Physiological and biochemical alterations in Vigna rdiate L. triggered by sesame derived elicitors as defense mechanism against Rhizoctonia and Macrophomina infestation

Improving agricultural products by the stimulation of plant growth and defense mechanisms by priming with plant extracts is needed to attain sustainability in agriculture. This study focused to consider the possible improvement in Vigna radiata L. seed germination rate, plant growth, and protection against the natural stress by increasing the defense mechanisms through the incorporation of Sesamum indicum phytochemical compounds with pre-sowing seed treatment technologies. The gas chromatography coupled with mass spectroscopy (GC–MS) analysis revealed that the methanol extract of S. indicum leaf extract contained eight major bioactive compounds, namely, 2-ethylacridine (8.24%), tert-butyl (5-isopropyl-2-methylphenoxy) dimethylsilane (13.25%), tris(tert-butyldimethylsilyloxy) arsane (10.66%), 1,1,1,3,5,5,5-heptamethyltrisiloxane (18.50%), acetamide, N-[4-(trimethylsilyl) phenyl (19.97%), 3,3-diisopropoxy-1,1,1,5,5,5-hexamethyltrisiloxane (6.78%), silicic acid, diethyl bis(trimethylsilyl) ester (17.71%) and cylotrisiloxane, hexamethyl-(4.89%). The V. radiata seeds were treated with sesame leaf extract seeds at concentrations 0, 10, 25, 50, and 100 mg/L. Sesame leaf extract at 50 and 100 mg/L concentrations was effective in increasing the germination percentage and the fresh and dry weights of roots and shoots. The increased peroxidase activity was noticed after treatment with S. indicum extract. In addition, disease percentage (< 60%) of both fungal pathogens (Rhizoctonia and Macrophomina) was significantly reduced in V. radiata plants treated with 100 mg/L of sesame leaf extract. These results revealed that physiochemical components present in S. indicum mature leaf extract significantly enhanced growth and defense mechanism in green gram plants.

Root and shoot length of V. radiata after treatment with S. indicum.The root and shoot length of leaf extract primed V. radiata seeds is represented in figure.After five days of germination, the result showed that sesame leaf extract significantly increased the root length and shoot length compared with the control seeds (Fig. 2).Seed priming treatment enhanced the seedling growth with increased effect from 50 mg/L and maximum at 100 mg/L leaf extract.On average, seed priming treatment effectively enhanced the shoot length Root to shoot ratio of V. radiata after treatment with S. indicum.The root to shoot ratio depends upon the accumulation of sesame leaf extract in seed treatments, which influences the stimulation of root and shoot growth.The seed treatment with increasing concentration of sesame leaf extract increased the root to shoot ratio from 0.0758 of 10 mg/L to 0.1678 of 100 mg/L, as against the control with a ratio of 0.06080 (F 4,20 = 21.67;P < 0.0001) (Fig. 3G).
Seed vigor index of V. radiata after treatment with S. indicum.respectively (F 4,20 = 18.07,P < 0.0001).The increase in the concentration of the leaf extract enhanced the growth parameters under consideration proportionately (Fig. 3H).
Peroxidase activity of V. radiata after treatment with S. indicum.The V. radiata treated leaves were assessed for alterations in peroxidase (POD) enzyme activity.The leaves exhibited increased POD activity when treated with 100 mg/L concentration of the sesame leaf extract (F 4,20 = 13.13;P < 0.0001).The POD activity in leaves was found to be 117.9min −1 g −1 FW when treated with 100 mg/L of the leaf extract.In contrast, the plants treated with 10 mg/l of the leaves extract exhibited POD activity of 79.27 min −1 gv −1 FW, which was higher than that of the control (76.24 min −1 g −1 FW).The POD activities at different concentrations of sesame leaf extract were significantly accelerated at higher concentration (Fig. 4).The increasing peroxidase activity was recorded from 24 to 144 h on control and leaf extract primed V. radiata plants at 0 h (F 4,20 = 1.06;P < 0.988-not significant) and 24 h (F 4,20 = 3.98; P < 0.006).However, the enzymatic responses were insignificant in all treatments except 100 mg/L.A stable and increased level of POD activity higher than the control was observed after 24 h.As against the control, the treated plants displayed a seven-fold increase in POD activity.

Discussion
Seed treatments with S. indicum extract increased germination percentage growth in V. radiata in a dose-dependent manner.Also, the stimulatory effects appeared more pronounced at 75 and 100 mg/L seed treatments.The plant extract acts as a bio-stimulant, accelerating crop productivity by enhancing germination, growth, and biomass and activating defense-related enzymes in plants 30 .In this study, sesame leaf extract was used as a biostimulant and as an elicitor to improve green gram production.The methanol extract of S. indicum L. leaves was screened by gas chromatography-mass spectrometry (GC-MS) analysis for qualitative phytochemical detection.The spectrum revealed the presence of eight major phytochemical compounds.In a similar study, 2-ethylacridine, silicic acid, and diethyl bis (trimethylsilyl) were identified by the mass spectrometric analysis of the Dicranopteris linearis stem extract, and the identified phytochemicals possessed significant antimicrobial and antioxidant potential 31 .The methanolic extract from the leaves of Centella asiatica contained the bioactive compound tert-Butyl (5-isopropyl-2-methylphenoxy) dimethylsilane that had antimicrobial properties 32 .The ethanolic extracts of Olea europaea isolates showed the presence of phenolic compounds tris (tert-butyldimethylsilyloxy) arsane, silicic acid, diethyl bis(trimethylsilyl) ester, and cyclotrisiloxane hexamethyl in the latex of the plants with www.nature.com/scientificreports/effective antimicrobial properties 33 .The bioactive compound 1,1,1,3,5,5,5-Heptamethyltrisiloxane was extracted from Allium atroviolaceum flowers that exhibited antibacterial activity against Bacillus subtilis and enhanced the photosynthetic capacity of the plants 34 .Ethyl acetate extract consisted 3,3-diisopropoxy-1,1,1,5,5,5-hexamethyltrisiloxane, cyclotrisiloxane, and hexamethyl isolated from the bark of Trichilia gilgian.The phytochemicals were inhibitory against the Sitophilus zeamaïs and Rhyzopertha dominica at high doses of the extract (1 g/10 L −1 and 0.5 g/10 L −1 ).The fruit extract of Salvadora persica L. was found to possess 3,3-diisopropoxy-1,1,1,5,5,5hexamethyltrisiloxane and cyclotrisiloxane hexamethyl compound that had antimicrobial activity against Streptococcus mutans 35 .The leaves and bark extracts derived from Dillenia scabrella contained the phytochemicals cyclotrisiloxane hexamethyl, silicic acid, and diethyl bis (trimethylsilyl) ester, which displayed antioxidant and anti-diabetic activities 36 .Acetone crude extract of Turbinaria decurrens seaweeds comprised cyclotrisiloxane hexamethyl with potential antimicrobial, antioxidant, and antibacterial properties 37 .S. indicum is a valuable oil seed plant known to be relatively rich in bioactive compounds.The results showed that seed treatment with sesame leaf extract have significant impact on V. radiata germination and plant growth.The bioactive compounds present in the extract improve the agricultural yield by pretreating the seeds 38,39 .
The sesame leaf extract-treated seeds significantly enhanced the percentage of germination when compared with untreated seeds.V. radiata seeds treated with sesame leaf extract (100 mg/L) showed a considerable increase in the percentage of germination when compared with the lower concentrations and the control.In an independent study, phenolic compounds were extracted from grape seeds and spruce bark, which showed a significant stimulatory effect on the radicle length of Phaseouls vulgaris seeds 40 .The seed treatment with an oil-based solution of tyrosol (10 mg/L) extracted from Phomopsis sp.displayed improved germination percentage of soybean plant 41 .The extract obtained from the seaweed, Laurencia obtuse, induced maximum seed germination at a concentration of 20 g/L 42 .
The root and shoot length varied significantly when compared to that of the untreated seed.The sesame leaf extract treated seeds showed significant increase in root and shoot length (12.6 cm and 23.4 cm in 100 mg/L) than that of control (2.9 cm and 12.4 cm).However, the root and the shoot lengths were comparable when treated with lower concentrations of the leaf extract and in the control.The bioactive compounds act as stimulators and have an important role in enhancement of the growth and development of the plants 43 .Application of trehalose (Tre) and salicylic acid (SA) on drought stress sweet basil (Ocimum basilicum L.) plant showed enhanced plant growth by increasing the root and shoot length 44 .The spruce bark, Asclepias syriaca and grape seeds extracts stimulated radicle elongation in Phaseouls vulgaris than the control solution 40 .
The effect of various concentrations of sesame leaf extracts in seed treatment could be seen in the vigor index.The increased vigor index was noticed in seeds treated with a higher concentration of sesame leaf extracts 100 mg/L and followed by 50 mg/ L, 25 mg/L, 10 mg/L, and control, whereas the decreased vigor index was recorded in 10 mg/L and control.At the concentrations of 25 mg/L and 50 mg/L, the leaf extract of Azadirachta indica and bark of Boswellia dalzielii extract significantly enhanced the germination rate and vigor index of cotton seeds 45 .
The result reported that sesame leaf extract of 100 mg/L had increased significantly in the growth of root and shoot fresh (74 mg and 550 mg) and dry weights (6.98 mg and 47.40 mg) than that of control fresh (30 mg and 244 mg) and dry weights (2.8 mg and 30 mg).Similarly, seed-priming of carrots with 50 and 100 mg/L with alpha-tocopherol (Toc) showed a significant level of plant biomass (p ≤ 0.001and 0.01) accumulation when exposed to drought conditions 46 .The polyphenolic extracts facilitated the assimilation of the fresh biomass in Phaseouls vulgaris germinated seeds 40 .
Seed soaking with sesame leaf extract induced maximum increase in root shoot ratio while an increase in concentration was observed by 10, 25, 50, and 100 mg/L seed treatment.
The sesame leaf extract induced the peroxidase (POD) enzyme activity, which has a key role in stimulating the plants defense mechanism.POD activity increased significantly (p ≤ 0.001) in carrot cultivars under normal and drought conditions 46 .Seed treatment with methyl salicylate (MeSA) at 50 and 100 mg/L concentrations significantly increased POD activity in IR 20, IR 50, IR 64, ASD 16, ASD 19, and ADT 46 rice (Oryza sativa L) varieties 20 .Phenolic compounds and its derivatives have been used to accelerate the defense mechanism against the pathogen attacks 38 .

Conclusion
Plant based material is a key source of bioactive compounds.The result clearly focus on the potential of bioactive compounds derived from the leaf extract to improve crop yield.Thus, seed treatment with methanolic sesame leaf extract effectively increased all the growth parameters under consideration, and also accelerated POD activity in plant.Further, we conclude that use of S. indicum in seed treatment has excellent potential for increasing ideal emergence and biomass accumulation, thus produce promising effects on the growth of V. radiata.

Materials and methods
Collection and preparation of leaf sample.This study was carried out in July to October 2021and the location of the experiment had coordinates latitude of 8.946481° N and longitude of 77.289979° E. During the experimental period, the climatic conditions were 22-32 °C day/night temperature fluctuation, 12-13 h photoperiod and 81-83% relative humidity (RH) during the day.The leaves of S. indicum were collected from the field, www.nature.com/scientificreports/Tenkasi, Tamil Nadu, India.Fresh leaves were collected from the matured plant during the ripening phase.The leaves were cleaned and shade dried for a period of 10 days at room temperature 28 ± 3 °C.The dried leaves were then powdered using a mixer grinder.Cultivars mentioned above were used for research purpose only and it does not come under endangered species of wild flora and fauna as per IUCN.Essential methods and guidelines are adopted from the IUCN.
Preparation of methanol plant extracts of sesame leaves.The powdered leaves (100 g) were extracted using a magnetic stirrer with methanol 500 L −1 for 48 h until the solvent was extracted as a clear substance.The extracts were filtered using Whatman filter paper no. 1, and the solvents were evaporated using a water bath at 60 °C to yield the crude extract.The yield of fine slurry extracts was 45.8% volume by the weight of the extract after evaporation of solvent and the weight of the plant powder.The plant extracts were stored in bottles for the further experiments 47 .The crude plant extracts (200 mg/ml) were further dissolved in 2 ml of 1% dimethyl sulfoxide (DMSO) to get standard stock solutions.The different concentrations were prepared by using the stock solutions (10, 25, 50, and 100 mg/L).

Phytochemical analysis.
The phytochemical analysis of the S. radiatum leaf extract was carried out to find the phytochemicals like alkaloids, saponins, flavonoids, terpenoids, tannins, steroids, quinones, and phenols by using the standard method.
Collection of seed and seed preparation.V. radiata seeds (green seeds) were primed with different concentrations of leaf extract.The seeds were obtained from the nearby agricultural store in Nannagaram, Courtallam, Tenkasi, Tamil Nadu, India with latitude 8.9449° N and longitude 77.2930° E. Seeds were selected with uniform size.To evaluate the effects of sesame leaf extract on seed germination, a petri plate experiment, and seedling vigor evaluation were conducted in the laboratory.For the experiment purpose, the seeds were surface sterilized using 2% mercuric chloride for 2 min and the seeds were rinsed repeatedly with distilled water.Seeds were soaked in prepared concentrations of plant extract as a test solution and water as a control for 5 h.

Seed germination test and seedling measurement of V. radiata after treatment with S. indicum.
A total of 50 seeds were placed on the petri dish for germination.The seeds were treated with different concentrations of plant extract, and the control seeds were treated with distilled water.After a period of 5 h, each petri dish was covered with a lid, and observed for seedling emergence and germination at regular intervals for five days 48 .For the statistical analysis, the percentage of germination was recorded once the formation of radicle length had reached 2 mm 49 .
Seedling vigour of V. radiata after treatment with S. indicum.The treated and control seeds were sown at a depth of 3 cm in plastic bags of 8 inches in length and 6 inches in width, containing soil with a mixture of two parts of silt loam soil and one part of sand.The plants were kept in greenhouse conditions and watered regularly.The seedling growth was recorded on the 11th day from the start of the experiment.The root and shoot lengths were recorded in centimeters using a ruler.Each treatment contained five replications, and for each replication, five seeds were used.
Seedling length and weight of V. radiata after treatment with S. indicum.The root and shoot lengths were measured by selecting five seeds randomly for each treatment.The shoot length was measured by a cut from the base of the hypocotyl, and root length was measured from the tip of the primary root to the base of the hypocotyl.The fresh and dry weights were weighed using an electric balance and expressed in grams for each replicate 27 .The shoot and root weights were examined using monopan balance.The seedling vigor percentage index was calculated by multiplying the germination rate with the seedling length.The root-to-shoot ratio was calculated by the dry weight of the root divided by the dry weight of the shoot for each treatment.
POD activity of V. radiata after treatment with S. indicum.S. radiatum treated V. radiata and control seeds were assayed alterations in the peroxidase (POD) activity.The collected leaves were homogenized and centrifuged at 10,000 rpm for 20 min at 4 °C.The supernatant was collected and used the determination of the enzyme activity.The leaves of V. radiata were extracted using the guaiacol method 50 .The mixture consisted of 12 mM of 0.01% H 2 O 2 , 50 mM of 0.1 M phosphate buffer (pH 5.8), and 7.2 mM of guaiacol containing a volume of 3L.The absorbance was measured continuously at 470 nm by a spectrophotometer at 25 °C.The phosphate buffer was used as a control.The results were determined by measuring the absorbance of tetra-guaiacol at 470 nm for 3 min at 30 s intervals.POD activity was expressed as unit per minute per gram fresh weight (min −1 g −1 Fresh Weight).
Determination of root infecting fungi.Different dosages (10, 25, 50, and 100 ug/mL) of the sesame extract were used for soil amendment.Surface sterilized seeds were sown in 8 cm diameter plastic pots, each containing 300 g soil, and watered regularly to maintain sufficient moisture required for the growth of V. radiata plants.The pots were kept in a screen house in a randomized complete block design with three replicates per treatment; seeds treated with sterile distilled water served as control.To determine the incidence of root rot fungi M. phaseolina (Gen Bank Accession No. MG434668) and R. solani (Gen Bank Accession No. KF312465), 1 cm long root pieces were washed in running tap water and surface sterilized with 1% Ca(OCl) 2 .The pieces were then transferred to PDA plates supplemented with penicillin (200 mg/L) and streptomycin (200 mg/L).The petri

Figure 1 .
Figure1.Days until 20%, 40%, 60% and 80% emergence of V. radiata under greenhouse condition after seed treatment with S. indicum extract.Mean (± SEM) followed by the same letter in an individual experiment indicates no significant difference (P < 0.05) in a Tukey's test (Treatment concentration in mg/L).

Figure 2 .
Figure 2. External morphology of V. radiata 11 days post treatment with S. indicum extract (A Control; B 10 mg/L, C 25 mg/L, D 50 mg/L, and E 100 mg/L).

1 Figure 3 .
Figure 3. Shoot and root length in cm (A, B), shoot fresh and dry weight in mg (C, D), root fresh and dry weight in mg (E, F), root to shoot ratio and seed vigour (G, H) of V. radiata 11 days post treatment with S. indicum extract.Mean (± SEM) followed by the same letter in an individual experiment indicates no significant difference (P < 0.05) in a Tukey's test (Treatment concentration in mg/L).

Figure 4 .Figure 5 .
Figure 4. Effects of S. indicum extract treatments on POD activity in V. radiata plant.Mean standard error ( ±) followed by the same letter within bar indicates no significant difference (P ≤ 0.05) in a Tukey test.

Figure 6 .
Figure 6.Incidence of root rot fungi M. phaseolina on mung bean post treatment with S. indicum.Mean standard error ( ±) followed by the same letter within bar indicates no significant difference (P ≤ 0.05) in a Tukey test.

Peak no RT Peak area % Compound name Molecular formula Molecular weight Chemical structure
The effect of different concentrations of sesame leaf extract on germination percentage, root, and shoot length was determined by the evaluation of seed vigor index.The highest vigor index of 3172 100 mg/L was observed when treated with 100 mg/ mL, followed by 2334, 1503, 1034.4,and 798 for the concentrations 50 mg/L, 25 mg/L, 10 mg/L, and control,

Table 1 .
Chemical composition of leaf extract of S. indicum.