Introduction

Peanut (Arachis hypogaea L.) is commonly known as groundnut in India belongs to the Fabaceae family. The peanut has tetraploid genome (AABB) evolved from a hybridization event of two diploid species, Arachis duranensis genome (AA) and Arachis ipaensis genome (BB) (Ozias-Akins and Breiteneder 2019). Peanut seeds are rich in edible oil and protein content (Suchoszek-Łukaniuk et al. 2011). Mature seeds contain about 45–50% oil, 25% protein, macronutrients, minerals and vitamin B, vitamin E and folate (Ozias-Akins and Breiteneder 2019). The total production of peanuts in the world is 49,535 thousand tonnes; India is the second leading country with 13% total production. Gujarat state contributes 42% of the total production of India (FAS 2022).

Diseases caused by different insects, nematodes, fungal, and viral pathogens affect the production and quality of peanuts. Various fungi including Sclerotium rolfsii, S. minor, Pythium spp., and Rhizoctonia solani damage above and underground parts of peanuts (Thiessen and Woodward 2012). Sclerotium rolfsii (Athelia rolfsii) is a necrotrophic, soil-borne pathogen infecting more than 500 plant species. S. rolfsii occurs commonly in the tropics, sub-tropics and other warm temperate regions and causes diseases such as root rot, stem rot, wilt and foot rot in planta (Deepthi and Reddy 2013). Stem rot disease is distributed worldwide and is predominant in warm, dry climates. S. rolfsii causes stem rot disease in peanuts with 10–40% yield loss (Bosamia et al. 2020).

The disease management strategies using various chemicals can reduce the yield loss but leads significant disadvantages. These drawbacks include high cost, pollution of soil, air and water, resistance to chemicals and adverse effects on plant, animal, and human health (Pal et al. 2014). Global pesticide usage increased by 36 % during 2000–2019 and reached 4.2 million tonnes in 2019 (FAO 2021). There is a significant need for ecologically safer and environmental friendly disease management practices, including intercropping, crop rotation, tillage, manuring and the use of biological control agents. There are several reports on the use of seaweed extracts as a valuable resource for mitigation of diseases by acting as bio-elicitors in the plants (Agarwal et al. 2021; Shukla et al. 2021).

Seaweed, a multicellular organism, belongs to three major phyla of algae such as Chlorophyta (green), Ochrophyta (brown) and Rhotophyta (red). The seaweed extract contains different compounds, which relayed bio-elicitors activity towards defense against different stress conditions in the plants. Different polysaccharides present in seaweed extracts have enormous potential for the plant defense (Stadnik and Fretias 2014; Shukla et al. 2019). The algal extract prepared from marine algae of phyla, Chlorophyta, Rhodophyta, and Ochrophyta exhibited antifungal activity (Sheikh et al. 2018; Agarwal et al. 2021). The extract obtained from brown algae contains laminarin and sulphated fucans, which induce antifungal compounds in alfalfa cotyledons (Kobayashi et al. 1993). Seaweed extracts obtained from brown algae, Padina gymnospora and Sargassum liebmannii elicited a defense response against the necrotrophic fungus Alternaria solani in tomato plants (Hernández-Herrera et al. 2014). In our previous study, we have shown the efficacy of S-extract (S. tenerrimum extract) for reducing charcoal rot disease in tomato (Lycopersicon esculentum) caused by Macrophomina phaseolina, a necrotrophic soil-borne fungus (Bosmaia et al. 2023; Khedia et al. 2020). The S-extract is enriched with phytohormones (IAA, zeatin, GA9) and active compounds like sulphated polysaccharide, terpenoids, sargachromenol, pheophytine, flavonoids and sterols (Prasad et al. 2010). Therefore, in the present study we aimed to evaluate the efficacy of S-extract for reducing stem rot disease in peanut caused by S. rolfsii, which is also necrotrophic soil-borne fungus like M. phaseolina. Here, we have evaluated physiological, morphological and biochemical parameters to understand the disease-reducing mechanism.

Materials and methods

Plant materials and treatments

The seeds of high-yielding groundnut variety TG37A were obtained from ICAR-Directorate of Groundnut Research, Junagadh, India for present study. TG37A was developed by a crossbreeding involving the parent TG 25 and TG 26 and matures in 114 days. TG37A is distinguished by its Spanish bunch growth habit, semi-dwarf stature, compact pod arrangement, and smooth-surface pods, each typically containing three seeds. This variety is susceptible to stem rot disease caused by S. rolfsii (Dodia et al. 2019).

Seeds were sown in earthen pot containing black soil, red soil and farmyard manure (2:1:1) and germinated plants were maintained under greenhouse conditions. Three plants were grown per pot and watered regularly. The S. rolfsii culture was procured from ICAR-Indian Agricultural Research Institute, New Delhi, India. Fresh culture was initiated on potato dextrose agar medium. Furthermore, the fungus was grown on autoclaved sorghum seeds for 15 days at 25 ºC. The procedure for preparing Sargassum tenerrimum extract (S-extract) is mentioned in Khedia et al. (2020). For the fungus treatment (F), 2 g of sorghum seeds infused with S. rolfsii mycelium were positioned at the base of plants. The seeds were covered with autoclaved wheat straw to facilitate the initial growth of fungus (Bosamia et al. 2020). Peanut plants were subjected to foliar application of S-extract (S, 10%), as well as the application of S. rolfsii, either individually (F) or in combination (S + F). Control (C) set of peanut plants was sprayed with only water.

Independent experiments were conducted at both vegetative and reproductive stages to evaluate the efficacy of different treatments (C, S, F and S + F). At vegetative stage, the 1st treatment (C, S, F and S + F) was applied to 17-d-old plants, and subsequently 2nd treatment applied after 7 days. At the reproductive stage, the 1st treatment (C, S, F and S + F) was applied to 57-d-old plants and subsequently 2nd application was administered after 7 days. In both sets of experiments, the fungal treatment (F) was given only once at the time of first foliar spray. In each experiment, 9 pots, each containing three plants were assigned to each treatment group. In both the vegetative and reproductive stage experiments, the third leaf from the apex of each plant of three pots was collected after 7 days of 1st spray and same day 2nd spray was administered and subsequently the collection of the tissues of 2nd spray was carried out after 7 days in three replicates for physio-biochemical analysis.

Growth parameters

For evaluating the efficacy of S-extract as a growth stimulant and towards enhancing disease resistance against S. rolfsii in peanuts, morphological parameters like plant height, number of primary branches and branch length were determined for both the vegetative and reproductive stage experiments.

Electrolyte leakage and membrane stability index

For the determination of electrolyte leakage (EL) and membrane stability index (MSI), young mature leaflets of similar size were collected in triplicates from each treatment. EL was determined following the method described by (Lutts et al. 1996). A single leaflet was incubated in 10 mL distilled water at 25 °C and 80 rpm for 24 h. The electrical conductivity of the solution (Lt) was measured. Subsequently, the solution was autoclaved at 120 °C for 20 min and cool. Conductivity (L0) was measured at room temperature and EL (%) was calculated using Eq. 1.

$$\text{EL} (\%)=\frac{Lt}{L0}\times 100$$
(1)

MSI was determined following the method described by Hayat et al. (2012). A single leaflet was immersed in 10 mL distilled water in 50 mL tubes in two sets. In the first set, the tube was heated at 40 °C for 30 min using a water bath, and conductivity of solution (C1) was measured at room temperature. In the second set, the tube was incubated in a boiling water bath for 10 min, and the conductivity of the cooled solution (C2) was measured at room temperature. MSI was calculated using Eq. 2.

$$\text{MSI }=1-\left(\frac{C1}{C2}\right)\times 100$$
(2)

Biochemical analysis

For the biochemical analysis, young leaves from the apex were collected, freezed in liquid nitrogen and stored at –80 °C for further use. The anti-oxidative enzymes were quantified as mentioned below, the absorbance readings were recorded using an Epoch microplate spectrophotometer (Biotek, USA).

Determination of pigments content

The pigment contents were estimated using acetone extract. The leaf sample was powdered in a chilled mortar and pestle using liquid nitrogen. The powdered sample was mixed with 3 mL of chilled acetone (80%), centrifuged to obtain clear supernatant and used for the determination of pigments content. The absorbance of the supernatant was measured at wavelengths of 470 nm, 646 nm and 663 nm. The determination of chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (Tchl) and total carotenoids (TC) content was carried out as per the procedure of Lichtenthaler (1987).

Estimation of phenols, total soluble sugars and total amino acids

To estimate the phenols, total soluble sugars (TSS) and total amino acids (TAA), the leaf sample was homogenized in 80% warm ethanol (60 °C) followed by centrifugation to obtain clear solution and further used for assays.

Total phenolic content was determined using the Folin-Ciocalteu method as described by Patel et al. (2018). In the reaction mixture, 2.5 mL of 0.2 M Folin-Ciocalteu reagent was added to 1 mL of the ethanol extract. After incubation for 5 min, 2 mL of 7.5% Na2CO3 was added and further incubated in the boiling water bath for 1 min. The absorbance was measured at 760 nm. A standard curve was prepared using gallic acid and phenolic content was quantified as milligrams per gram fresh weight (mg g−1 FW) in terms of gallic acid equivalent.

The estimation of soluble sugars was carried out using the Anthrone method as described by Shukla et al. (2012). The reaction was initiated by adding 100 µL of ethanol extract to a tube containing 3 mL of anthrone reagent (prepared in 72% H2SO4), incubated in the boiling water bath for 10 min. After cooling the reaction mixture, absorbance was read at 620 nm. The standard curve was obtained using D-glucose.

Total amino acid content was determined following the method described by Shukla et al. (2012). In the reaction mixture, 1 mL ethanol extract was mixed with 0.2 M citrate buffer (pH 5), 80% ethanol and 1% ninhydrin reagent followed by incubation in boiling water bath for 15 min. After cooling the mixture, absorbance was read at 570 nm. Standard curve was prepared using glycine.

Antioxidative enzymes

Phosphate buffer extract of leaf samples was prepared following the standard procedure (Khedia et al. 2020). For the preparation of the extract, 100 mg tissue was homogenized in potassium phosphate buffer (50 mM, pH 7.8), vortexed for 2 min and centrifuged to obtain the clear supernatant. The supernatant was further used for enzyme assays. Enzyme activity was expressed as units per milligram of protein (U mg−1 protein). The protein content in the samples was estimated using the standard Bradford method (Bradford 1976).

Catalase (CAT), EC 1.11.1.6, activity was determined by measuring the initial rate of H2O2 degradation as described by Dhindsa et al. (1981). The reaction mixture (3 mL) contained 1.5 mL of 0.1 M phosphate buffer (pH 7.5), 950 µL of distilled water, 50 µL of enzyme extract and 500 µL of 75 mM H2O2. Absorbance was measured immediately after adding H2O2. Kinetic measurements were performed at 240 nm for 1 min at 10 s intervals, and enzyme activity was expressed as the degradation of one mole H2O2 per minute (mol H2O2 min−1).

Superoxide dismutase (SOD), EC 1.15.1.1 activity was measured by measuring the inhibition of photoreduction of NBT as described in (Dhindsa et al. 1981). The reaction mixture (3 mL) contained 1 mL of distilled water, 1.5 mL of 100 mM L-methionine, 0.1 mL of 3 mM EDTA, 0.1 mL of 2.25 mM NBT, 0.1 mL of enzyme extract and 0.1 mL of 60 µM riboflavin. The reaction was initiated after adding riboflavin in the dark. The reaction mixture was incubated under fluorescent light (15 W) for 15 min and the reaction was terminated by switching off the light source. Absorbance was measured at 560 nm.

Guaiacol peroxidase (GPOX), EC 1.11.1.7. activity was measured following the method described (Hammerschmidt et al. 1982). The reaction mixture (3 mL) contained 2.75 mL of 0.1 M phosphate buffer (pH 5.8), 75 µL of 0.2 M guaiacol, 75 µL of H2O2 (3%), and 100 µL of enzyme extract. The reaction was initiated immediately after adding the enzyme extract and the absorbance was recorded at 470 nm after 1 min. One unit of enzyme activity is defined as a change in absorbance of 0.01 at 470 nm.

Polyphenol oxidase (PPO), EC 1.10.3.1 activity was determined following the method described by Oktay et al. (1995). The reaction mixture (3 mL) comprised 2.3 mL of phosphate buffer (pH 7), 600 µL of 20 mM catechol, and 100 µL of enzyme extract. Absorbance readings were taken initially and after 1 min at 420 nm. One unit of enzyme activity is defined as an increase in absorbance of 0.001 per min (Ho 1999).

Quantification of ROS

The superoxide radical (O2•−) content was determined using the phosphate buffer extract as described by Elstner and Heupel (1976). The reaction mixture contained 900 µL of 65 mM potassium phosphate buffer (pH 7.8), 1 mL phosphate buffer extract and 100 µL of 10 mM hydroxylamine hydrochloride. This mixture was incubated at 25 ºC for 1 h. For colour development, 1 mL each of 17 mM hydroxylamine hydrochloride and 7 mM α-napthylamine were added and incubated at 25 °C for 20 min. The absorbance was measured at 530 nm. A standard curve was generated using sodium nitrite (NaNO2) and the O2•− content was expressed as micromoles per gram fresh weight (µmol g−1 FW).

H2O2 content was determined using the method described by (Mukherjee and Choudhuri 1983). In the assay, 1 mL of acetone extract was mixed with 0.5 mL titanium dioxide reagent (0.1% TiO2, prepared in 20% v/v H2SO4) and subjected to centrifuge at 6000 rpm for 15 min. The absorbance of the supernatant was measured at 415 nm. The standard curve was obtained using H2O2 and the content in sample was expressed as micromoles per gram fresh weight (µmol g−1 FW).

Yield

Yield parameters such as pod size (cm), number of pods per plant, 100 seeds weight (g) and seed weight (g plant−1) were observed at the time of maturity of plants from both the treatments at vegetative and reproductive stages (about 120 days after sowing). For each parameter, nine pots comprising 3 plants each were considered as independent biological replicate.

Statistical analysis

Statistical analysis was performed using InfoStat statistical software. One-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) post-hoc test was performed to determine significant differences in multiple comparisons. The Fischer’s LSD test was carried out with a significance level set at 0.05 (alpha = 0.05). Each assay was replicated three times with nine biological replicates for the assessment of growth parameters.

Results

Growth parameters

In both the vegetative and reproductive experiments, the S treatment showed improved plant growth (Fig. 1a, b). In the vegetative experiments, the plant height increased 1.28-fold and 1.12-fold with S treatment as compared to C in the first and second applications, respectively (Fig. 1c, d). Further, the S + F treatment demonstrated 1.44-fold and 1.10-fold increase in plant height as compared to F with the first and second applications, respectively (Fig. 1c, d). The branch length also showed 1.39-fold and 1.07-fold increase with the S treatment compared to the C after the first and second applications, respectively (Fig. 1e, f). The number of branches was significantly reduced in F as compared to S and S + F treatment after the first application (Fig. 1g), however, after the second application all treatments showed almost similar number of branches (Fig. 1h).

Fig. 1
figure 1

Morphology of peanut plants in response to different treatments after 7 days of 2nd spray at vegetative stage (A) and reproductive stage (B). Plant height at vegetative stage (C, D) and reproductive stage (I, J); Branch length at vegetative stage (E, F) and reproductive stage (K, L); Number of branches at vegetative stage (G, H) and reproductive stage (M, N). Values are represented as means ± SD (n = 9) and marked with different letters to indicate significant difference at p ≤ 0.05

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Similarly, in the reproductive experiment, plant height exhibited a significant decrease with the F treatment on both the first and second applications (Fig. 1i, j). The branch length increased significantly with S treatment on both the applications as compared to C, F and S + F treatments (Fig. 1k, l). The number of branches was similar to the first application for all treatments, however, on the second application of the treatments at reproductive stage, S treatment showed a significant increase of 1.15-fold as compared to C (Fig. 1m, n).

Pigments

In vegetative experiment, with both applications, the F treatment exhibited the lowest pigment accumulation (Fig. 2a–h). The pigments, Chl a, Chl b, TChl and TC showed significantly higher accumulation with S treatment as compared to C and F treatment (Fig. 2a–g). The S + F treatment showed 2.05- and 1.29-fold higher TChl and TC pigments accumulation, respectively, as compared to F in the 2nd spray of vegetative stage (Fig. 2f, h). In the reproductive experiment, higher accumulation of Chl a, Chl b, TChl and TC were observed with the second application of C and S treatment as compared to the first application (Fig. 2i–p). Furthermore, all the pigments with first application showed statistically similar result in C/S and F/S + F treatments (Fig. 2i, k m, o). However, the second treatment showed statistically higher pigments in S treatment compared to other treatments (Fig. 2j, l, n, p).

Fig. 2
figure 2

Pigments content of peanut leaf analysed after 7 days of treatment. Chl a content at vegetative stage (A, B) and reproductive stage (I, J); Chl b content at vegetative stage (C, D) and reproductive stage (K, L); Total Chl content at vegetative stage (E, F) and reproductive stage (M, N); Total carotenoid content at vegetative stage (G, H) and reproductive stage (O, P). Values are represented as means ± SD (n = 3) and marked with different letters to indicate significant difference at p ≤ 0.05

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Electrolyte leakage and membrane stability index

The F treatment resulted in the highest EL, during both applications at the vegetative stage (17.67% and 12.25% with the first and second applications, respectively) and reproductive stage (14.73% and 26.64% with the first and second application, respectively, Fig. 3a, b, e, f). The S treatment significantly reduced EL at vegetative stage (Fig. 3a, b). During the first application in vegetative experiment, the S treatment resulted in a 1.24-fold decrease in EL compared to the C, while the S + F treatment exhibited a notable 1.39-fold reduction compared to the F (Fig. 3a). Similarly, during the second application, both the S and S + F treatment groups exhibited a 1.06-fold decrease in EL levels as compared to C and F treatments, respectively (Fig. 3b). Moreover, in the reproductive experiment during the second application, the S + F treatment displayed a remarkable 3.96-fold decrease in EL levels compared to the F treatment (Fig. 3f).

Fig. 3
figure 3

Electrolyte leakage (EL) and membrane stability index (MSI) of peanut leaf analysed after 7 days of treatment. EL at vegetative stage (A, B) and reproductive stage (E, F); MSI at vegetative stage (C, D) and reproductive stage (G, H). Values are represented as means ± SD (n = 3) and marked with different letters to indicate significant difference at p ≤ 0.05

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Interestingly, during the vegetative experiment the MSI was also significantly reduced in the F treatment compared to other treatments (Fig. 3c, d). In the reproductive experiment, 1.02-fold and 1.04-fold higher MSI was observed with S + F treatment as compared to F treatment with first and second applications, respectively (Fig. 3 g, h).

Non enzymatic antioxidants and osmolytes

In vegetative experiment, the highest TAA accumulation was observed in S treatment (5.59 mg g−1 FW) and S + F treatment (4.26 mg g−1 FW) during the first and second applications, respectively (Fig. 4a, b). The S treatment showed 1.29-fold increase in TAA content as compared to C with the first application (Fig. 4a), and 1.16-fold increase in S + F treatment as compared to F with the second application, (Fig. 4b). In reproductive experiments, highest TAA accumulation was observed in S + F treatment (3.78 mg g−1 FW) with the first application, whereas, with the second application maximum accumulation was in the F treatment (3.89 mg g−1 FW, Fig. 4g, h).

Fig. 4
figure 4

Non enzymatic antioxidants and osmolytes content in leaf analysed after 7 days of treatment. Total amino acids (TAA) content at vegetative stage (A, B) and reproductive stage (G, H); Total soluble sugars (TSS) content at vegetative stage (C, D) and reproductive stage (I, J); Total phenol content vegetative stage (E, F) and reproductive stage (K, L). Values are represented as means ± SD (n = 3) and marked with different letters to indicate significant difference at p ≤ 0.05

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In vegetative experiments, with both applications, the maximum TSS accumulation was observed with the S treatment (1.44 and 0.83 mg g−1 FW, respectively), while the lowest accumulation was observed with the F treatment (1.10 and 0.39 mg g−1 FW, respectively, Fig. 4c, d). The S treatment showed 1.17- and 1.47-fold increased TSS accumulation as compared to C treatment with both applications, respectively (Fig. 4c, d). Interestingly, S + F treatment showed 1.13- and 1.60-fold increased TSS accumulation as compared to F treatment with both applications, respectively (Fig. 4c, d). In reproductive experiment, TSS content decreased with F and S + F treatments as compared to C/S treatments during both applications (Fig. 4i, j).

In vegetative experiment, maximum phenol accumulation was observed in S + F (1.61 and 3.24 mg g−1 FW) with both applications (Fig. 4e, f). During the second application phenol content increased by 1.70-fold as compared to F (Fig. 4f). The reproductive experiment resulted minimum phenol accumulation in S treatment (1.29 and 1.17 mg g−1 FW, respectively) with both the applications (Fig. 4k, l).

Reactive oxygen species

In vegetative experiment, O2•− production was highest (21.74 and 4.58 µmol g−1 FW) in the F treatment during both applications (Fig. 5a, b). The S + F treatment showed significantly reduced O2•− content (1.28-fold) compared to the F. However, the O2•− content decreased by 2.70- and 1.28-fold in S and S + F treatments compared to C and F, respectively, during the second application (Fig. 5b). In reproductive experiments, minimum O2•− content was observed in S treatment during the first application, whereas, with second application S + F showed minimum O2•− accumulation (10.38 µmol g−1 FW, Fig. 5e, f).

Fig. 5
figure 5

ROS content of leaf analyzed after 7 days of treatment. O2– content at vegetative stage (A, B) and reproductive stage (E, F); H2O2 content at vegetative stage (C, D) and reproductive stage (G, H). Values are represented as means ± SD (n = 3) and marked with different letters to indicate significant difference at p ≤ 0.05

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In vegetative experiment, H2O2 production was minimal in S + F (20.67 µmol g−1 FW) and S treatments (69.37 µmol g−1 FW) during first and second applications, respectively (Fig. 5 c, d). The S + F treatment significantly decreased H2O2 accumulation by 1.34- and 1.21-fold compared to F during first and second application, respectively (Fig. 5c, d). In reproductive experiments, the S + F treatment showed a minimum accumulation of H2O2 (99.37 and 56.09 µmol g−1 FW, respectively) during both applications (Fig. 5g, h). S + F treatment showed significantly reduced H2O2 accumulation as compared to F during the first application (Fig. 5g).

Antioxidative enzymes

In vegetative experiment, after the first application the S treatment showed an increase in CAT (1.33-fold), SOD (1.17-fold), GPOX (1.86-fold) and PPO (1.73-fold) activities as compared to C treatment (Fig. 6a, c, e, g). The S + F treatment showed a maximum accumulation of SOD, GPOX and PPO enzymes on the first application (Fig. 6c, e, g). In S + F, the activities of CAT, SOD, GPOX and PPO were increased 1.57-, 1.16-, 1.38- and 1.83-fold, respectively, compared to F (Fig. 6a, c, e, g). With the second application, CAT, GPOX and PPO increased 1.52, 1.13 and 1.63-fold with S + F treatment as compared to F treatment (Fig. 6b, f, h). Interestingly, maximum SOD accumulation (37.50 U mg−1 protein) was observed with F treatment on the second application (Fig. 6d).

Fig. 6
figure 6

Antioxidative enzymes activity of leaf analysed after 7 days of treatment. CAT activity at vegetative stage (A, B) and reproductive stage (I, J); SOD activity at vegetative stage (C, D) and reproductive stage (K, L); GPOX at vegetative stage (E, F) and reproductive stage (M, N); PPO at vegetative stage (G, H) and reproductive stage (O, P). Values are represented as means ± SD (n = 3) and marked with different letters to indicate significant difference at p ≤ 0.05

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In reproductive experiments, the CAT showed statistically similar accumulation with S and S + F treatments during both applications (Fig. 6i, j). The SOD showed 1.07-fold higher accumulation with F treatment as compared to S + F during the first application, whereas, it was similar with second application (Fig. 6k, l). During the second application, the GPOX showed maximum accumulation (1394.15 U mg−1 protein) with S treatment and PPO showed maximum accumulation (777.20 U mg−1 protein) with F treatment (Fig. 6n, p).

Yield

In vegetative experiment, S-extract did not improve seed weight per plant compared to control (Table 1). Only the pod and seed number marginally increased. The F treatment revealed a significant decrease in the pod number, seed number as well as seed weight per plant. However, the S + F treatment showed improved parameters compared to F except pod length and width (Table 1).

Table 1 Yield parameters of peanut plants under different treatments
Full size table

In reproductive experiment, the S treatment resulted in increased pod number, seed number and seed weight per plant compared to control. The F treatment showed decreased pod number, seed number and seed weight per plant across all the treatments, however, statistically at par. Further, addition of S-extract along with F improved the yield parameters compared to F alone except pod length and width (Table 1).

Discussion

Seaweed extract is known to enhance plant growth and crop yield and improve tolerance to biotic and abiotic stresses (Hussain et al. 2021; Khan et al. 2009; Khedia et al. 2020). Seaweed extracts are gaining significance in agriculture as there can be used as organic fertilizers and bio-stimulants, which can support in sustainable farming for better growth and productivity, promoting environmental sustainability and reducing the dependence on synthetic chemicals. Seaweeds contain natural compounds, such as phytohormones, polysaccharides and bioactive substances that have been found to enhance tolerance against different plant diseases and pests (Agarwal et al. 2021). Algal extracts when applied as a foliar spray or as a soil amendment to induce the plant’s innate defense mechanisms (Agarwal et al. 2021; Shukla et al. 2021).

Our previous study has shown that S-extract enhances M. phaseolina tolerance in tomato plants by regulating phytohormones and antioxidative activity (Khedia et al. 2020). However, there is no report on S-extract towards reducing stem disease in peanuts, therefore, we aimed to evaluate S-extract towards disease tolerance in peanut plants. The S-extract contains different plant growth-promoting hormones like IAA, zeatin and GA9 (Prasad et al. 2010). In the present study, the application of S-extract showed increased plant height and branch length in both the stages compared to control, this may be attributed by the presence of phytohormones in S-extract. Similarly, the red and brown seaweed extracts revealed increased shoot length in mung bean (Di Filippo-Herrera et al. 2019). The S-extract also showed overall improved growth of peanuts via improving photosynthesis, leaf chlorophyll content, main stem height, lateral branch length and dry matter accumulation (Meng et al. 2022).

In the current study, the application S and S + F during second spray of vegetative stage showed increased chlorophyll content compared to C and F alone, respectively. The result indicated the application of S-extract during vegetative stage help to reduce the disease severity in terms of chlorosis in peanut plants. In the reproductive stage, the chlorophyll and carotenoid contents increased in S compared to C. This aligns with previous research findings, where Rosenvingea intricate seaweed extract, was found to enhance the growth and pigments in cluster beans (Thirumaran et al. 2009). The application of A. nodusum extract increased chlorophyll content, attributed to enhance chloroplasts biogenesis and reduced chlorophyll degradation. This effect was associated with the up-regulation of genes related to photosynthesis, cell metabolism, stress response, as well as sulphur and nitrogen metabolism in Brassica napus (Jannin et al. 2013). Application of aqueous alkaline extract of A. nodosum to the soil resulted in higher concentration of chlorophyll in the leaves as compared to control plants (Blunden et al. 1997). The application of Kappaphycus alvarezii sap resulted in significant increased photosynthesis pigments such as TChl and carotenoids at both the vegetative and reproductive stages (Patel et al. 2018). The Sargassum polycystum extract enhanced pigments (Chl a, Chl b, TChl, and carotenoids) content in red gram (Erulan et al. 2009). The liquid extracts of Sargassum wightii improved photosynthetic pigments in cluster bean plants (Vijayanand et al. 2014). The aqueous extract of Sargassum johnstonii enhanced pigment contents in tomato plants (Kumari et al. 2011). The A. nodosum extract showed increased chlorophyll content in cucumber cotyledons (Whapham et al. 1993). The Durvillaea potatorum and A. nodosum were found to significantly increase the chlorophyll levels in the tomato leaves at the flowering stage (Hussain et al. 2021).

Electrolyte leakage assay reveals loss of cell membrane integrity and death of the cells in response to various biotic and abiotic stresses (Demidchik et al. 2014). The application of A. nodosum extract has been found to lower the EL in onion (Gomah et al. 2015) and faba bean (Elshalakany 2020). The present study revealed a reduction in EL in S + F treatment compared to F in both the stages suggesting that S-extract has a protective role against S. rolfsii infection. MSI in plants provides information about the ability of plant cell membranes to maintain their structural integrity and functionality during environmental challenges (Nijabat et al. 2020). S-extract increased MSI in S + F treatment compared to F, showing the efficiency of S-extract for maintaining membrane integrity which can be attributed to the accumulation of enzymatic and non-enzymatic antioxidants (Burguieres et al. 2007). There is a positive correlation between enzymatic and non-enzymatic antioxidants and the membrane stability index. Higher levels of enzymatic and non-enzymatic antioxidants are often associated with improved membrane stability (Slabbert and Krüger 2014). A. nodosum extract has been reported to protect membrane integrity in Arabidopsis during freezing stress (Nair et al. 2012). Similarly, the K. alvarezii sap also showed enhanced membrane integrity in wheat (Patel et al. 2018).

The increased phenol content in the S and S + F treatments after second spray during the vegetative stage suggests the induction of phenolic compounds, which likely contribute to enhanced disease resistance against S. rolfsii. The presence of free phenolic pool in plants plays a pivotal role in fungal resistance due to its antifungal properties, formation of physical barriers, initiation of defense responses, generation of ROS, and the chelation of nutrients (Battacharyya et al. 2015). The total phenolic content was also found higher in A. nodosum extract treated carrots compared to control (Jayaraj et al. 2008). The enhanced accumulation of phenols in S + F treatment to plants might be related to the increased activity of enzymes such as PPO and GPOX, which are involved in phenol oxidation.

Osmoprotectants, such as total proteins, proline, amino acids and soluble sugars, are found increased with the application of K. alvarezii sap (Patel et al. 2018). The S + F plant showed increased amino acid content compared to F in vegetative stage suggesting that amino acids could have contributed in disease resistance in peanuts. Amino acids are primary metabolites that play vital roles in plant immunity. They serve as precursors for the production of defensive compounds and facilitate communication between the regulatory hormones of defense pathways, such as SA and JA (Zeier 2013). The free amino acids present during plant-endophyte interaction, either independently or in conjunction with pathogenic fungi, have the potential to induce enhanced resistance against pathogenic infections (Waqas et al. 2015).

The application of seaweed extract improved the sugar content in sugarcane (Chen et al. 2021; Deshmukh and Phonde 2013; Karthikeyan and Shanmugam 2017). It has been demonstrated that the presence of sucrose and monosaccharides enables plants to stimulate efficient defense mechanisms against fungal pathogens (Jeandet et al. 2022). Increased soluble sugars content in S + F plants during vegetative experiment suggests that S-extract might have mitigated S. rolfsii infection by enhancing the accumulation of soluble sugars. Increased levels of soluble sugars can provide a readily available carbon source for various plant defense responses, including the synthesis of antimicrobial compounds and strengthening of the cell wall (Morkunas and Ratajczak 2014; Trouvelot et al. 2014). In the reproductive stage, the total amino acid and total soluble sugar were decreased compared to vegetative stage. This is because production of osmolytes may be more pronounced during certain developmental stages when plants are actively responding to environmental stresses. It also reflects that during maturation plant energy is diverted towards reproductive processes rather than stress response mechanisms.

ROS are produced in plants during fungal infection and participate in plant defense mechanisms (Torres et al. 2006). It is essential to regulate the levels of ROS in plants to avoid damage caused by excessive ROS activity (Tripathy and Oelmüller 2012). In the peanut plants, ROS accumulation was found reduced in S + F treatment compared to F treatment during both stages. The fungus S. rolfsii is a necrotrophic organism that invades plants by killing plant tissue and thriving on the dead or dying tissue by releasing toxic compounds (Cho 2015; Rajasekhar et al. 2019; Shao et al. 2021). The reduced ROS generation observed in the S + F treated plants likely prevented cell death and inhibited the further invasion of S. rolfsii. The reduced accumulation of ROS in S + F as compared to F during both stages can be attributed to the increased activities of antioxidative enzymes.

During vegetative stage CAT, GPOX and PPO activities were increased significantly in S + F treatment compared to F treatment. However, enzyme activities were decreased in reproductive stage of S + F treatment compared to F treatment. The observed decrease in ROS generation with the S + F treatment group compared to F treatment indicates the role of these antioxidative enzymes in modulating ROS levels in plants during disease caused by S. rolfsii. Similarly, tomato plants showed significantly increased activity of defense-related proteins, such as PPO, GPOX and proteinase inhibitors after spraying with brown algae extracts, including Padina gymnospora and Sargassum liebmannii (Hernández-Herrera et al. 2014). Increased activities of certain defense-related enzymes, including peroxidase (PO), PPO, phenylalanine ammonia lyase (PAL), chitinase and β-1,3-glucanase, were also observed upon application of A. nodosum extract and SA application in plant. (Jayaraj et al. 2008). Similarly, commercial extract from A. nodosum was reported to enhance the activities of chitinase, glucanase, PPO, PAL, PO and lipoxygenase (LOX) enzymes in cucumber leaves (Jayaraman et al. 2011). Laminaran-containing A. nodosum preparations have also been reported to up-regulate the expression of defense-related genes, such as PAL, caffeic acid O-methyl transferase, LOX and SA, in tobacco plants (Klarzynski et al. 2000; Patier et al. 1995; Potin et al. 1999).

Conclusion

The present study provides significant insights into the beneficial effects of S-extract on plant growth and stress response. The findings demonstrate positive influence on growth parameters, pigment accumulation, membrane stability, non-enzymatic antioxidants, osmolytes, ROS regulation, and antioxidative enzyme activities in the peanut (Fig. 7). The damage caused by S. rolfsii infection was reduced with combinatorial stress S + F as was evident by different physio-biochemical parameters. The presence of active compounds in S-extract including fucans, laminarin and phytohormones positively regulate the stress responses of peanut, and enhance disease resistance. The findings will contribute to the development of effective strategies aimed at improving peanut productivity and resilience in the face of environmental challenges. However, further research through transcriptomics and metabolomics studies is warranted to gain a deeper understanding of the molecular and metabolic dynamics underlying the plant’s defence response. In future, the detailed study for the efficacy of S-extract should be attempted in large scale field condition for the evaluation of disease tolerance in different crops.

Fig. 7
figure 7

Graphical representation elucidating the effect of S-extract on peanut plants during stem rot disease infection by modulating physio-biochemical response. VS vegetative stage; RS reproductive stage; first and second sign in each stage denoted as 1st and 2nd spray, respectively. The upwards and downwards arrows depict the significantly higher or lower mean values in different comparison groups. Dash represents that mean values are not significantly different in the comparison group

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