.1 M H2O2, 2 mL of phosphate buffer (pH 7.0), and 50 μL of enzyme extract.
The MDA content was determined by the method of thethiobarbituric acid (TBA), as described by Dhindsa et al. [40]. In total, 1 mL of supernatant was mixed with 2 mL of 0.6% TBA and heated in boiling water for 15 min. Then, the reaction mixture was cooled and centrifuged at 3000× g for 10 min. The absorbance of the supernatant was measured at 450, 520, and 600 nm.

2.3.3. Root Activity

Determination of root activity was done according to the reduction of TTC (triphenyl tetrazolium chloride) at 485 nm, as described by Brouwer [41]. First, 0.5-g root tip samples were submerged in a mixed solution of 0.1 M phosphate buffer (pH 7.0) and 0.4% TTC in the dark at 37 °C for 3 h, and after that the reaction was terminated by adding 1 M H2SO4. At the same time, a blank contrast was set, where H2SO4 was added firstly, then root tip samples were submerged; the other operations were the same as above. Then, the root tip samples were homogenized in ethyl acetate and centrifuged at 15,000× g for 10 min. The absorbance of the extract was recorded at 485 nm.

2.3.4. Photosynthetic Pigments

The contents of photosynthetic pigments were determined according to the method of Amon [42]. Photosynthetic pigments were extracted from fresh leaf tissue with 80% acetone. The extract was centrifuged at 5000× g for 3 min. The optical density of the supernatant was measured by spectrophotometry at 470, 645, and 663 nm. The contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were determined by adjusting the extinction coefficient.

2.3.5. Chlorophyll Fluorescence Parameters

Chlorophyll fluorescence measurements were performed on a top fully expanded leaf using a portable fluorescence spectrometer system (PAM–2500, WALZ, Effeltrich, Germany). After dark adaptation for 30 min, leaves were illuminated by red light. Measurement light and saturation pulse light were set as 0.1 μmol of photon m−2 s−1 and 4000 μmol of photon m−2 s−1, respectively. Fv/Fm, qp, ETR, ΦPSII, and Fv/Fo were automatically acquired by the instrument.

2.3.6. Gas-Exchange Parameters

The net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration were measured using a portable photosynthetic system (GFS–3000, WALZ, Effeltrich, Germany) from 9:00 to 11:00 a.m. Air temperature, photosynthetically active radiation, CO2 concentration, and relative humidity (RH) inside the leaf chamber were set to 25 °C, 800 μmol m−2 s−1, 400 μmol mol−1, and 80%, respectively. When the RH and atmospheric CO2 concentration reached a stable value, the top fully expanded leaf was measured.

2.3.7. Dry Matter Accumulation and Root/Shoot Ratio

In each treatment, four sugar beets were divided into roots and overground tissues, then oven-dried at 80 °C until a constant weight was measured to determine the dry matter accumulation and root/shoot ratio.

2.3.8. Yield and Sugar Content

Ten sugar beets were randomly selected in each plot on 28 September in 2017 and 2018. Roots were washed under running water and weighed to determine yield (g plant−1). Sugar content (%) was measured by a refractometer (RM–40, Meitler Toledo, Zurich, Switzerland).

2.4. Statistical Analysis

Data preparation was performed using Microsoft Excel 2003. Analysis of variance (ANOVA) and Duncan’s multiple range test were performed using SPSS 21.0 software (IBM Inc., Chicago, IL, USA) to compare the means results from each treatment at the p < 0.05 significance level. All figures were drawn using SigmaPlot 14.0 (Systat Software lnc., San Jose, CA, USA). Since the results in 2018 showed a similar pattern in 2017, we only analyzed the results of 2017, except for the yield and sugar content of root.

3. Results

3.1. Activities of Nitrogen Assimilation Enzymes in Root

Nitrogen assimilation enzymes’ (NR, NiR, GS, and GOGAT) activities of roots firstly increased but then decreased with the progression of sugar beet growth and development (Figure 1A–D). Saline-alkaline stress caused decreases in the activities of NR, NiR, GS, and GOGAT. However, the biochar-based organic fertilizer treatment under saline-alkaline conditions maintained higher NR, NiR, GS, and GOGAT activities compared to the SA treatment at the five stages. In addition, the extent of the increase was greater at 50 DAS than that during other stages. Compared with SA, SA + B increased the NR activity of roots by 111.1% at 50 DAS, by 47.7% at 70 DAS, by 16.0% at 90 DAS, by 35.0% at 110 DAS, and by 42.9% at 130 DAS. SA + B enhanced the Nir activity of roots: 250.0%, 64.3%, 20.0%, 46.7%, and 66.7% for the above five stages of sugar beet. The GS activity of roots in SA + B was 41.7%, 16.1%, 11.6%, 24.1%, and 33.2% higher than that of SA at 50, 70, 90, 110, and 130 DAS, respectively. Similar improvements were observed in the GOGAT activity of roots.

3.2. Activities of Antioxidant Enzymes and Content of MDA in Root

The SOD, CAT, and POD activities of roots firstly increased and then decreased with the progression of sugar beet growth and development (Figure 2A–C). The SOD, CAT, and POD activities of roots were reduced by saline-alkaline stress in all periods. However, the decline in SOD, CAT, and POD activities of roots was lower in the SA + B treatment as compared to the SA treatment. From 50 to 130 DAS, these values decreased by 26.5–70.4%, 28.3–46.7%, and 23.9–45.2%, respectively, in the SA treatment, whereas they decreased by 2.2–20.7%, 2.1–34.1%, and 9.4–13.8%, respectively, in the SA + B treatment.
The MDA content in roots firstly increased but then decreased with the progression of sugar beet growth and development (Figure 2D). The MDA content in roots was increased by saline-alkaline stress at each DAS. However, the application of biochar-based organic fertilizer effectively reduced the increase of the MDA content in saline-alkaline-stressed sugar beet root at the late growth stage. The MDA content of the SA + B treatment was 27.2% and 26.0% lower than that of the SA treatment at 110 and 130 DAS, respectively.

3.3. Root Activity

In this study, the root activity of sugar beet gradually increased throughout the growth period (Figure 3). Saline-alkaline stress reduced root activity at each DAS. Compared with the CK treatment, SA and SA + B decreased the root activity by 66.7% and 18.5%, respectively, at 50 DAS; by 63.6% and 15.2%, respectively, at 70 DAS; by 50.0% and 11.1%, respectively, at 90 DAS; by 42.5% and 7.5%, respectively, at 110 DAS; and by 42.9% and 4.8%, respectively, at 130 DAS. There was a lack of significant differences in the root activity between the CK and SA + B treatments at each DAS.

3.4. Contents of Photosynthetic Pigments

Chlorophyll a, chlorophyll b, chlorophyll (a + b), and carotenoids contents firstly increased but then decreased with the progression of sugar beet growth and development (Figure 4A–D). The photosynthetic pigments contents were reduced by saline-alkaline stress in all periods. From 50 to 130 DAS, the above parameters in SA decreased by 19.4–27.9%, 24.0–34.6%, 21.8–28.1%, and 9.2–33.6%, in comparison with CK, respectively. The application of biochar-based organic fertilizer significantly increased these parameters under saline-alkaline stress, especially for the chlorophyll b content. Compared with SA, SA + B significantly increased the chlorophyll b content by 23.9% at 50 DAS, by 41.2% at 70 DAS, by 36.1% at 90 DAS, by 33.8% at 110 DAS, and by 27.2% at 130 DAS. At the same time, similar trends were observed in the chlorophyll a and chlorophyll (a + b) contents. The carotenoids content of SA + B was 3.4%, 40.0%, 10.5%, 21.7%, and 17.2% higher than that of the SA at 50, 70, 90, 110, and 130 DAS, respectively, but there were no significant differences between the treatments at 50 and 90 DAS.

3.5. Gas-Exchange Parameters

The gas-exchange parameters (photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration) firstly increased but then decreased during the growth stage (Figure 5A–D). Saline-alkaline stress negatively affected the gas-exchange parameters of sugar beet at all growth stages. Compared with the CK treatment, the SA treatment significantly decreased the photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration by 22.8%, 33.1%, 33.2%, and 23.8%, respectively, at 50 DAS; by 56.9%, 32.5%, 37.6%, and 42.1%, respectively, at 70 DAS; by 43.2%, 30.3%, 32.4%, and 18.7%, respectively, at 90 DAS; by 44.2%, 36.1%, 40.2%, and 16.5%, respectively, at 110 DAS; and by 41.1%, 40.2%, 66.4%, and 37.0%, respectively, at 130 DAS. The photosynthetic rate of SA + B was always higher than that of SA throughout the growth period, but there was no significant difference between the treatments at 50 DAS. Stomatal conductance in SA + B was 29.8%, 32.6%, 25.4%, 45.3%, and 55.0% higher than that of SA at 50, 70, 90, 110, and 130 DAS, respectively. Similar betterments were observed in the transpiration rate. The difference of the intercellular CO2 concentration between the SA and SA + B treatments exhibited the approximate trend of the transpiration rate throughout the growth period, except that there was no significant difference at 90 and 110 DAS.

3.6. Chlorophyll Fluorescence Parameters

The obtained results in Table 2 show that chlorophyll fluorescence parameters were significantly decreased in the saline-alkaline stressed sugar beets at all stages. The maximum decrease in fluorescence parameters was determined in the SA treatment at each DAS. Compared with the CK treatment, the SA treatment significantly decreased Fv/Fm, qp, ETR, ΦPSII, and Fv/Fo (five-DAS average) by 18.7%, 24.7%, 21.3%, 22.3%, and 49.3%, respectively. However, fluorescence parameters were significantly promoted by the application of biochar-based organic fertilizer in saline-alkaline conditions. From 50 to 130 DAS, the SA + B treatment significantly increased the above indicators by 11.3–33.2%, 14.0–49.7%, 12.7–34.1%, 5.7–30.0%, and 46.9–124.2%, respectively, compared with the SA treatment.

3.7. Dry Matter Accumulation and Root/Shoot Ratio

It is evident from Figure 6A,B that the dry matter accumulation and root/shoot ratio of sugar beer were significantly reduced by saline-alkaline stress at each DAS. Dry matter accumulation of sugar beet under the SA treatment was 18.5%, 46.5%, 28.6%, 10.6%, and 23.6% lower than that of the CK treatment at 50, 70, 90, 110, and 130 DAS, respectively. At the same time, the root/shoot ratio of sugar beet under the SA treatment was 26.3%, 44.8%, 53.1%, 50.9%, and 41.9% lower than that of the CK treatment at 50, 70, 90, 110, and 130 DAS, respectively. However, biochar-based organic fertilizer treatment could promote the dry matter accumulation and root/shoot ratio of sugar beet under saline-alkaline stress throughout the growth period. Compared with the SA treatment, the SA + B treatment significantly increased dry matter accumulation at 50, 70, 90, 110, and 130 DAS, by 21.7%, 42.1%, 29.6%, 9.8%, and 3.6%, respectively. Similar improvements were also observed in the root/shoot ratio at each DAS.

3.8. Yield and Sugar Content of Root

Figure 7A–D shows the reductions in sugar beet root yield and sugar content due to saline-alkaline stress. Compared with the CK treatment, the SA treatment reduced the sugar beet yield and sugar content by 47.2% and 7.8%, respectively, in 2017 and by 49.6% and 5.9%, respectively, in 2018. Biochar-based organic fertilizer treatment could positively affect these indicators under saline-alkaline conditions. Compared with the SA treatment, the SA + B treatment significantly increased the sugar beet yield and sugar content by 48.3% and 12.7%, respectively, in 2017 and by 49.8% and 11.2%, respectively, in 2018.

4. Discussion

It has been recognized that nitrogen is the most important nutritional factor that drives the growth and development of plants [43]. NR and NiR are considered to be key enzymes that promote the reduction of nitrate to ammonia in plants [44]. Ammonium is mounted on the carbon skeleton by the GS-GOGAT (glutamine synthetase and glutamate synthetase) pathway, which converts inorganic nitrogen to organic nitrogen [45]. These enzymes are inducible enzymes whose activity in roots can be increased by adequate inorganic nitrogen [46]. In this study, the activities of these enzymes in sugar beet roots were significantly reduced by saline-alkaline stress throughout the growth period (Figure 1), indicating that nitrogen assimilation of sugar beet roots was inhibited by saline-alkaline stress, as previously reported by Cordovilla et al. [47]. Under saline-alkaline conditions, the application of biochar-based organic fertilizer improved the activities of NR and NiR (Figure 1A,B), especially during the late growth stages (110–130 DAS). Moreover, compared to the SA treatment, the SA + B treatment showed higher GS and GOGAT activities at all stages (Figure 1C,D). A possible reason for this positive effect is that the decomposition of organic matter in biochar-based organic fertilizer maintains a relatively high level of soil inorganic nitrogen throughout the growth period [48], which might improve the absorption of inorganic nitrogen by sugar beet roots to avoid the suppressive nitrogen assimilation caused by saline-alkaline stress. Another potential reason may be that both biochar and animal manure could increase the abundance of nitrifying bacteria and ammonifying bacteria in the soil, thereby promoting the conversion of organic nitrogen to inorganic nitrogen, which improves the nitrogen assimilation of sugar beet roots under saline-alkaline conditions [49].
Similar to other abiotic stresses, saline-alkaline stress induces the generation of excessive ROS in plants, which inflicts oxidative damage on lipids, hence increasing the MDA content [4]. Under environmental stress, the enzyme defense system of plants provides a foundation for maintaining their growth, as the system is closely related to plants’ antioxidant capacity [50]. SOD, POD, and CAT are important components of the antioxidant enzyme system, which plays an important role in removing excessive ROS. In our experiment, the activities of SOD, POD, and CAT in the roots were significantly reduced by saline-alkaline stress at all stages (Figure 2A–C). Meanwhile, the content of MDA was increased in the roots (Figure 2D). The application of manure and biochar can effectively alleviate oxidative damage to lipids, improve the activities of antioxidant enzymes, and reduce the MDA content in roots under abiotic stresses [51]. This conclusion is confirmed by the results of the current study, since the SOD, POD, and CAT activities of SA + B were higher than those of SA, accompanied by a reduction in the MDA content at each stage. This result may be due to the positive effects of biochar-based organic fertilizer on the pH and cation exchange capacity of saline-alkaline soil [52], which could protect the sugar beet roots from saline-alkaline stress. Another reason may be that biochar could upregulate pathways and genes associated with plant defense, thereby reducing the negative effects of saline-alkaline stress on sugar beet roots [53]. Previous studies have shown that environmental stress inhibits the antioxidant enzyme activity and respiration rate of roots, leading to a decrease in root activity, which negatively affects nutrient absorption by the plant root and yield [54]. Similar results were observed in our study. However, there was no difference in root activity at each DAS between the CK and the SA + B treatments (Figure 3), suggesting that the application of biochar-based organic fertilizer greatly improves root activity under saline-alkaline conditions. Another reason for this result may be that biochar-based organic fertilizer could reduce the saline-alkaline soil bulk density, leading to improved porosity and aeration in saline-alkaline soil, which could restrain anaerobic root respiration and improve the root activity of sugar beet in saline-alkaline soil.
Photosynthetic pigments play an important role in photosynthesis. In the current study, the contents of chlorophyll a, chlorophyll b, chlorophyll a + b, and carotenoids were reduced by saline-alkaline stress during the growth of sugar beet (Figure 4A,D). A similar result was reported by Kreslavski et al., who suggested that saline-alkaline stress could reduce the activity of pigment synthase and damage the chloroplast structure to inhibit the formation of photosynthetic pigments [55]. In the present work, the photosynthetic pigment contents of sugar beet at each DAS were increased by applying biochar-based organic fertilizer to saline-alkaline soil. This finding agrees with a study by Karanatsidis et al., who reported that the application of organic fertilizer may reduce the activity of chlorophyll catabolic enzyme and Mg2+ precipitation caused by high pH to promote photosynthetic pigment synthesis [56]. The activities of PSII and PSI together determine the photoreaction process, which greatly affects photosynthesis [57]. The reduction in photosynthetic pigments content inhibit the light energy capture of plants, which reduces PSII activity [58]. The absorption and utilization of light energy can be reflected by the chlorophyll fluorescence parameters, which can contribute to the analysis of photosynthetic organ damage caused by stress [59]. Decreased chlorophyll fluorescence parameters indicate that PSII is damaged and inactivated by saline-alkaline stress [60]. Saline-alkaline stress significantly decreased Fv/Fm, qp, ETR, ΦPSII, and Fv/Fo of sugar beet throughout the growth process (Table 2). The above parameters of sugar beet in the SA + B treatment were significantly higher than those in the SA treatment at each DAS, which suggests that the application of biochar-based organic fertilizer may protect the PSII activity of sugar beet from saline-alkaline soil. This result may also prove that the photosynthetic pigment contents of sugar beet could be increased by applying biochar-based organic fertilizer to saline-alkaline soil.
In addition to the contents of photosynthetic pigments and PSII activity, stomatal conductance is also crucial for plants’ photosynthesis [61]. Saline-alkaline stress negatively affects the photosynthesis of plants, and stomatal conductance decreases with saline-alkaline stress increasing; this is a physiological response of plants to saline-alkaline stress [62]. Under saline-alkaline conditions, the exchanges of H2O and CO2 between the environment and plants are inhibited by closed stomata, thus reducing the transpiration rate and intercellular CO2 concentration and eventually resulting in a significant decrease in the photosynthetic rate [63]. This effect can explain why the gas-exchange parameters of CK were higher than those of SA during the whole growth period (Figure 5). SA + B showed higher gas-exchange parameters than SA at each DAS, indicating that the application of biochar-based organic fertilizer promotes the opening of stomata to effectively increase the exchanges of H2O and CO2 between the environment and sugar beet, thus alleviating the damage to photosynthesis in sugar beet caused by saline-alkaline stress.
Previous studies have demonstrated that the application of biochar or manure improves the nutrient absorption and photosynthesis of plants under environmental stress, leading to an improved root/shoot ratio and plant growth, which enhances plant yield [4,11]. Similar results were observed in the current study. Compared with SA, SA + B caused greater improvements in the dry matter accumulation and root/shoot ratio of sugar beet at each DAS (Figure 6), which laid a solid foundation for SA + B to obtain a higher root yield (Figure 7A). After 110 DAS, the growth of sugar beet was basically terminated by saline-alkaline stress. This result is consistent with a previous study by An et al. [4]. They found that saline-alkaline stress caused premature plant senescence and inhibited the transport of photoassimilated carbon to the root. However, the root/shoot ratio of the SA + B treatment was higher than that of the SA treatment during late growth (130 DAS), suggesting that although the application of biochar-based organic fertilizers could not prevent the premature senescence in sugar beet caused by saline-alkaline stress, it may effectively promote the transport of photoassimilated carbon to the root at the late growth stage under saline-alkaline conditions. Another potential reason may be that more photoassimilated carbon was necessary to maintain higher root activity in SA + B, which might stimulate the transport of photoassimilated carbon to the root. This may explain why SA + B showed the highest sugar content (Figure 7B).
The inadequacy of this study was that it did not evaluate the effect of biochar-based organic fertilizer on improving saline-alkaline soil. Biochar-based organic fertilizer, as an environmentally friendly product, needs more attention in agricultural production in the future and its improvement effect of physical properties, such as bulk density, porosity, and agglomerate structure, and chemical properties, such as pH, salt content, cation exchange capacity, nutrient status, organic matter content, enzyme activity, abundance of bacteria, and microbial diversity, of saline-alkaline soil should be explored under field conditions, which will helpfully reveal t mechanism that alleviates the adverse effect of saline-alkaline stress on plants.

5. Conclusions

Under saline-alkaline conditions, the activities of nitrogen assimilation enzymes in the root were improved by applying biochar-based organic fertilizer, which provided a foundation for the normal growth of sugar beet. At the same time, the application of biochar-based organic fertilizer could significantly enhance antioxidant enzymes’ activity to improve root activity, thus alleviating the adverse effect of saline-alkaline stress on root growth. Moreover, the application of biochar-based organic fertilizer could promote the synthesis of photosynthetic pigments, ensure PSII activity, encourage stomatal opening, and improve photosynthesis. Actually, these positive effects were the key mechanisms by which the application of biochar-based organic fertilizer significantly improved the yield and sugar content of sugar beet under saline-alkaline conditions. The current study was a pot experiment that did not reflect the conditions that would be encountered when sugar beet is planted in actual fields. The results obtained in the pot experiment may not be viable in field conditions. Therefore, in the future, we will verify whether biochar-based organic fertilizer could alleviate the adverse effects of saline-alkaline stress on sugar beets yield under field conditions.

Author Contributions

Conceptualization, C.L.; Data curation, P.Z.; Formal analysis, P.Z. and F.Y.; Funding acquisition, C.L.; Investigation, F.Y.; Methodology, Y.W. and C.L.; Project administration, C.L.; Resources, C.L.; Supervision, Y.W. and C.L.; Validation, X.L., J.C., and X.W.; Writing—original draft, P.Z.; Writing—review & editing, P.Z., H.Z., L.L., and C.L. All authors have read and agreed to the published version of the manuscript.

Funding

We are grateful for the support from the grants from the National Natural Science Foundation of China (31671622) and China Agriculture Research System (CARS-170201).

Acknowledgments

We are grateful for the high efficiency and valuable comments by the editor and reviewers that improved this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The activities of nitrogen assimilation enzymes of sugar beet root at different DAS (days after sowing) in 2018. (A) Nitrate reductase (NR), (B) nitrite reductase (NiR), (C) glutamine synthetase (GS), and (D) glutamate synthase (GOGAT). CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
Figure 1. The activities of nitrogen assimilation enzymes of sugar beet root at different DAS (days after sowing) in 2018. (A) Nitrate reductase (NR), (B) nitrite reductase (NiR), (C) glutamine synthetase (GS), and (D) glutamate synthase (GOGAT). CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
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Figure 2. The activities of antioxidant enzymes and MDA (malondialdehyde) content of sugar beet root at different DAS in 2018. (A) Superoxide dismutase (SOD), (B) catalase (CAT), (C) peroxidase (POD), and (D) malondialdehyde (MDA) content. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
Figure 2. The activities of antioxidant enzymes and MDA (malondialdehyde) content of sugar beet root at different DAS in 2018. (A) Superoxide dismutase (SOD), (B) catalase (CAT), (C) peroxidase (POD), and (D) malondialdehyde (MDA) content. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
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Figure 3. Root activities of sugar beet root at different DAS in 2018. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
Figure 3. Root activities of sugar beet root at different DAS in 2018. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
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Figure 4. The contents of photosynthetic pigments of sugar beet at different DAS in 2018. (A) chlorophyll a, (B) chlorophyll b, (C) total chlorophyll, and (D) carotenoids. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
Figure 4. The contents of photosynthetic pigments of sugar beet at different DAS in 2018. (A) chlorophyll a, (B) chlorophyll b, (C) total chlorophyll, and (D) carotenoids. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
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Figure 5. The gas–exchange parameters of sugar beet at different DAS in 2018. (A) Net photosynthetic rate, (B) stomatal conductance, (C) transpiration rate, and (D) and intercellular CO2 concentration. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
Figure 5. The gas–exchange parameters of sugar beet at different DAS in 2018. (A) Net photosynthetic rate, (B) stomatal conductance, (C) transpiration rate, and (D) and intercellular CO2 concentration. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
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Figure 6. The dry matter accumulation and root/shoot ratio of sugar beet at different DAS in 2018. (A) dry matter accumulation and (B) root/shoot ratio. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
Figure 6. The dry matter accumulation and root/shoot ratio of sugar beet at different DAS in 2018. (A) dry matter accumulation and (B) root/shoot ratio. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level at each DAS. Error bars represent standard deviations of the means (n = 4).
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Figure 7. The root yield and sugar content of sugar beet in 2017 and 2018. (A,C) Root yield and (B,D) sugar content in 2017 and 2018. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level.
Figure 7. The root yield and sugar content of sugar beet in 2017 and 2018. (A,C) Root yield and (B,D) sugar content in 2017 and 2018. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. Different letters indicate significant differences between treatments at the p < 0.05 level.
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Table
Table 1. Basic chemical properties of the experimental soil.
Table 1. Basic chemical properties of the experimental soil.
pHElectrical Conductivity EC1:5 (mS cm−1)Available Nitrogen (mg kg−1)Available Phosphorus (mg kg−1)Available Potassium (mg kg−1)Organic Matter (g kg−1)
Neutral soil7.370.26123.12117.26171.5837.33
Saline-alkaline soil9.081.12114.3162.95127.4118.75
Table
Table 2. The chlorophyll fluorescence parameters of sugar beet at different DAS in 2018.
Table 2. The chlorophyll fluorescence parameters of sugar beet at different DAS in 2018.
TreatmentsDays after Sowing (d)
507090110130
CK0.747 ± 0.019 a0.775 ± 0.022 a0.765 ± 0.033 a0.793 ± 0.017 a0.782 ± 0.002 a
Fv/FmSA0.574 ± 0.022 b0.552 ± 0.024 b0.656 ± 0.038 b0.674 ± 0.012 c0.679 ± 0.004 c
SA + B0.713 ± 0.002 a0.735 ± 0.015 a0.751 ± 0.029 a0.762 ± 0.004 b0.756 ± 0.002 b
CK0.855 ± 0.007 a0.821 ± 0.004 a0.846 ± 0.005 a0.824 ± 0.009 a0.783 ± 0.006 a
qpSA0.663 ± 0.089 b0.579 ± 0.084 b0.535 ± 0.022 c0.693 ± 0.029 b0.634 ± 0.023 c
SA + B0.817 ± 0.003 a0.727 ± 0.039 a0.801 ± 0.005 a0.797 ± 0.026 a0.748 ± 0.011 b
CK55.5 ± 0.8 a56.4 ± 0.4 a54.0 ± 0.4 a52.7 ± 1.3 a48.1 ± 0.3 a
ETRSA45.3 ± 0.5 b48.7 ± 1.1 c42.2 ± 3.9 b41.4 ± 0.9 c33.1 ± 0.7 c
SA + B54.2 ± 0.6 a54.9 ± 0.5 b51.9 ± 0.5 a50.2 ± 0.8 b44.4 ± 1.4 b
CK0.649 ± 0.006 a0.618 ± 0.047 a0.641 ± 0.005 a0.592 ± 0.007 a0.545 ± 0.007 a
ΦPSIISA0.478 ± 0.021 c0.429 ± 0.057 b0.464 ± 0.021 c0.566 ± 0.008 b0.427 ± 0.029 b
SA + B0.582 ± 0.009 b0.539 ± 0.016 a0.603 ± 0.011 b0.592 ± 0.007 a0.513 ± 0.007 a
CK2.86 ± 0.29 a3.48 ± 0.45 a3.31 ± 0.66 a3.84 ± 0.22 a3.61 ± 0.04 a
Fv/FoSA1.35 ± 0.13 c1.24 ± 0.12 c1.93 ± 0.32 b2.07 ± 0.12 c2.11 ± 0.04 c
SA + B2.45 ± 0.03 b2.78 ± 0.22 b3.06 ± 0.52 a3.21 ± 0.06 b3.10 ± 0.04 b
Fv/Fm, maximum quantum yield of PSII; qp, photochemical quenching; ETR, electron transport rate; ΦPSII, PSII actual photochemical efficiency; Fv/Fo, PSII potential photochemical activity. CK, chemical fertilizers were applied to neutral soil; SA, chemical fertilizers were applied to saline-alkaline soil; SA + B, biochar-based organic fertilizer was applied to saline-alkaline soil. All data are means ± SE of four repetitions (n = 4). Different letters within the same column represent significant differences (p < 0.05).

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MDPI and ACS Style

Zhang, P.; Yang, F.; Zhang, H.; Liu, L.; Liu, X.; Chen, J.; Wang, X.; Wang, Y.; Li, C. Beneficial Effects of Biochar-Based Organic Fertilizer on Nitrogen Assimilation, Antioxidant Capacities, and Photosynthesis of Sugar Beet (Beta vulgaris L.) under Saline-Alkaline Stress. Agronomy 2020, 10, 1562. https://doi.org/10.3390/agronomy10101562

AMA Style

Zhang P, Yang F, Zhang H, Liu L, Liu X, Chen J, Wang X, Wang Y, Li C. Beneficial Effects of Biochar-Based Organic Fertilizer on Nitrogen Assimilation, Antioxidant Capacities, and Photosynthesis of Sugar Beet (Beta vulgaris L.) under Saline-Alkaline Stress. Agronomy. 2020; 10(10):1562. https://doi.org/10.3390/agronomy10101562

Chicago/Turabian Style

Zhang, Pengfei, Fangfang Yang, He Zhang, Lei Liu, Xinyu Liu, Jingting Chen, Xin Wang, Yubo Wang, and Caifeng Li. 2020. "Beneficial Effects of Biochar-Based Organic Fertilizer on Nitrogen Assimilation, Antioxidant Capacities, and Photosynthesis of Sugar Beet (Beta vulgaris L.) under Saline-Alkaline Stress" Agronomy 10, no. 10: 1562. https://doi.org/10.3390/agronomy10101562

APA Style

Zhang, P., Yang, F., Zhang, H., Liu, L., Liu, X., Chen, J., Wang, X., Wang, Y., & Li, C. (2020). Beneficial Effects of Biochar-Based Organic Fertilizer on Nitrogen Assimilation, Antioxidant Capacities, and Photosynthesis of Sugar Beet (Beta vulgaris L.) under Saline-Alkaline Stress. Agronomy, 10(10), 1562. https://doi.org/10.3390/agronomy10101562

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