Sustainable Agronomic Strategies for Enhancing the Yield and Nutritional Quality of Wild Tomato, Solanum Lycopersicum (l) Var Cerasiforme Mill

Abstract

Urbanization and global climate change have constrained plant development and yield. Utilization of wild gene pool, together with the application of sustainable and eco-friendly agronomic crop improvement strategies, is being focused on to tackle mounting food insecurity issues. In this aspect, the green seaweed, Ulva flexuosa, was assessed for plant biostimulant potential on cherry tomato, in terms of seed priming effects, nutrition and yield. SEM-EDX analysis of U. flexuosa presented the occurrence of cell wall elements (O, Na, Mg, S, Cl, K and Ca). The phytochemical analyses of liquid seaweed extract (EF-LSE) revealed the presence of carbohydrates, protein, phenols, flavonoids, saponins, tannins and coumarins. The EF-LSEs were found to stimulate seed germination in a dose-dependent manner, recording higher seed germination, and biomass and growth parameters. The seedlings of treated seeds altered the biochemical profile of the fruit, in terms of TSS (93%), phenol (92%), lycopene (12%) and ascorbic acid (86.8%). The EF-LSEs positively influenced fruit yield (97%). Henceforth, this investigation brings to light the plant biostimulant potential of the under-utilized seaweed source, U. flexuosa, to be useful as a bio fertilizer in agronomic fields for a cumulative enhancement of crop vigour as well as yields to meet the growing food demands.

1. Introduction

Agriculture is facing various crises that are worsening with time. Increasing food production to meet or feed the mounting population is a foremost challenge. This can be accomplished by further use of farm lands for an overall hike in food production or technically enhancing the yields from pre-existing lands by application of fertilizers or implementation of novel approaches; for instance, precision farming systems viz., cutting-edge irrigation arrangements, and ecologically accomplishable crop revolutions [1]. Crop diseases decrease yield, resulting in a prominent crisis to food security, creating a global malnutrition spree affecting nearly 815 million people [2]. Henceforward, natural fertilizers are well thought out as probable as well as safe alternatives to chemical fertilizers [3]. Additionally, the presence of several horticultural important traits in the wild gene pool makes them suitable as potential breeding candidates for crop improvement [4]. In this aspect, plant secondary metabolites are being emphasized for their disease regulator competences and combined in more than a few defense control programs [5].

The marine ecosystem serves as a rich source of bioactive compounds, such as sulfated polysaccharides, terpenoids, phenolics, lactones, sterol and fatty acids, possessing pharmacological and plant growth-stimulating properties [6]. Seaweeds form a key portion of these bioactive natural composites, with over 9000 species, known for their biostimulator potentials. Additionally, seaweed products as biostimulants that can enhance crop production are also being focused on. Biostimulants are materials supplementary to fertilizers, which endorse plant growth at lower concentrations [7]. In addition, seaweeds are extensively applied in the fields of agriculture and horticulture to improve quality and quantity, and the results are promising [8]. Innumerable seaweeds are being applied as liquid fertilizers to upsurge crop yields, as they are rich in macro-nutrients, besides trace elements essential for the development and enrichment of plants. Commercial seaweed products are also being marketed successfully [9]. Besides being inexpensive, the seaweed extracts have surplus allelopathic chemicals that promote seed germination as well as emergence rates. Seaweed extracts are known to have a positive impact on prime stages of plant ontogenesis—starting from seed germination to seedling growth [10]. Furthermore, seaweeds are reported with higher amounts of growth hormones, attributed to their plant biostimulant activities [11].

Seed germination is a decisive procedure in plant growth, and the enrichment of germination potentialities of a seed can eventually enable a surge in crop yields, and is dependent on numerous chemical factors (soil moisture salinity, metal, mineral composition) [12]. The emergence of seed is promoted by various methods for enhanced agricultural yields, like exposing them to biostimulants or growth promoting hormones by the process of seed priming. As the very first stage of plant growth, germination is defined as an outcrop of the radicle from the tissues enfolding the seed [13]. As germination rates may vary among species, the analyses of germination rates might be directly proportional to the growth rates and consequently, their yields [14,15].

Cherry tomato, Solanum lycopersicum (L.) var. cerasiforme Mill. is a widespread, table purpose tomato variety, bearing bright red color, and small fruits. It is a probable ancestor of a cultivated tomato variety with small fruits bright red in color, resembling a cherry and tasting excellent [16]. They are also favorable candidates in breeding programs for their genetic diversity, offering the selection of parental traits along with extensive geographic ranges. [17]. With the debarring effects of crop growth promoting chemicals that alter soil ecology and have hazardous environmental and health impacts, researchers are concentrating on the allegation of naturally benign substitutes to increase yields, while offering effective crop protection. The favorable agronomic traits of cherry tomato (an intermediary genetic admixture flanked by wild currant-type tomatoes and domesticated garden tomatoes), such as higher nutrient composition, offer plans for balanced utilization to unravel indigenous complications encompassing crop adaptation to climatic variations or, to endorse functional food consumption [18].

Seed priming of native seed species can evoke ecological restoration, stimulating the expression of dormant genes responsible for the expression of favorable agronomic traits. Since the sources of natural varieties are collected from the wild and their sources are limited, there is a persistent requirement for novel methods for seed-based restoration technologies. In this aspect, seed priming could be a strategy for sustainable seedling establishment, plant growth, and restoration of native seed. Hence, this research intended to discover the bio stimulator potentials of liquid seaweed extract of green alga, U. flexuosa, along with screening of their phytochemical and elemental composition on cherry tomato.

2. Materials and Methods

2.1. Seed Collection and Preparation

Cherry tomato seeds, variety ATL-01-19, were purchased from Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Seeds of undeviating dimensions and hue were carefully chosen for the study, surface sterilized with 0.1% mercuric chloride, washed thrice in sterile distilled water. The experiment was carried out at Sri Paramakalyani Centre for Excellence in Environmental Science, Manonmaniam Sundaranar University, Alwarkurichi, from July to August, 2018.

2.2. Seaweed Collection

The seaweed, Ulva flexuosa (Ulvaceae), was collected from the rocks in coastal areas at Colachel beach (Figure 1), Kanyakumari (8°14′5168″ N and 77°14′35.209″ E) during a low tide period (August 2018). It was washed in seawater several times to remove impurities, sand particles, and epiphytes, and brought to the research laboratory in taped up polythene bags. The seaweed was washed thoroughly in tap water several times, wearied and spread on blotting paper to remove excess water, and shade dried for 2 to 3 hours.

2.3. Preparation of Starter Fertilizer Solution (SFS)

Ammonium dihydrogen phosphate (Merck) was used as a starter fertilizer solution by mixing 1 mg of NH₄H₂PO₄ in sterile distilled water (10 mL), designated as positive control.

2.4. Preparations of Liquid Seaweed Extract (EF-LSE)

The washed seaweed was then cut into minor fragments, boiled in distilled water (100 gms/1 L) for one hour in an autoclave (121 °C, 15 psi of pressure), and filtered through a cheese cloth (double layered), yielding 890 ml of LSE. The LSE was stored at 4 °C in a refrigerator until further use. The test concentrations of EF-LSE were prepared by diluting the extract with distilled water (20%, 40%, 60%, 80% and 100%).

2.5. EF-LSE Analysis

2.5.1. Physicochemical Analysis

Physicochemical structures of the EF-LSEs—pH, electrical conductivity, and color appearance—was determined. pH was determined using a pH meter (ELICO LI 120, Hyderabad, India), whereas conductivity was done with the help of a conductivity meter (Microprocessor EC Meter 1615, Parwanoo, Himachal Pradesh, India) and expressed in ds/m. The colour of the EF-LSE was visually observed and noted.

2.5.2. Elemental Composition of EF-LSE Using X-ray–Energy Dispersive Spectroscopic (ED) Analysis

The elemental composition of U. flexuosa was performed using EDAX (BRUKER) to elucidate the components present in the seaweed cell wall.

2.5.3. Phytochemical and Biochemical Screening

Phytochemical [19] and biochemical screening of U. flexuosa was performed to analyse the quantitative amounts of phenol [20], chlorophyll [21] and protein [22] by standard procedures.

2.6. Preparation of Seeds

Tomato seeds, tomato wild relative, Solanum lycopersicum (L.) var. cerasiforme Mill, were used for all the tests. Tomato seeds, without any visible signs of infection, of uniform size, shape and colour, were carefully chosen, surface sterilized using 0.1% mercuric chloride, before and after rinsing in sterile distilled water. The seeds were used for further analysis.

To investigate the possible effects of UF-LSEs on tomato plant’s vegetative growth and yield, small pot field experiments containing sterilized soil were conducted by sowing the primed seeds in a tray. The seedlings (2-3 true leaf stage) were transplanted into autoclaved pot mixture (red soil: cow dung: vermiculate at 2:1:1, w/w/w) in the surface sterilized (1% mercuric chloride) pots (15 cm diameter, 750 mL volume) at 1 seedling/pot. The pots were labeled based on the treatments. Vegetative growth parameters were analyzed by sampling from seedlings selected from randomized block designs.

2.7. Biostimulant Assays

2.7.1. Seed Bioassay

Seed Germination Test

Seed germination assay was conducted using five surface sterilised seeds per assay, replicated five times. The seeds were exposed to EF-LSE extracts (10 mL), SFS in sealed and labelled conical flasks and kept in a shaker for 12 hours. Seeds in 10 mL sterile distilled water were used as control. The seeds were then removed and spread on a filter paper to blot out the solutions at room temperature for 24 hours. The treated seeds were placed in pre-labelled sterile petri dishes (9 cm) over filter paper (Whatman No. 5) that was moistened (sterile distilled water), instantaneously taped up with parafilm (Merck) to prevent moisture loss, and incubated (25 ± 2 °C/alternative 16 h light-8 h dark). The plates were checked for radicle protrusion (>2 mm) on a daily basis (hint of germination). Ten seeds were tested for each concentration of EF-LSE.

Germination was recorded every day by counting the emerging hypocotyls. The mean germination time (MGT) was premeditated [23] by counts made on the time taken for 1%, 10%, 25%, 50%, 75% and 100% of the seeds to germinate and expressed as days.

(1)$\mathrm{MGT}=\frac{\Sigma \left(n\mathrm{T}\right)}{\Sigma n}$

where,

• n = number of newly germinated seeds at time T (25 °C)

• T = hours from the beginning of the germination test

• Σ n = final germination

• *100% will refer to the total number of seeds germinated after exposure to the highest EF-LSE concentration.

The germination percentage (GP) was calculated using the following formula:

(2)$\mathrm{GP} = \frac{\mathrm{Number} \mathrm{of} \mathrm{seeds} \mathrm{germinated}}{\mathrm{Total} \mathrm{number} \mathrm{of} \mathrm{seeds}} \times 100$

Seed Germination Energy (GE) was calculated according to the following formula:

(3)$\mathrm{GE} = \frac{\mathrm{Number} \mathrm{of} \mathrm{germinating} \mathrm{seeds}}{\mathrm{No}. \mathrm{of} \mathrm{total} \mathrm{seeds} \mathrm{per} \mathrm{test} \mathrm{post} \mathrm{germination} \mathrm{for} 3 \mathrm{days}}\times 100$

Seedling vigour index (SVI) SVI was calculated [24] by the following formula:

(4)$\mathrm{SVI}=\mathrm{Seedling} \mathrm{length} \left(\mathrm{cm}\right)\times \mathrm{germination} \%$

Seed Imbibition

The biomass (wet and dry weight, mg) of the seeds primed in EF-LSEs and SFS solutions for 24 hours were determined with the help of an electronic balance after oven-drying at 40 °C for two days. Seed imbibition was determined by measuring the weight of seeds (100 seeds/treatment) before and during priming with SFS and EF-LSEs at 6, 12, 24, 36 and 48 hours and plotting the water imbibition curve by determining the seed moisture content (MC) and through means of which, the seed imbibition time was calculated [25]. Seeds primed in distilled water served as control. The MC was calculated by the formula:

(5)$\mathrm{Moisture} \mathrm{content} = \frac{\mathrm{Fresh} \mathrm{weight}-\mathrm{Dry} \mathrm{weight}}{\mathrm{Dry} \mathrm{weight}} \times 100$

2.7.2. Growth Parameter Assay

Growth parameters such as lengths of total plant, plumule and radicle length (cm) and root-shoot lengths and ratio were measured with a Vernier calliper post 5 days and 20 days for petri plate and glasshouse tests, respectively.

2.7.3. Fruit Yield and Quality Parameter assay

The seedlings derived from primed seeds were cultured as per recommended standard procedures [26] were selected on the basis of randomised block design, performing yield and quality parameter assays, repeating each test five times. The effect of EF-LSE on the yield of cherry tomato plants was estimated by fruit weight. The quality parameters of treated cherry tomato plants were calculated in terms of total soluble solids (TSS) [27], ascorbic acid [28], lycopene [29] and phenol [20] contents.

2.8. Statistical Analysis

All the tests were repeated five times. The effect of EF-LSE on seeds was determined by analysis of variance, one-way (ANOVA), and the treatment means were compared by Tukey-family error test (p < 0.05) by using Minitab^®17 software package (LEAD Technologies Inc., Charlotte, NC, USA).

3. Results

3.1. Seaweed Collection and Identification

The seaweeds collected from Colachel beach (Figure 1) were subjected to microscopical (Nikon Phase Contrast, Japan) and macroscopical analyses and confirmed to be Ulva flexuosa Wulfen (Ulvaceae), based on morphological characteristics and organoleptic features (Table 1). The seaweed belonging to the green alga, phylum Chlorophyta, is tubular and branched (Figure 2). The cells were observed to be arranged in transverse rows.

3.2. EF-LSE Analyses

3.2.1. Physicochemical Screening

The EF-LSEs appeared as pale greenish yellow in colour. The pH of the EF-LSEs of doses 20%, 40%, 60%, 80% and 100% was measured and found to be 7.160, 7.240, 7.34, 7.4 and 7.58, respectively. The pH of control was 7 and that of SFS was 4.2. The electrical conductivity (EC) of the respective EF-LSE doses was recorded as 0.96 ds m⁻¹, 1.01 ds m⁻¹, 1.08 ds m⁻¹, 1.54 ds m⁻¹ and 2.8 ds m⁻¹, respectively (Table 2).

3.2.2. Elemental Composition of EF-LSE Using X-ray–EDS Analysis

The elemental composition of the seaweed elucidated via EDX analysis (Figure 3) revealed the presence of seven compounds on seaweed cell surface—oxygen, Na, Mg, S, Cl, K and Ca. Oxygen was present in higher quantities (56.25%), followed by chlorine (13.4%), sulphur (7.79), and potassium (7.52%). Magnesium (5.3%), calcium (4.89%) and sodium (4.84%) were also recorded.

3.2.3. Phytochemical Screening

The phytochemical analysis of EF-LSE revealed the presence of carbohydrates, protein, phenols, flavonoids, saponins, tannins and coumarins (Table 3).

3.2.4. Biochemical Screening

The seaweed examined qualitatively revealed the presence of 1 mg/g of phenol, 6.1% protein, and 0.9 mg/g of total chlorophyll contents.

3.3. Biostimulant Assays

The cherry tomato seeds purchased were sown in a greenhouse, according to standard horticultural methods (Figure 4).

3.3.1. Effect of EF-LSEs on Cherry Tomato Seeds

Germination of cherry tomato seeds was initiated on day 2 in seeds treated with 80% and 100% of EF-LSEs. The EF-LSEs were able to initiate germination of the seedlings in a dose-dependent manner (Figure 5). EF-LSE treated seeds emerged early when compared to the control (Figure 6). In addition, the EF-LSE treated seeds in doses 20%, 40%, 60%, 80% and 100% exhibited lower MGT (Figure 7) of 4, 3.4, 3, 2.6 and 2.2 days (F_6,28 = 3.23; p < 0.0001), respectively, compared with seeds treated with SFS, 4 days (F_6,28 = 3.23; p < 0.0001) as well as that of the control, 4.9 days (F_6,28 = 3.23; p < 0.0001).

The germination time course for control seeds was longer (Figure 6), taking 86.54, 93.2, 110.64, 116.4 and 125.80 hours (F_4,20 = 30.33; p < 0.0001) for 1%, 10%, 25%, 50%, 75% and 100% of seeds to germinate. Seeds treated with SFS exhibited a time course almost similar to that of control, 85.54, 91.8, 109.34, 114.51 and 125.37 hours (F_4,20 = 30.33; p < 0.0001). The germination time course decreased with increase in EF-LSE concentrations. The time taken for emergence of 1% of seeds decreased from 76.4 (F_4,20 = 44.77; p < 0.0001) to 56.8 (F_4,20 = 36.81; p < 0.0001) and 50.8 (F_4,20 = 31.39; p1 < 0.000) hours in seeds treated with 20%, 40% and 60% EF – LSEs, respectively.

Similarly, the rate of emergence of seeds increased, resulting in a decreased germination time course, further to 44.6 (F_4,20 = 15.97; p < 0.0001) and 40 (F_4,20 = 13.66; p < 0.0001) hours in 80% and 100% EF-LSE treatments, respectively. As a series, the time taken for the emergence of 100% of seeds in treatment decreased from 163.2 (F_4,20 = 30.33; p < 0.0001) to 148.8 (F_4,20 = 38.16; p < 0.0001) hours in the respective control and SFS treated seeds. The EF-LSE treated seeds took 132 (F_4,20 = 44.77; p < 0.0001), 112.8 (F_4,20 = 36.81; p < 0.0001), 100.8 (F_4,20 = 31.39; p < 0.0001), 91.1 (F_4,20 = 15.97; p < 0.0001) and 76.8 (F_4,20 = 13.66; p < 0.0001) hours for 100% emergence at treatment concentrations of 20%, 40%, 60%, 80% and 100%, respectively.

The seeds in the petri dishes treated with distilled water exhibited 84.8% GP (F_6,28 = 6.4; p < 0.0001) at the end of seven days. SFS treated seeds showed a GP of 86.6% GP (F_6,28 = 6.4; p < 0.0001). Seeds treated with 20%, 40%, 60%, 80% and 100% EF-LSE, displayed 93%, 94%, 95%, 96% and 97% (F_6,28 = 6.4; p < 0.0001) GP, respectively (Figure 8). The number of days taken for all the EF-LSE treated seeds to germinate also decreased to 5.5, 4.7, 4.2, 3.6 and 3.2 days (F_4,20 = 13.66; p < 0.0001).

The germination energy (Figure 9) of the seeds treated with EF-LSEs were higher, in the range of 55.6, 85.4, 91.6, 94.6 and 97.8 (F_6,28 = 31.34; p < 0.0001) exposed to 20%, 40%, 60%, 80% and 100% EF–LSEs, respectively, which was very high in comparison with the control (10%) and that of SFS-treated (20%) (F_6,28 = 31.34; p < 0.0001) seeds.

The seedling vigour index of the control seeds and SFS treated seeds were 457.18 (F_6,28 = 32.86; p < 0.0001) and 743.75 (F_6,28 = 32.86; p < 0.0001), respectively. However, the SVI of seeds treated with EF-LSEs (Table 4) of dosages 20%, 40%, 60%, 80% and 100% were enhanced to 1003.59, 1047.04, 1060.24, 1236.96 and 1281.31 (F_6,28 = 32.86; p < 0.0001), respectively (Table 4).

The biomass of the cherry tomato seeds was determined after 48 hours of priming. Seeds that were not treated with any extracts unveiled respective dry and wet weights of 0.013 mg (F_6,28 = 32.08; p < 0.0001) and 0.113 mg (F_6,28 = 19.23; p < 0.0001). The dry weights of the tomato plants treated with SFS, EF-LSE extracts – 20%, 40%, 60%, 80% and 100% were found to be 0.0218 mg, 0.0262 mg, 0.0296 mg, 0.0316 mg, 0.042 mg and 0.0528 mg (F_6,28 = 32.08; p < 0.0001) respectively. The wet weights of the tomato plants treated with SFS, EF-LSE extracts—20%, 40%, 60%, 80% and 100% were found to be 0.1396 mg, 0.1434 mg, 0.144 mg, 0.152 mg, 0.2 mg and 0.308 mg (F_6,28 = 19.23; p < 0.0001), respectively (Table 4).

The moisture content of the dry seeds was 7.8%, which increased after six hours in all SFS and EF-LSE treatments. The moisture content of control seeds increased to 8%, 9%, 9.4%, 9.7% and 9.9% at 6, 12, 24, 36 and 48 hours. A comparatively higher imbibition occurred in EF-LSE treated seeds, with a minimum imbibition of 8.5% and a maximum of 9.5 after six hours, in 20% and 100% primed seeds (Figure 10).

3.3.2. Effect of EF-LSEs on Growth Parameters of Cherry Tomato

The effect of EF-LSEs on the growth of tomato seedlings tested exhibited significant differences in total seedling length (Figure 11), radicle, and plumule length (Figure 12).

The height of the tomato seedlings at the end of five days was 5.4 cm (F_6,28 = 30.98; p < 0.005), with radicle and plumule lengths of 4 cm (F_6,28 = 42.16; p < 0.005) and 3 cm (F_6,28 = 42.94; p < 0.005), respectively (Figure 12), with 1.26 radicle: plumule ratio (F_5,24 = 32.20, p < 0.005) (Figure 13).

The SFS was able to induce the seedling length to 8.6 cm (F_6,28 = 30.98; p < 0.005) with a corresponding radicle and plumule lengths, ratio of 4.2 (F_6,28 = 42.16; p < 0.005) and 3.72 cm (F_6,28 = 42.94; p < 0.005), 1.36 (F_5,24 = 32.20, p < 0.005), respectively. The EF-LSEs had a positive effect in stimulating the seedling height and their respective radicle and plumule lengths to 10.8 cm (F_6,28 = 30.98; p < 0.005), 5.12 cm (F_6,28 = 42.16; p < 0.005) and 4.12 cm (F_6,28 = 42.94; p < 0.005), respectively, at 20% concentration, exhibiting radicle: plumule ratio of 1.36 (F_5,24 = 32.20, p < 0.005).

The EF-LSEs had a positive effect in stimulating seedling height and their respective radicle and plumule lengths to 10.8 cm (F_6,28 = 30.98; p < 0.005), 5.12 cm (F_6,28 = 42.16; p < 0.005) and 4.12 cm (F_6,28 = 42.94; p < 0.005), respectively, at 20% concentration, exhibiting radicle: plumule ratio of 1.36 cm (F_5,24 = 32.20, p < 0.005). Similarly, the 40% EF-LSE treated seeds exhibited respective seedling height, radicle and plumule lengths, radicle: plumule ratio of 12.07 cm (F_{6, 28} = 30.98; p < 0.005), 5.72 cm (F_6,28 = 42.16; p < 0.005) and 5 cm (F_6,28 = 42.94; p < 0.005) and 1.59 (F_5,24 = 32.20, p < 0.005). Likewise 60% and 80% EF-LSE treated seeds were observed with seedling lengths of 13.09 cm and 13.84 cm (F_6,28 = 30.98; p < 0.005), with corresponding radicle and plumule lengths of 7.6 cm (F_6,28 = 42.16; p < 0.005) and 5.8 cm (F_6,28 = 42.94; p < 0.005) as well as 8.2 cm (F_6,28 = 42.16; p < 0.005) and 6.4 cm (F_6,28 = 42.94; p < 0.005) besides the radicle: plumule ratios of 1.76 and 1.9 (F_5,24 = 32.20, P < 0.005), respectively. The seeds treated with 100% EF-LSE exhibited the highest seedling length of 14.1 cm (F_6,28 = 30.98; p < 0.005) with radicle and plumule lengths of 8.2 cm (F_6,28 = 42.16; p < 0.005) and 6.4 cm (F_6,28 = 42.94; p < 0.005), respectively. They revealed a radicle: plumule ratio of 2.04 (F_5,24 = 32.20, p < 0.005).

The effect of EF-LSEs on the growth of tomato plants tested exhibited significant differences in total plant height (Figure 11), root, and shoot lengths (Figure 14).

The height of the tomato plant at the end of 20 days was 16 cm (F_6,28 = 40.45; p < 0.003), with root and shoot lengths of 6 cm (F_6,28 = 30.87; p < 0.005) and 10 cm (F_5,24 = 32.40; p < 0.005), respectively, with 0.68 root: shoot ratio (F_6,28 = 18.76; p < 0.000). The SFS was able to induce plant height to 19 cm (F_6,28 = 40.45; p < 0.003) with a corresponding root, shoot lengths and ratio of 7 cm (F_6,28 = 30.87; p < 0.005) and 12.2 cm (F_5,24 = 32.40; p < 0.005), and 0.82 (F_6,28 = 18.76; p < 0.00001), respectively. The EF-LSEs had a positive effect in stimulating tomato plant height and their respective root and shoot lengths to 23.4 cm (F_{6, 28} = 40.45; p < 0.003), 9.9 cm (F_6,28 = 30.87; p < 0.005) and 13.22 cm (F_5,24 = 32.40; p < 0.005), respectively, at 20% concentration, exhibiting root: shoot ratio 0.88 (F_6,28 = 18.76; p < 0.0001). Similarly, the 40% EF-LSE treated seeds exhibited respective plant height, root and shoot lengths, root: shoot of 24.6 cm (F_6,28 = 40.45; p < 0.003), 10.1 cm (F_6,28 = 30.87; p < 0.005) and 14.6 cm (F_5,24 = 32.40; p < 0.005) and 1 (F_6,28 = 18.76; p < 0.000). Likewise, 60% and 80% EF-LSE treated seeds were observed with total plant heights of 28.38 cm and 31cm (F_6,28 = 40.45; p < 0.003), with corresponding root and shoot lengths of 12.38 cm (F_6,28 = 30.87; p < 0.005) and 15.8 cm (F_5,24 = 32.40; p < 0.005) as well as 13.82 cm (F_6,28 = 30.87; p < 0.005) and 16 cm (F_5,24 = 32.40; p < 0.005) besides the root: shoot ratios of 1.41 and 1.606 (F_6,28 = 18.76; p < 0.0001), respectively (Figure 13). The seeds treated with 100% EF-LSE exhibited the highest plant height of 32 cm (F_6,28 = 40.45; p < 0.003) with root and shoot lengths of 14.54 cm (F_6,28 = 30.87; p < 0.005) and 17.48 cm (F_5,24 = 32.40; p < 0.005), respectively. They revealed a root: shoot ratio of 1.776 (F_6,28 = 18.76; p < 0.0001).

3.3.3. Effect of EF-LSEs on Fruit Quality Parameters of Cherry Tomato

The treated plants displayed a yield of 2.617 kg/plant compared with that of the control: 1.07 kg/plant (F_1,8 = 40.92; p < 0.0001). Further, the quality parameters analyses revealed the presence of increased amounts of 6.54 (Brix) TSS, 13.2 mg/100 g FM ascorbic acid, 88.45 µg g⁻¹ FM lycopene and 7.26 mg g⁻¹ DM phenol contents, compared with that of the control, which recorded 5.2 (Brix) TSS (F_1,8 = 57.1; p < 0.002), 10.48 mg/100 g FM ascorbic acid (F_1,8 = 53.52; p < 0.0001), 55.206 µg g⁻¹ FM lycopene (F_1,8 = 61; p < 0.003) and 5.72 mg g⁻¹ DM phenol (F_1,8 = 47. 8; p < 0.0001) contents (Table 5).

4. Discussion

The indiscriminate application of fertilizers has not only intoxicated the environment, but also lost their efficiency. Alternative naturally benign bases of fertilizers, sourced from biological sources such as plants, animals and micro-organisms, have paved the way for the practice of “organic farming”. Many eco-friendly bioactive compounds from seaweeds have been widely used in the agricultural field as plant growth promoters. Seaweeds are reported for their copious amounts of novel as well as assorted range of marine secondary metabolites [30]. Global population growth has seen leaps and bounds in the recent years, posing food insecurity [31]. With the foremost necessity of augmentation of crop production, farmers are in stress to improve yields of agriculturally important crops. As an imperative crop, the germination capability of tomato seed is valued to be around 70%. Seed emergence is mainly prejudiced by the equipoise, flanked by the growth skills of the embryo, in addition to the mechanical resistance of the endosperm, which should be debilitated for germination [32]. Seaweeds are being sought out as potential enhancers of crop growth and yield and are replacing chemical fertilizers owing to higher efficiencies, broader action range, eco-friendly nature, and cost-effective feature. Seaweeds, reported with outstanding plant growth promoting potentials, increased plant height, root as well as shoot lengths, consequently, are designated as plant growth biostimulants, as reviewed by Khan et al. [7] and Craigie [33] As a crucial and initial plant growth activity, the evaluation of a seed’s germination and associated parameters can help in determining the rate of a crop success, in terms of yield and economy [34]. As contemplation, the current investigation was performed to determine the plant growth stimulant activities of green seaweed U. flexuosa (Chlorophyceae).

As the germination of a seed counts on various physical aspects, together with nutrient composition [12], preliminary tests of the extracts were performed by analyzing the pH and electrical conductivity (EC) of the extracts. The nutrient content of a solution, in terms of salts and electrolyte concentration can be determined by measuring their EC. The EF-LSE of U. flexuosa was found to possess a neutral pH and an optimum EC that indicates the presence of salts, for instance, boron, zinc, magnesium, calcium and other essential plant nutrients in a nutritive solution [35]. Higher rates of EC of nutrient or fertilizer solutions are proven with the stimulation of favourable agronomic traits, such as increase in nutritional quality, colour gradient and quality of tomato fruits [36,37]. However, solutions outside the optimum EC had an inhibitory effect on plant growth activities [38]. A nutrient solution within optimum EC was found optimal for the growth stimulation of lettuce in glasshouse conditions [39]. Henceforth, the EF-LSEs were designated as ideal to be tested for biostimulant potential by means of seed priming.

Additional experiments were carried out to determine the phytochemical as well as the elemental composition of the EF-LSEs. A preliminary phytochemical screening of the EF-LSE was done, which exposed the existence of more than a few compounds, such as carbohydrates, protein, phenols, flavonoids, saponins, tannins, and coumarins. Carbohydrates from different seaweeds were found to act as growth promoters of several crops such as tomato, soybean, duckweed and mung bean [40,41,42]. Proteins from seaweeds are recorded for their enhanced plant biostimulant activities in mung bean [43], and cherry tomato plants [44]. Proteins help plants to alleviate stress and increase their tolerance levels against abiotic stress like heat, cold, salt and even heavy metals [45].

Seaweeds are rich in phenolic compounds with varied bioactive properties [46,47]. Rajauria et al. [48] identified and characterized eight phenolic compounds from brown Irish seaweed Himanthalia elongate, which exhibited strong antioxidant activities. Chanthini et al. [6] correlated the levels of phenolic compound concentration with their antifungal potential. U. flexuosa had a considerate amount of phenols (1 mg/g of dry weight). Farasat et al. [49] detected higher phenolics as well as flavonoid levels from U. flexuosa and other edible green seaweeds. Besides, Ascophyllun nodosum extracts were able to increase the levels of phenols and flavonoids together, post application [50]. Saponins showcase a wide array of biological activities that play a pivotal role in plant growth as well as defense [51]. Coumarins also play a crucial part in plant development. These compounds have been proven with plant growth promotion capabilities alone and also in combination with phytohormones in faba bean [52]. Besides, the coumarin compounds were able to stimulate seed germination and seedling growth of wheat and sorghum seeds at optimum concentrations [53].

Tuhy et al. [54] testified that plant biomass surged by treatment with seaweed-derived micronutrients. The composition and functioning, together with the yield of all the plants, are reliant on their chlorophyll contents [55]. The chlorophyll content of U. flexuosa ranged up to 0.9 mg/g, which is relatively high among several other green seaweeds. This was also in agreement with the results published by Rathod [56]. The elemental composition performed revealed the presence of seven elements (O, Na, Mg, S, Cl, K and Ca) present on cell wall surface. The seaweed was 56.25% w/v of oxygen and along with high phenol content may be regarded as excellent candidates of antioxidizing agents [57]. Plant growth promoting elements such as sodium and potassium present in the cell wall of the seaweed makes them appropriate biofertilizers, besides a broad-spectrum of applications in the agricultural sector [58]. Several other mineral compounds such as chlorine, magnesium and calcium that are critical plant micronutrients are extant in the cell surface.

Tomato seeds treated with EF-LSEs displayed a positive response with respect to early germination, mean germination time, germination percentage, energy as well as better seed vigour index. Seed priming treatments achieved with quite a few plant derivatives such as plant hormones have been operational in the enhancement of seed germination of Angelica glauca, a threatened medicinal herb [59] as well as endive and chicory [60]. Seaweed extracts have been proven to show development enhancing properties on plant as well as seeds of various plants [61,62]. Also, priming seeds promoted early emergence of brinjal and tomato seeds compared with un-primed [63].

The seeds primed with EF-LSEs of U. flexuosa was analysed in different concentrations, comparing with the standard SFS solution and the control. The EF-LSE treated seeds displayed an increased germination percentage, exhibiting a lower mean germination time (MGT), taking only 2.2 days to emerge. Codium tomentosum, a green seaweed extract-treated aubergine seeds displayed lower MGT [64]. The potential of EF-LSEs to stimulate seed germination was reported long back in ornamental plants [65], green Chilies and Turnip [66]. Kavipriya et al. [67] also reported that priming of green gram seed with different seaweed extracts such as Ulva lactuca and Caulerpa scalpelliformis induced faster seed germination. Rapid seed emergence was recorded by priming the red gram seeds with the extracts of Sargassum myriocystum [68]. Furthermore, the positive effects of seaweed on the germination of green [69] and black gram [70] were also noted.

The comparative increase in seed emergence is correlated with seed eminence that is appeared to be augmented by the treatment of EF-LSEs. In addition, Amabika and Sujatha [68] proved that seed quality can be assessed by determining their seedling vigour index. Higher SVI of EF-LSE treated seeds implies an upliftment in seed quality. This was also evident from the results of other parameters of EF-LSE treated seeds, exhibiting higher seedling-plant height, radicle-root, as well as plumule-shoot lengths and dry-wet weight, in comparison with the control. Similar results of increased SVI from seed priming with EF-LSEs of U. lactuca, U. reticulata, Padina pavonica, S. johnstonii were correlated with that of increased seed germination and growth rates of brinjal and tomato, along with chilli [63].

As the primary developmental plant growth phase, the radicle and plumule are of prominent importance to determine the foundation of a plant. Seeds with an eminent radicle and plumule grow hastily, besides having an amplified competence [71]. Longer radicle lengths are also indicators of greater plant establishment efficiency. Seeds that produce shortened radicle-plumule might have issues in nutrient conduction to the embryo [72]. The EF-LSE primed seeds exhibited higher lengths of radicle and plumule, which improved with higher concentrations. EF-LSEs of seaweeds, S. wightii and U. lactuca, were demonstrated in their latent seed germination, besides plant growth promotion capabilities [73,74]. Similar increase of radicle-plumule lengths of tomato seeds was observed with the EF-LSEs of C. sertularioides and S. liebmannii [75].

The growth enhancement displayed by the EF-LSE primed seeds is owed to the occurrence of essential plant macro- and micro-nutrients, in addition to phytohormones. Di Filippo-Herrera et al. [76] reported the biostimulant activity of red seaweeds (Acanthophora spicifera, Gelidium robustum, and Gracilaria parvispora) and brown seaweeds (Macrocystis pyrifera, Sargassum horridum and Ecklonia arborea) primed on seeds of mung bean, which is primarily due to their nutritional and hormonal constituents. The fact that seaweeds stimulate plant growth has been documented by various researchers worldwide [77,78].

Similarly, EF-LSEs were earmarked for plant growth promotion by amplifying the growth of root and shoot of the tomato seeds, thereby displaying increased heights, compared to the control. Plant height was higher compared with seeds treated with SFS. Seaweed-treated seeds exhibiting higher root shoot lengths and ratio were previously reported in many studies [74,79,80]. Unlike the plant assessment results, comparatively developed shoot lengths remained. This suggests that the distribution of photosynthates and other compounds that aid in plant growth has shifted towards the shoot or increased in the above ground area. This could pave the way for the increase of plant yield [76].

Seed weight is considered an ecologically crucial character in plant progress, by way of influencing the establishment capacity of a seedling, as well as plant height and yield. This was proved by Wuff [81], who found that the Desmodium paniculatum seeds of higher biomass produced a high yield. In addition, the EF-LSE treated seeds displayed increased wet and dry weights equated with control, besides improving with an EF-LSE concentration. This was in agreement with the results published by Karthikeyan and Shanmugam [82], who studied the effect of Kappaphycus alvarezii extract on peanuts. Vijayakumar et al. [83] also reported the increase in seed weight of Capsicum annum by treatment with Codium decorticatum EF-LSE. Increased seed weights produced plants with higher height, shoot mass, and yield [84]. Seed weight increase might be due to the production and accumulation of storage oils and several proteins that might promote plant growth abilities [85]. Seeds take in surrounding water, protoplasmic macromolecules by imbibition. The EF-LSEs are actively imbibed in the seed through capillary action, thus increasing biomass. Seeds with higher biomass have been reported to have better seedling growth. Sun et al. [86]. reported that maize seeds with increased biomass had high yields.

The biomass of EF-LSE treated seeds increased with time, thus revealing that the higher imbibing rate occurs at later hours of priming. This is attributed to the active process of enzymatic breakdown and mitosis essential for emergence. The increased imbibing rate of seeds exposed to EF-LSEs is primarily due to the abundance of plant essential nutrients and phytohormone composition of the EF-LSEs. U. lactuca and P. gymnospora primed tomato seeds exhibited higher imbibition rates during later stages of priming and hence were reported as more successful and better candidates for developing effective biostimulants to improve the growth of tomato plants (Hernández-Herrera [9]).

With respect to the biostimulant potentials of seaweed extract, the yield and quality parameters analyzed presented favorable results. The EF-LSE primed seeds observed an increase in yield, attributed to the presence of phyto-hormones and various plant growth promoting elements, as evident from the EDX analysis of the seaweed. Furthermore, the flavour of the cherry tomato fruit is directly proportional to the amount of TSS [87]. The nutritional value of these tomatoes is based on ascorbic acid content, which was also enhanced by the EF-LSE. Lycopene content, which is the reason for the ripening of fruits at an optimum stage, was also enhanced by EF-LSE treatment. Phenolic compounds, an indication of plant innate defense system, were augmented on treatment with seaweed extracts. These results are in agreement with that of Murtic et al. [88].

Since the cherry tomato gene pool, a wild relative of tomato, provides an opportunity to produce more nutritive and resilient tomato cultivar varieties, an attempt to preserve and conserve this inclusive gene pool in gene banks is critical [89]. Additionally, these wild relative crop types are nutritional repositories, whose cultivation shall be enhanced to meet the increasing global food security targets. The establishment of these species can be further stimulated by seed priming techniques. The application of seaweed extracts as a seed priming agent towards the improvement of agronomic traits of cherry tomato have resulted in positive responses to amplified seed germination capabilities, germination energy, and augmented seedling establishment. In addition, seed priming effects have induced a long-lasting priming effect by altering plant and fruit physiology in a favorable way, by increasing their biochemical constituents and fruit yield. Hence, this study proves the potential of U. flexuosa as a potential agricultural biostimulant that is both economic and effective.

Author Contributions

K.M.-P.C. and S.S.-N.; methodology, K.M.-P.C.; software, K.M.-P.C. and S.S.-N.; validation, V.S.-R., A.T., S.K., and R.S.; formal analysis, S.S.-N.; investigation, K.M.-P.C., resources, R.P., and A.T.; data curation, S.S.-N.; writing—original draft preparation, K.M.-P.C.; writing—review and editing, K.M.-P.C., and S.S.-N.; visualization, K.M.-P.C.,V.S.-R., N.S.S., H.S. and R.P.; supervision, S.S.-N.; project administration, R.S.; funding acquisition, S.S.-N.

Funding

This research was funded by the Department of Biotechnology, Ministry of Science and Technology grant number BT/IN/Indo-US/Foldscope//39/2015 dated 20 April 2018.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

LSE	Liquid seaweed extract
EF	Ulva flexuosa
SFS	Starter Fertilizer Solution
EDS	Energy Dispersive Spectroscopy
MGT	Mean Germination Time
SVI	Seedling Vigour Index
GE	Germination Energy
GP	Germination Percentage

Figures and Tables

Figure 1. Sample collection site of seaweed from Colachel beach, Kanyakumari, Tamil Nadu, India.

Figure 2. Macroscopic and microscopic images of U. flexuosa. ((A): Sample collection site, Colachel beach; (B): U. flexuosa; (C,D): Macroscopic image; Microscopic image of U. flexuosa-(E): 10×; (F): 40× and (G): 100×, showing blue coloured pigments on cell wall).

Figure 3. SEM-EDX—Energy Dispersive Spectrum of U. flexuosa showing the presence of various elements in their cell wall.

Figure 4. Cherry tomato plants.

Figure 5. Effect of LSEs on growth of tomato seedlings.

Figure 6. Effect of EF-LSEs on germination of tomato seeds—germination time course.

Figure 7. Effect of LSEs on germination of tomato seeds—mean germination time.

Figure 8. Effect of LSEs on germination percentage of cherry tomato seeds.

Figure 9. Effect of LSEs on germination energy of cherry tomato seeds.

Figure 10. Effect of EF-LSEs on seed imbibition capabilities of tomato seeds.

Figure 11. Effect of EF-LSEs on growth parameters of tomato seedling and plant height.

Figure 12. Effect of EF-LSEs on growth parameters of tomato seeds (plumule-radicle length).

Figure 13. Radicle-plumule as well as root-shoot ratio of tomato seedlings.

Figure 14. Effect of EF-LSE on root-shoot lengths of tomato plants.

Table 1

Organoleptic features of Ulva flexuosa.

Feature	U. flexuosa Appearance
Habitat	Highly cosmopolitan in shallow marine or brackish habitats
Shape	Long, filamentous hollow tube thallus
Size	20 cm
Colour	Light green
Odour	Foul smelling
Taste	Salty
Base	Divided
Blade	Expanding above the stalk, which ends in a rounded tip

Table 2

Physico-chemical properties of LSEs, pH and EC (dS m⁻¹).

Treatments	pH	EC (dS m−1)
C	7.02 ± 0.004 a	-
SFS	4.2 ± 0.89 b	0.756 ± 0.03 a
EF 20%	7.160 ± 0.30 c	1.0180± 0.10 b
EF 40%	7.240± 0.26 c	1.0800 ± 0.15 c
EF 60%	7.340 ± 0.21 cd	1.540 ± 0.36 c
EF 80%	7.400 ± 0.35 cd	2.880 ± 0.54 c
EF 100%	7.580 ± 0.37 d	3.700 ± 0.57 d

Columns denoted by a different letter are significantly different at p ≤ 0.05.

Table 3

Phytochemical composition of EF-LSE.

Phytochemicals	Inference
Alkaloids	-
Carbohydrates	+
Carboxylic acid	-
Coumarin	+
Flavonoids	+
Phenolics	+
Protein	+
Quinone	-
Saponin	+
Steroid	-
Tannin	+
Xanthoprotein	-

Table 4

Seedling vigour index and biomass (wet and dry weight) of tomato seeds treated with LSEs.

Treatments	Seedling Vigour Index (SVI)	Seed Weight (mg)
		Wet Weight	Dry Weight
C	457.18 ± 2.76 a	0.113 ± 0.007 a	0.013 ± 0.007 a
SFS	743.75 ± 3.02 b	0.1396 ± 0.003 b	0.0218 ± 0.003 b
Ef 20%	1003.59 ± 1.98 c	0.1434 ± 0.009 c	0.0262 ± 0.009 c
Ef 40%	1047.04 ± 3.01 d	0.144 ± 0.005 c	0.0296 ± 0.001 d
Ef 60%	1060.24 ± 2.45 e	0.152 ± 0.003 d	0.0316 ± 0.003 e
Ef 80%	1236.96 ± 1.43 f	0.2 ± 0.003 e	0.042 ± 0.003 f
Ef 100%	1281.31 ± 2.65 g	0.308 ± 0.002 f	0.0528 ± 0.002 g

Columns denoted by a different letter are significantly different at p ≤ 0.05.

Table 5

Yield, TSS, Ascorbic acid, lycopene and phenol contents of treated cherry tomato fruits.

Parameters	C	EF-LSE Treated
Yield (kg/plant)	1.07 ± 0.96 a	2.617± 1.02 b
TSS (Brix)	5.2 ± 1.1 a	6.54 ± 1.07 b
Ascorbic acid (mg/100g FM)	10.48 ± 1.8 a	13.2 ± 1.5 b
Lycopene (µg g−1 FM)	55.206 ± 3.9 a	88.45 ± 4.3 b
Phenol (mg g−1 DM)	5.72 ± 1.47 a	7.26 ± 1.15 b

Rows denoted by a different letter are significantly different at p ≤ 0.05.