Effect of crude extract and polysaccharides derived from Fucus spiralis on radish plants Raphanus sativus L. agrophysiological traits under drought stress

Abstract

Drought is a significant environmental stressor that induces changes in the physiological, morphological, biochemical, and molecular traits of plants, ultimately resulting in reduced plant growth and crop productivity. Seaweed extracts are thought to be effective in mitigating the effects of drought stress on plants. In this study, we investigated the impact of crude extract (CE), and polysaccharides (PS) derived from the brown macroalgae Fucus spiralis (Heterokontophyta, Phaeophyceae) applied at 5% (v/v) and 0.1% (w/v) respectively on radish plants Raphanus sativus L. subjected to varying levels of drought stress, specifically 80% of field capacity (FC) for no stress, 60% FC for moderate stress, and 40% FC for severe stress. Our examination of growth parameters, along with physiological and biochemical characteristics, revealed that both CE and PS increased the fresh weight over the control by 47.43% and 64% at 40% FC and 12.5% and 38% at 60% FC respectively. Under stress (40% FC), the application of CE and PS decreased proline content of radish leaves by 23.45% and 6.46% respectively in comparison with the control. Furthermore, PS treatment caused an increase of the alkaline phosphatase and urease activity in the soil by 182.5% and 34.6% respectively. CE and PS treatments led to decreased sugar content and total phenolics levels. Notably, lipid peroxidation was reduced in stressed plants treated with both CE and PS, with PS treatment yielding lower concentrations (3.75 nmol MDA.g^− 1 FW at 40% FC). Overall, F. spiralis extracts interacted through several mechanisms using various compounds to mitigate the negative effects of drought stress on radish plants. These results demonstrate that seaweed extracts could be adopted in integrated production systems to boost food productivity under harsh climatic conditions.

Introduction

In a world where climate change is posing serious challenges, the agriculture sector has been severely affected and is under increasing pressure to develop new strategies that guarantee food security [1]. One of agriculture’s most pressing challenges today is the impact of drought on crop productivity. Drought is one of the main abiotic stresses that affect most cropped plants worldwide [2, 3]. From 2008 to 2018, drought accounted for approximately 34% of crop and livestock production losses in low- and middle-income as well as least developed countries, resulting in an estimated cost of USD 37 billion to the agricultural sector [4].

To deal with water deficits, plants have developed acclimation and adaptation mechanisms. Understanding these mechanisms by which plants respond to water stress is a challenge to enhancing crop drought tolerance [5]. Water stress tolerance is seen in almost all plant species, but its difference varies from one species to another. Reactive oxygen species (ROS) accumulation in plant cells is one of the physiological and biological changes caused by drought, which also causes oxidative stress in plants. Reactive oxygen radicals are detrimental and can damage plant metabolism in many ways. Lipid peroxidation and malondialdehyde (MDA) accumulation by ROS damages photosynthetic components, inactivates proteins and enzymes, and destroys the structure and permeability of cell membranes [6]. To counteract the negative effects of drought stress plants have developed various mechanisms including morphological and structural modifications, the expression of genes related to drought resistance, as well as the synthesis of hormones and osmotic regulators, etc [7].

Various strategies have been developed to deal with the detrimental effects of drought on the growth and development of plants. These methods include seed priming, fertilizers, biochar, plant growth regulators, plant growth promoting microorganisms, and seaweed extracts [8,9,10]. Seaweed extracts are considered plant biostimulants, defined as “any substance or microorganism applied to plants with the aim of enhancing nutrition efficiency, abiotic stress tolerance, and/or crop quality traits, regardless of its nutrients content.” [11]. Seaweed extracts have gained wide acceptance nowadays as plant biostimulants in agricultural production [12]. It can increase crop resistance to stress, enhance crop quality, and promote crop growth. It mostly consists of natural hormones such as cytokinin, auxin, betaines, polysaccharides, and phenolic compounds [2, 3]. The biochemical composition of seaweed extracts is complex and because of the numerous interactions between the significant number of bioactive compounds contained in a single extract, comprehending their mechanism of action is therefore extremely complex and frequently requires a multidisciplinary approach [11]. Additionally, it has been demonstrated that seaweed extracts can mitigate several abiotic stresses, including drought [13]. Plant antioxidant activity can be increased by applying seaweed extracts via foliar application under drought stress [14]. Additionally, Seaweed polysaccharides have been proven to enhance plant growth and productivity [15]. Seaweed poly- and oligosaccharides can improve plant vigor, increase the uptake of soil nutrients, and protect plants against several abiotic and biotic stresses, by stimulating the production of plant’s secondary metabolites and managing its defense pathways [16].

Radish (Raphanus sativus L.) is an annual vegetable that belongs to the family of Brassicaceae (Cruciferae), order Capparidales, genus Raphanus, and species sativus [17]. Radish holds significant importance and is commercially cultivated in countries such as China, Japan, the USA, Korea, across Europe, Pakistan, India, Yemen, and throughout Southeast Asia. It is widely consumed globally for its roots, which include the hypocotyls and primary root [17]. However, radish growth and productivity could be impacted negatively by water limitation [18]. It was demonstrated that Drought impacts radish photosynthetic capacity by disrupting water balance and compromising membrane integrity, which leads to reduced biomass accumulation, particularly in globular roots [19].

Fucus spiralis is a brown macroalga that belongs to the family Fucaceae occupying the littoral zone of the Atlantic coast of Europe and North America, with the genus Fucus frequently exhibiting high abundance in rocky intertidal, temperate ecosystems [20]. F. spiralis is highly adapted to tidal zones due to its water-absorbing polysaccharides and efficient photosynthetic rates in aerial conditions during low tide [21]. F.spiralis has garnered attention for its bioactive compounds and potential applications. It contains an abundance of bioactive components, including phlorotannins, fucoxanthin, different vitamins, phenolic compounds, lipids, and polysaccharides [2]. The prospective applications of algal biomass include its role in biofuel production and the use of its by-products in cosmetics, pharmaceuticals, wastewater treatment, and agriculture [22]. Many authors were interested in evaluating the effect of F.spiralis extracts and polysaccharides on plant growth and development. The application of F.spiralis in combination with Ulva rigida and PGPR were able to enhance Faba bean fresh weight, proteins, carbohydrates and chlorophyll content [23]. Moreover, the application of polysaccharides extracted from F.spiralis enhanced tomato growth, productivity and quality [15].

This study aimed to elucidate the role of seaweed crude extracts and polysaccharides derived from the macroalgae F.spiralis in mitigating drought stress in (Raphanus sativus L.) radish plants. Given the limited understanding of interactions between polysaccharides and other compounds within seaweed extracts in the context of abiotic stress tolerance, we sought to investigate how these bioactive compounds influence plant responses under water-deficient conditions. By assessing a range of morphological, physiological, and biochemical parameters, this research provides insight into the mechanisms through which seaweed extracts may enhance plant resilience to drought, contributing valuable knowledge to sustainable agriculture practices in stress-prone environments.

Materials and methods

Seaweed extracts preparation

Crude extract

F. spiralis brown algae sample was collected in February 2023 from El Jadida, Sidi Bouzid, Morocco (33.23° N, 8.55° W). Seaweeds were harvested, washed in seawater to remove epiphytes and pollutants, and kept in refrigerated boxes throughout transport. Algal samples were properly cleaned with tap water upon arrival at the laboratory, they were oven dried at 60 °C for 72 h, ground, and sieved to obtain particles less than 1 mm in diameter.

The extraction was done in an alkaline solution as described by El Boukhari et al. (2023) [10] with modifications, the pH was adjusted by adding 1 M KOH to distilled water until pH 10 was reached. Seaweed was added to the alkaline solution at a ratio of 1/10 g/ml (seaweed/water alkaline solution). The mixture of seaweed and alkaline water was subjected to an ultrasound pretreatment using an ultrasound processor (Sonics and materials, inc, VCX1500, USA) at (20% amplitude, frequency of 20 Khz, 1500 W) for 5 min. Then it was heated at 60 °C for 3 h with agitation in an induction plate (Heidolph instruments, MR Hei-Tec, Germany). Afterwards, the mixture was filtered using centrifugation for 10 min at 3000 rpm. The resultant extract was considered as 100% extract concentration (pH 6.34; EC 16.62 dS.m^− 1) and was applied at a dilution of 5% (v/v). This concentration was chosen based on some preliminary tests (data not shown).

Seaweed polysaccharides

Crude polysaccharides were extracted as described by Song et al. (2018) [24] with some modifications. Seaweeds were added to distilled water at a ratio of 1/10 g/ml (seaweed to water). This mixture was then heated at 90 °C for 3 h. After the extraction step, the mixture was filtered using centrifugation for 10 min at 3000 rpm. Ethanol was added to the filtered solution at a ratio of 3:1. The mixture was then kept at 4 °C overnight. The resultant precipitate was obtained by filtering the solution using muslin cloth. After filtration, the residue was washed with a mixture of chloroform and acetone at a ratio of 3:1 (chloroform: acetone) to remove lipids. Then, the resultant crude polysaccharides were fresh dried using a fresh dryer (Labconco, Freezone 2.5, USA). The result was considered 100% polysaccharides concentration and applied at a dilution of 0.1% (w/v). This concentration was chosen based on the results of Mzibra et al. (2018) [25]and taking into consideration its potential applicability for on farm application.

Extract biochemical profile

The resulting crude seaweed extract was analyzed to determine its biochemical composition. Total phenolic compounds were determined as described by Ainsworth and Gillespie (2007) [26] and were expressed µg gallic acid.ml^-1. Total proteins were determined according to Bradford (1976) [27], and values were expressed as µg bovine serum albumin (BSA).ml^-1. Soluble sugars were determined according to Yemm and Willis (1954) [28] and values were given as mg glucose.ml^-1. Proline was quantified using the method described by Bates et al. (1973) [29] and was reported as µmol.ml^-1.

Experimental conditions

The experiment was conducted at Mohammed VI Polytechnic University’s experimental farm in Benguerir, Morocco (32° 13′ 6.67′′ N, 7° 53′ 14.44′′ W) during August 2023. Mean temperatures and solar radiation during this period are shown in Fig. 1. The experiment took place in a controlled greenhouse, a cooling and shading systems were used to control temperature and radiation during the day. Sowing was done on August 03,2023. A standard variety of radish seeds “NATIONAL” was used in this experiment. Seeds were purchased from BADRA company (Casablanca, Morocco). Radish seeds were sown in a 180 mL pots filled with a substrate mixture of soil: peat: sand 1:1:1 (164 g). Radish seedlings were irrigated with different amounts of water to achieve different percentages of field capacity at different stages of the experiment. Field capacity was determined by measuring the weight difference of a soil sample at saturation and dryness statuses using the following formula:

$$\begin{aligned}&Field\:Capacity\cr&\quad=\left(\left(Wet\:Weight-Dry\:Weight\right)\div Dry\:Weight\right)\cr&\qquad\times 100\end{aligned}$$

$$\begin{aligned}&Field Capacity\cr&\quad=\left(\left(206\:g-164\:g\right)\div164\:g\right)\cr&\qquad\times\:100=25.61\%\end{aligned}$$

All seedlings were irrigated with 80% of field capacity (35 ml) for the first 13 days after sowing. Afterwards, drought stress was applied as follows: the first group of radish seedlings was irrigated with 40% the field capacity (18 ml), the second was irrigated with 60% of field capacity (26 ml), and the remaining seedlings were irrigated with 80% of field capacity representing high stress, mild stress, and no stress treatments respectively. Plants were harvested after achieving 4 leaves growth stage equivalent to 20 days after sowing.

Fig. 1

External Mean temperature and solar radiation at the experimental farm of Mohammed 6 Polytechnic University during august 2023

Treatment application

Immediately after sowing, treatments were administered, each designed to assess their impact on plant growth. These treatments consisted of three groups: seaweed crude extracts (CE), polysaccharides extract derived from seaweed (PS), and a control group (Ct). Every single treatment was conducted in 3 independent replicates. Each radish seedling received an application of 40 mL of the respective treatments as a soil drench.

Physiological and biochemical parameters determination

After a 20-day growth period, seedlings were collected at the 4-leaves stage, fresh weight FW was determined, and various parameters were assessed to evaluate their response to drought stress conditions.

Relative water content (RWC)

Relative water content was determined following the methodology outlined by Luís González and Marco González-Vilar (2001) [30]. Fresh leaves were carefully weighed immediately after taking the samples to obtain their fresh weight (FW). Subsequently, these leaves were submerged in distilled water within Eppendorf tubes and kept overnight at a temperature of 4 °C. After this soaking period, the leaves were retrieved, the extra water on the surface was removed, and their turgid weight (TW) was recorded. Finally, the samples were subjected to an oven-drying process to determine their dry weight (DW). RWC was then calculated using the following formula:

$$\:(RWC\:in\:{\%})=[(\text{F}\text{W}-\text{D}\text{W})\div(\text{T}\text{W}-\text{D}\text{W})]\times\:100$$

Chlorophyll content

Chlorophyll a, and chlorophyll b contents were determined according to the method outlined by Lichtenthaler (1987) [31]. To begin, 8 mg of finely ground leaf biomass was added to 2 mL of 80% (v/v) acetone. The mixture was then subjected to centrifugation at 12,000 g for 30 min at room temperature. The supernatant was carefully recovered for analysis. The absorbance of the recovered supernatant was measured at three specific wavelengths: 662 nm,642 nm and 470 nm using a microplate reader (BMG Labtech, Fluostar Omega, Germany). The values for chlorophyll a, chlorophyll b and total carotenoids were calculated using the following formulas [31]:

$$\:\:\text{C}\text{h}\text{l}\text{o}\text{r}\text{o}\text{p}\text{h}\text{y}\text{l}\text{l}\:\text{a}\:\left({\mu\:}\text{g}\cdot\:\text{m}\text{L}^{-1}\right)=12.5\times\:{A}_{662}-\:2.79\times\:{A}_{642}$$

$$\:\text{C}\text{h}\text{l}\text{o}\text{r}\text{o}\text{p}\text{h}\text{y}\text{l}\text{l}\:\text{b}\:\left({\mu\:}\text{g}\cdot\:\text{m}\text{L}^{-1}\right)=\:21.5\times\:{A}_{642}\:-\:5.10\times\:{A}_{662}$$

$$\begin{aligned}&Total\:Carotenoids\:\left({\mu\:}\text{g}.\:\text{m}\text{L}^{-1}\right)\cr&\quad =\frac{1000{A}_{470}-1.82ch\left(a\right)-85.02ch\left(b\right)}{198}\end{aligned}$$

Plant samples extraction

Plant extracts were prepared by grinding 100 mg of fresh material using a mortar and pestle. Then, 2 mL of distilled water was added to the ground material. The mixture was subjected to sonication (Elma, S100 Elmasonic, Germany) for 2 h and subsequently incubated at room temperature for 24 h. After the incubation period, the mixture was centrifuged at 12,000 g for 30 min. The resulting supernatant was collected and used for the subsequent analysis.

Proline content

The determination of proline content was conducted following the method of Bates et al. (1973) [29]. Initially, 500 µL of the plant extract was mixed with a combination of 500 µL of glacial acetic acid and 6 M orthophosphoric acid (in a 3:2 v/v ratio), along with 25 mg of ninhydrin. The tubes were incubated in a water bath for 1 h at 100 °C and allowed to cool, then 5 mL of toluene was added. After thoroughly mixing, the toluene phase was recovered, and the absorbance was determined at 520 nm. Proline was used as a standard and values were expressed in micromoles per milligram fresh weight (µmol.mg^− 1 FW).

Total phenolic compounds

The quantification of phenolic compounds in the plant extract was carried out following the method outlined by Ainsworth and Gillespie (2007) [26]. Initially, 100 µL of the plant extract was mixed with 200 µL of 10% Folin Ciocalteu’s Phenol Reagent and thoroughly vortexed. Subsequently, 800 µL of 700 mM Na₂CO₃ was added to the mixture, and it was allowed to stand for 2 h at room temperature. The absorbance was measured at 765 nm. Gallic acid served as a standard. The phenolic content in the plant extract was expressed in gallic acid equivalent (GAE) per milligram of fresh weight (µg GAE·g^− 1 FW).

Total protein content

The total protein content was determined using the method by Bradford (1976) [27]. Initially, 200 mg of plant material was ground together with 2 mL of a 50 mM phosphate buffer at pH 6. The mixture was then subjected to centrifugation at 12,000 g for 30 min to isolate the protein extract. To measure the protein content, 100 µL of distilled water was mixed with 100 µL of the protein extract, followed by the addition of 2 mL of Bradford’s reagent. After 1 min, the optical density was measured at 595 nm. Bovine serum albumin (BSA) protein was used as standard. The protein content in the samples was reported in micrograms of equivalent BSA per milligram of fresh weight (µg equivalent BSA.mg^− 1 FW).

Soluble sugars content

Soluble sugars were quantified using the Anthrone reagent method, as described by Yemm and Willis (1954) [28]. First, Anthrone reagent was prepared by dissolving 0.2 g of anthrone in 100 mL of sulfuric acid. To analyze soluble sugars, 200 µL of plant extract was mixed with 1 mL of the prepared Anthrone reagent and subsequently incubated at 100 °C for 10 min. The optical density was measured at 620 nm. Soluble sugar contents were determined by referencing a standard curve established using glucose solutions. The results were reported in micrograms of glucose per milligram of fresh weight (µg glucose.mg^− 1 FW).

Malondialdehyde (MDA)

Leaf lipid peroxidation was assessed using the Thiobarbituric Acid (TBA) method, following the procedure outlined by Chu et al. (2010) [32]. In this assay, 200 µL of plant extract was combined with 500 µL of a 0.5% (w/v) TBA solution prepared in 20% Trichloroacetic Acid (TCA). The mixture was incubated in hot water at 95 °C for 30 min, after which the reaction was stopped using an ice bath. Subsequently, the mixture was centrifuged at 15,000 g for 30 min. The absorbance of the chromogenic product formed, known as the TBA-MDA (thiobarbituric acid-malondialdehyde) complex, was measured at both 532 nm and 600 nm wavelengths. To determine the quantity of malondialdehyde (MDA), a marker of lipid peroxidation, the non-specific absorption at 600 nm was subtracted from the absorption at 532 nm, considering an absorbance coefficient of extinction (ε) of 155.0 mM^− 1.cm^− 1. The results were reported in nanomoles per milligram of fresh weight (nmol.g^− 1 FW).

Enzyme activity determination

Soil enzyme activity

Urease activity

To quantify urease activity in soil samples, 2.5 g of soil was placed in each of two tubes. In the first tube (the test sample), 1.25 mL of a substrate solution (720 mM) and 10 mL of borate buffer (0.1 M, pH 10) were added, while the second tube (the control sample) received only 10 mL of borate buffer (0.1 M, pH 10). Both tubes were sealed and incubated at 37 °C for 2 h. After incubation, 1.25 mL of the substrate solution was added to the control, and then 15 mL of potassium chloride (2 M)–hydrochloric acid (0.01 M) solution was added to all samples. The samples were vigorously shaken on a rotatory shaker for 30 min to ensure thorough mixing. Following shaking, the samples were centrifuged at 5000 rpm for 5 min. The supernatant containing the reaction products was carefully collected. For the quantification of ammonium released, 500 µL of the filtrate was mixed with 4.5 mL of water. A color reaction was initiated by adding 5 mL of sodium salicylate–sodium hydroxide solution and 2 mL of sodium dichloroisocyanurate solution. After vortexing and incubating for 30 min at room temperature to allow color development, the absorbance of the resulting color was measured at 660 nm. Ammonium chloride was used as standard. Urease soil activity was expressed in (µg NH₄–N. g^− 1. 2 h^− 1) [33].

Alkaline phosphatase activity

In individual 2 mL Eppendorf tubes, 125 mg of soil was weighed, and 500 µL of Modified Universal Buffer (MUB) was added at pH 11. Subsequently, 125 µL of 10 mM p-Nitrophenyl Phosphate (pNPP) was added into each tube. The mixture was then subjected to incubation at 37 °C for 1 h. Then, 500 µL of 0.5 M NaOH was added to stop the enzymatic reaction. The samples were then centrifuged at 5000 rpm for 5 min. A control sample was prepared alongside each soil sample, following the same protocol but introducing 125 µL of p-nitrophenyl phosphate (10 mM) after the addition of 500 µL of 0.5 M NaOH. The color intensity of each sample was subsequently measured at 405 nm. p-nitrophenol (pNP) solution was used as standard. The enzyme activity was quantified as µmol pNP.h^− 1.g^− 1 [34].

Statistical analysis

Results are expressed as mean ± standard error (SE). The experiment was conducted in a split plot design with stress levels and biostimulants as mean and sub plots respectively. Each experimental unit was replicated three times and was conducted in three independent replicates. The data were analyzed using One way ANOVA test, with a P-value of ≤ 0.05 followed by Tukey’s multiple tests using the Minitab 20 statistical Software.

Results

Seaweed extract biochemical compounds

Table 1 outlines the biochemical composition of the crude extract from F.spiralis. Sugars were found to be the most abundant group of molecules in the extract, at 1 mg glucose per ml. Biochemical analysis indicated the presence of phenols, proteins, and proline.

Table 1 Fucus spiralis crude extract biochemical content

Fresh weight

Drought stress significantly reduced the fresh weight of radish plants (p ≤ 0.05). However, at 40% soil field capacity, the application of CE and PS significantly increased the plant’s fresh weight in comparison with the control by 47.43% and 64% respectively. At 60% soil field capacity the CE and PS application increased the plant’s fresh weight by 12.5% and 38% respectively in comparison with the control (Fig. 2) and (see Fig. 3).

Fig. 2

Fresh weights of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are means ± SE (n = 3)

Fig. 3

Radish seedlings. (A) Crude extract treatment at 40%, 60% and 80% field capacity (B) Polysaccharides treatment at 40%, 60%, and 80% field capacity (C) Control plants at 40%, 60% and 80% field capacity

Relative water content (RWC)

Water stress significantly reduced the relative water content (RWC) of radish leaves (Fig. 4). With the application of seaweed crude extract CE, RWC increased by 17.07%, 40.42%, and 14.33% respectively in 40%, 60%, and 80% stress levels compared to controls. For PS treatment, the RWC increased by 29.75% and 23.79% respectively in 60% and 80% stress levels compared to untreated plants.

Fig. 4

Relative Water Content of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are means ± SE (n = 3)

Chlorophyll and total carotenoids content

The chlorophyll and total carotenoids content of controlled plants slightly increased over the treated plants with CE and PS in all water regimes. No statistical differences were recorded between extracts and water regimes (Table 2).

Table 2 Chlorophyll a, b and total carotenoids of radish plants treated with F.spiralis CE and PS under different irrigation regimes 40%, 60% and 80% FC

Proline content

Drought stress caused a significant increase in proline content in radish plants (Fig. 5). The highest concentrations were recorded in the severe stress level of 40% FC. The application of seaweed extract CE decreased the proline content by 23.45% and 37% in 40% and 60% FC respectively in comparison with the control, while PS decreased the proline content by 6.46% and 52.8% in 40% and 60% FC respectively. Whereas, in 80% FC no difference was recorded between the treatments.

Fig. 5

Proline Content of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Total phenolic content

Drought stress caused an increase in total phenolics, and the application of CE and PS reduced the total phenols content respectively by 13% and 27.6% for the 40% FC, and by 30% and 10.9% for the 60% FC compared to the controls of each treatment. No statistical differences were shown among the treatments (Fig. 6).

Fig. 6

Total phenolic content of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Protein content

Drought stress did not impact the protein content of radish plants (Fig. 7). The interaction between the water regime and the extract was statistically significant with a p-value = 0.009. Applying extracts increased protein content by 31.6% for CE and 62.14% for PS in the 60% soil field capacity, and by 9% for CE and 65.45% for PS in the 80% soil field capacity compared to the controls. While for 40%, the control had the highest mean compared to CE and PS.

Fig. 7

Proteins content of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Soluble sugar content

Drought stress significantly impacted soluble sugars content of radish plants. The application of CE and PS induced a significant reduction in soluble sugar content respectively by 44% and 40% in the 40% FC in comparison with the controls. The interaction between the water regime and the extracts showed significant differences between various combinations (Fig. 8).

Fig. 8

Soluble sugars Content of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant difference treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Malondialdehyde MDA

As a response to drought stress the radish plants were significantly impacted by an increase in MDA levels. At 40% FC, where MDA concentrations were the highest, the application of CE and PS significantly decreased the MDA levels compared to control by 27.36% and 34.36% respectively. For the 60% and 80% FC, the application of CE and PS treatment caused a slight decrease with no significant differences in comparison with the controls (Fig. 9).

Fig. 9

MDA Content of radish plants treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant difference treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Alkaline phosphatase activity

Drought stress conditions led to a reduction in the alkaline phosphatase activity of the soil, while the application of CE and PS increased this activity in all stress levels. No statistical difference was recorded among treatments, although the highest concentrations were recorded for CE and PS compared to the control. In 40% and 60% FC, the PS treatment showed the highest concentrations with 279.88 and 340.9 µmol pNP.h^-1.g^− 1 respectively (Fig. 10).

Fig. 10

Alkaline phosphatase activity of radish plant’s soil treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Urease activity

Under drought stress conditions the urease activity was decreased in controls compared to treated plants with CE and PS respectively by 8.74% and 34.6% in the 40% FC and by 30.3% and 17% in the 60% FC (Fig. 11).

Fig. 11

Urease activity of radish plant’s soil treated with seaweed extracts, CE Crude Extract, PS Polysaccharides, and Control under different irrigation regimes 40%, 60%, and 80% FC soil field capacity. Different letters represent the significant differences between treatments at p ≤ 0.05. Values are mean ± SE (n = 3)

Discussion

Drought represents a critical threat to food production worldwide. Many strategies and innovations have been implemented to reduce the negative impact of drought on crops production. Yet, still there is a need for sustainable innovations to deal with this issue. In this context, seaweed extract biostimulants are considered as a promising technology for mitigating drought stress on plants [35]. Therefore, This study was conducted to elucidate the role of seaweed crude extracts and polysaccharides derived from the brown macroalgae F.spiralis in mitigating drought stress in (Raphanus sativus L.) radish plants and mechanisms involved in this process.

At 40% and 60% field capacity levels, water stress led to a decrease in fresh weight. Whereas, when CE and PS were applied, they resulted in an increase in fresh weight when compared to the control group. The activation of abscisic acid (ABA) response genes and the limitation of stomatal opening, which eventually restricts water loss, could be responsible for the improvement of plant’s fresh weight under water stress [36]. Furthermore, it was revealed that Kappaphycus alvarezii extract improved nutrient and water uptake, thus promoting general plant vigor and growth [37]. Along with amino acids and minerals, seaweed products also contain growth regulators such as auxins, cytokinins, and gibberellins. These elements contribute to plant growth and cell division [38].

Moreover, the application of CE and PS on radish seedlings impacted the biosynthesis of proline during mild and high stress which could be another mechanism involved in attenuating the negative impact on plant growth under water limitation status. As a response to drought, proline accumulates in plant cells [39]. Leading to the protection of membrane integrity, ROS scavenging, regulating cellular osmotic pressure, and ensuring proteins /enzymes stability [40]. In our study, the application of both CE and PS reduced the concentration of proline in radish plants during stress in comparison with stressed non-treated plants. These findings indicate that treated plants were probably less impacted by drought compared to the control. Similar results were reported by Elansary et al. (2016) [41] who recorded a reduction in proline biosynthesis in medicinal plants following the application of an Ascophyllum nodosum preparation in 50% and 100% ET irrigation scenarios. However, other studies suggest that seaweed extracts application enhance proline concentrations in plant tissues during drought [10, 42]. For example, application of Ascophyllum nodosum extracts significantly enhanced the concentration of proline in tomato leaf samples in comparison with the control following 7 days of water shortage [42]. The same authors suggested that the differentiation in the kinetics of proline accumulation as a response to the application of SE could be credited to the kind of biostimulant used, application mode, and/or crop type.

Even if not significant, the application of F.spiralis crude extract increased the RWC of treated plants in all stress levels. Similarly, plants treated with Ascophyllum nodosum had higher relative water content and higher stomatal conductance under drought stress [43]. Moreover, treating orange trees growing in a restricted water regime with Ascophyllum nodosum extract as a soil drench impacted positively plant growth and resulted in a higher stem water potential than control trees [44]. Additionally, the application of an Ecklonia maxima-derivate extract on chicory plants increased their RWC during moderate and severe drought stress conditions [45].

The chlorophyll content of radish plants was slightly higher in treated plants with CE and PS with no statistical significance. This result is in concomitance with those of El Boukhari et al. (2023) [10] who revealed that different seaweed extracts applications did not impact the leave’s chlorophyll index of Faba bean (Vicia faba L.) during both water stress and full irrigation phases.

MDA is a compound formed from membrane lipids in response to ROS, and serves as an indicator of drought stress, helping to assess the extent of plasma membrane damage and plants’ tolerance to drought conditions [46]. In our experiment, MDA levels in radish plants increased in response to stress conditions. However, the application of both CE and PS decreased the MDA levels in water stress conditions (40% and 60% FC) in comparison with control plants. Similar results were recorded by Jacomassi et al. (2022) [14] who found that seaweed extract application on sugarcane enabled the reduction of MDA levels in treated plants. The application of F. spiralis treatment on Salvia officinalis showed a reduction in MDA content, suggesting a decreased accumulation of reactive oxygen species (ROS) [47].

Plants use polyphenols to react to biotic and abiotic stresses, Polyphenols originate from the phenylpropanoid biosynthetic pathway, and they are well known for participating in scavenging reactive oxygen species (ROS) by acting as powerful antioxidants [48]. However, in this study, CE and PS didn’t impact phenolics content in radish plants at all stress levels.

During drought stress, soluble sugars are increasingly accumulated in plants [49]. Their accumulation reduces photosynthesis rate, induces the closure of stomates, maintains leaf water status and osmotic pressure, preserves cell membranes from ROS, and promotes plant’s adaptability [50]. Surprisingly, the concentration of soluble sugars in radish plants followed the same trend as proline content and decreased after the application of CE and PS during stress conditions in comparison with non-treated plants. This result proves again that CE and PS applications were able in attenuating drought stress on radish plants through probably an indirect effect related to the enhancement of soil physical, chemical and/or biological conditions. This hypothesis might be strengthened by the fact that CE and PS were applied solely in the soil, and they enhanced to some extent soil enzymatic activity (phosphatase and urease), especially PS treatment. Polysaccharides consist of sugar molecules that can serve as carbon sources to support microbial growth [51]. Moreover, they could improve soil properties by increasing water retention and enhancing aggregate stability [52].

Phosphatase enzymes have been studied extensively and are known for catalyzing the hydrolysis of ester-phosphate bonds, releasing phosphate (P) which can be taken up by plants [53]. Urease serves as an important soil enzyme that plays an important role in the occurrence of accessibility of N for plant growth within the nitrogen cycle and in the extensive usage of urea as a fertilizer [54]. The application of CE and PS increased the alkaline phosphatase activity in the soil in all stress levels (40% and 60% FC). Moreover, urease activity was higher in plants that received PS treatment mainly at the 60% FC water regime. According to Wang et al. (2016) [55] seaweed extract application to Malus hupehensis Rehd. can increase the activity of invertase, urease, proteinase, and phosphatase enzymes in the soil.

This study confirms again the complexity of seaweed extract biostimulants as their biochemical content wasn’t correlated with the growth and physiological traits recorded. This conclusion aligns with the results of Goñi et al. (2018) [42] who reported that the biochemical profiles of three extracts originating from the same seaweed Ascophyllum nodosum weren’t correlated with the positive effect recorded on tomato plants subjected to drought stress. Consequently, the complexity in understanding the compositional biochemistry of seaweed extracts drives to the heterogeneity of results within this category of plant biostimulants.

Conclusion

This study investigated the impact of F.spiralis crude extract and polysaccharides on the growth and physiology of radish plants (Raphanus sativus L.) under varying levels of drought stress. The results indicated improvements in plant fresh weight, relative water content, and soil enzyme activities. However, CE and PS applications reduced Proline, soluble sugars and MDA contents. The observed changes in growth and physiological traits did not correlate with the biochemical composition of the extracts, suggesting that the bioactive compounds within may act synergistically or influence the soil-plant system indirectly. This confirms that seaweed extract biostimulants represent a complex field, as their effects seem to be influenced by numerous factors. This complexity poses a challenge in determining the optimal combination of extracts dosage, application timing, crop type, and environmental conditions. Therefore, further research is needed to optimize the application of seaweed extracts as biostimulants for different plant species and growing conditions, aiming to enhance drought stress tolerance. Moreover, the continuous evidence of polysaccharides enhancing plant growth under various environments should invite researchers to explore their chemical composition and potential correlations they could have with the positive traits recorded.