1. Introduction
Salinity is a serious problem in global agriculture, affecting nearly 1 billion hectares of land, which is approximately 20%, almost half of the irrigated arable land worldwide [
1]. The low quantity and quality of water available for irrigation makes it necessary to use groundwater and unconventional sources with high concentration of salts. As such, a progressive salinization of the soil develops, especially in arid areas with irrigated crops [
2]. The excessive use of fertilizers and the reduced leaching capacity of certain soils have also contributed to this problem [
3]. The salinity of irrigation water and soil hinders the growth of most crops due to the inhibition of water uptake through the osmotic effects caused by the increased salt concentration in the root zone. It also leads to an excessive uptake of Na
+ and Cl
−, resulting in specific ionic toxicity [
4]. Furthermore, the high concentrations of these ions, Na
+ and Cl
−, in the root zone, disrupt the uptake of cations such as K
+ and Ca
2+, as well as anions such as NO
3− and PO
43− [
5]. The high concentrations of Na
+ and Cl
− also affect other processes, including water relations, light capture, CO
2 assimilation and antioxidant capacity, among others, ultimately resulting in reduced growth, biomass, and crop yield [
6,
7,
8,
9,
10,
11].
The use of biostimulants could prove to be an effective tool in reducing the toxic effect of salinity in plants, partly due to a reduction in the uptake and accumulation of Na
+ and Cl
− ions [
12]. Thus, the application of chitosan-based salicylic acid nanocomposite in a vineyard (
Vitis vinifera cv ‘Sultana’) [
13], the foliar application of 24-epibrassinolide to pepper (
Capsicum annuum L.) [
14], the treatment of basil (
Ocimun basilicum L.) with a hydrolyzed animal protein-based biostimulant [
15], the application of a graminaceae-derived protein hydrolysate and its fractions to lettuce (
Lactuca sativa L.) [
16], the addition of a protein hydrolysate of plant origin to spinach (
Spinacia oleracea L.) [
17], the application of the hydroalcoholic extracts of brown algae (
Sargassum spp.) to tomato (
Solanum lycopersicum L.) [
18], and the addition of
Ulva intestinalis (L.) extract to bean (
Phaseolus vulgaris L.) [
19] have been found to improve production and quality under saline conditions. Corn steep liquor (CSL) is a byproduct obtained from the cleaning and maceration of corn during wet milling. Although its use requires appropriate treatment to avoid environmental issues [
20], it holds great promise in terms of the circular economy and sustainability [
21]. It contains high amounts of proteins, amino acids, minerals, vitamins, reducing sugars, organic acids, enzymes, and other substances that promote plant growth. Its application to soil improves the utilization of macronutrients by promoting the growth of bacteria that contribute to nitrogen (N) fixation and phosphorus (P) solubilization [
22]. In lettuce hydroponics, the use of CSL favored microbial development that protected the root system [
23,
24]. In soybean (
Glycine max) crops, treatment with 1% CSL favored germination, growth and precocity due to an increase in the uptake and transport of nutrients [
25].
To meet the expectations generated by the biostimulant sector, it is necessary to establish the composition and mechanism of action of each product. In a previous study on the role of two CSL products, with different stabilization methods, in pepper (
Capsicum annuum L.) plants, a better response was found with the so-called CSL-B, which undergoes filtration to remove suspended solids and stabilization [
26]. Its composition (%, g g
−1 fresh product) includes free amino acids (5.0–6.0), total organic matter (40), total humic extract (30), fulvic acids (30), total N (3.0), ammoniacal N (0.3), organic N (2.7), potassium, K
2O, (2.5) and P, P
2O
5, (3.0). Its mode of action is related to regulation, hormone synthesis, and the stimulation of C and N metabolism [
26]. Despite being a widely studied product with many beneficial applications, it is unknown whether the application of CSL induces adaptive responses in pepper plants grown under challenging conditions such as salt stress. This study examines the mechanisms of action of this type of CSL in this crop under saline conditions and the effect of the application method, either root or foliar.
4. Discussion
The corn steep liquor (CSL) treatment, especially via root application, significantly improved the growth of pepper (
Capsicum annuum L.) plants treated with 100 mM NaCl under the prevailing experimental conditions. The application of CSL to the roots of bean (
Phaseolus vulgaris L.) plants was found to enhance plant growth [
25]. The application of biostimulants with different proportions of humic acids and fulvic acids in tomato (
Solanum lycopersicum L.) under saline stress conditions improves growth, increasing fresh and dry matter [
37]. The application of a biostimulant amino acid to a salt-resistant variety of basil (
Ocimun basilicum L.) improved production under saline conditions [
15]. Treatment with a hydrolyzed protein of plant origin enhanced the lettuce (
Lactuca sativa L.) and spinach (
Spinacia oleracea L.) production under moderate salinity, although its effect varied depending on its molecular fraction [
16,
17].
Under environmental stress, the significant inhibition of photosynthesis is commonly observed [
6,
7,
9,
10,
11]. However, in some plant species, the application of biostimulants can reverse this inhibition and restore normal plant growth [
12,
13,
38,
39]. In certain cases, these treatments with biostimulants can prevent the complete closure of stomata under stress conditions, thereby promoting the maintenance of photosynthetic activity in plants [
38]. The results obtained here verify that the CSL product, particularly via root application, would act in this manner, as its use increased A, E, and WUE, while reducing stomatal resistance (r) compared to plants under salinity stress (
Table 2). This effect can also be observed, although to a lesser extent and with less significance, in stressed plants receiving the foliar application of CSL (
Table 2). Similar findings were reported for tomato seedlings treated with a hydroalcoholic extract of
Sargassum spp. under saline conditions [
18].
When there is metabolic disturbance, the plants produce fluorescence to dissipate excess energy and prevent damage [
27]. The photosynthetic efficiency can be deduced from the value of the quantum yield of primary photosynthesis (Fv/Fm). In healthy plants or those not subjected to intense stress, the Fv/Fm value is typically around 0.85 [
27]. In the experimental conditions used here, the control plants had an Fv/Fm value of 0.849. The salinity treatment caused a reduction in the Fv/Fm values, indicating increased chlorophyll a fluorescence and thus high salt stress [
27]. The analysis of chlorophyll a fluorescence provides several indices that define plant vitality. The RC/ABS ratio is an essential parameter to evaluate the electron transport chain in photosystems. High values of this indicator correspond to a higher proportion of active reaction centers [
27]. The PI
ABS index represents the photosynthetic performance functionality of the photosystems. The 1-Vj value denotes electron leakage, particularly from photosystem II [
27]. Plants treated with CSL under saline conditions show a mitigated decrease in Fv/Fm, with values above 0.8, suggesting improved adaptation (
Table 3). This is due to the enhanced protection and activation of the photochemical process under stress conditions. Intracellular CO
2 availability would have also increased due to reduced stomatal closure, leading to the greater availability of the endogenous electron acceptor, NADP, and thereby reducing the electron transfer to oxygen and, consequently, ROS formation. Additionally, the net photosynthesis rate increased, which contributed to increased biomass production under these stress conditions (
Table 1). The rest of the photochemical activity and vitality indices of the plants suggest that, under saline stress conditions, the application of the CSL product, both foliar and via the roots, improved the coupling of the different components of the photochemical stage, hence improving the efficiency of the conversion of light energy into chemical energy, therefore increasing plant vitality. Thus, the RC/ABS and PI
ABS indices had their highest values in the control plants and those treated with CSL under saline conditions (
Table 3). These results also indicate that the electron loss in the photochemical phase of photosynthesis was reduced by CSL application to plants suffering salt stress, decreasing the formation of reactive oxygen species (ROS) [
27].
Saline stress also results in the accumulation of ROS, such as H
2O
2 and O
2−, which drastically disrupt metabolic homeostasis and affect the integrity of the cell membrane [
6,
8,
10,
18]. The reduction in ROS accumulation is crucial for plant survival under saline stress conditions; thus, oxidative metabolism has long been used as an indicator of the resulting damage [
6,
7,
8,
9,
10,
11]. Plants possess ROS detoxification mechanisms to prevent damage, which can be categorized into enzymatic systems and non-enzymatic systems consisting of antioxidant compounds (phenols, glutathione, flavonoids, ascorbic acid, anthocyanins, etc.) The increase in antioxidant enzymes and compounds under abiotic stress, such as water and salinity stress, depends on the species, cultivar, plant development stage, and metabolic state, as well as on the intensity and length of the stress [
7,
9,
11]. In the face of the abiotic stresses, biostimulants have been shown to reduce cellular oxidative damage, including the peroxidation of cell membrane lipids, in many plant species. This is achieved through the regulation of antioxidant defenses and the decrease in ROS levels in plants [
12,
38,
39]. In this regard, the concentration of MDA serves as an indicator of lipid membrane peroxidation, and an increase in its values suggests the excessive presence of ROS [
7,
9,
11]. The control plants exhibited the highest values of aerial biomass and leaf area, as well as the lowest levels of leaf MDA (
Table 1 and
Table 4). The plants treated with CSL under saline conditions had slightly higher MDA values, while the highest values were found in the plants experiencing saline stress without CSL application. These findings are in agreement with the foliar concentrations of H
2O
2 and O
2− (
Table 4). When different proportions of humic acids and fulvic acids were applied to tomato plants suffering saline stress, it was found that a reduction in the MDA content improved the response to salinity [
37].
One of the mechanisms by which biostimulants improve resistance to abiotic stresses is the induction of the plant’s antioxidant defenses, both enzymatic and non-enzymatic [
12,
38,
39]. In this study, the salt treatment induced a significant rise in the abundance of the analyzed antioxidant compounds (phenols, ascorbate, glutathione), especially for plants that received the CSL product via foliar application (
Table 5). These results can be interpreted as an attempt to mitigate the oxidative damage caused by adverse growth conditions in pepper plants [
7,
9,
11].
Compounds such as proline are often good indicators of resistance to saline stress, as they frequently play a role as osmoprotectants, osmoregulators, and antioxidants that protect against the generation of ROS [
7]. The degradation of proline by the enzyme proline dehydrogenase involves oxygen (O
2) consumption, which reduces the likelihood of ROS generation. This may have happened when CSL was applied to salt-stressed plants, as the levels of ROS were lower than when CSL was not applied (
Table 4). Therefore, CSL could act at the root level, enhancing ion selectivity by regulating the uptake of Na
+ and Cl
− and/or their accumulation in root tissues. As such, the translocation of these ions to the aerial part would be reduced, leading to an increased accumulation of K
+ (
Table 6). These results suggest that, under the conditions of this experiment, proline was an indicator of plant stress, rather than inducing resistance to saline stress. In a study conducted with tomato and commercial formulations of biostimulants containing different proportions of humic and fulvic acids, an increase in the proline content was observed under salinity [
37]. Similar results were obtained for wheat (
Triticum aestivum L.) seedlings treated with the extracts of the brown alga
Macrocystis pyrifera [
40] and in tomato seedlings treated with a hydroalcoholic extract of Sargassum spp. under saline conditions [
18].
According to some authors, the additional supply of organic constituents (e.g., amino acids) and/or hormones (e.g., cytokinins) may enhance ionic selectivity in roots, leading to a reduction in the ionic toxicity of NaCl in the aerial part of plants [
12]. The reduction in the foliar concentrations of Na
+ and Cl
− ions (due to CSL application to leaves) and the increase in the foliar concentration of K
+ (when applied to roots), along with the activation of other resistance processes, would explain the enhanced growth of plants receiving CSL under saline stress. The formulations of microalgae and cyanobacteria extracts promoted salinity tolerance in tomato by enhancing the enzymatic antioxidant activity, root growth, and nutrient uptake [
41]. The application of a plant-based protein to lettuce reduced the ionic toxicity under moderate salinity when its molecular fraction was intermediate (between 1 and 10 kDa) [
16].