Effect of Seaweed Liquid Fertilizer on Sorghum bicolor and Pennisetum glaucum

Millets are small-seeded grasses belonging to the family Poaceae that help in human and animal nourishment. They are mostly grown by using chemical fertilizers which are depleting soil fertility. Organic fertilizers are eco-friendly and help to enhance the growth and development of plants thereby maintaining soil health. Seaweed liquid fertilizer (SLF) are organic fertilizers that can be used to control the side effects caused by harmful chemical fertilizers. The present study was carried out to check the effects of seaweed liquid fertilizer on the growth and development of millets. Different concentrations of seaweed liquid fertilizer (0.25%, 0.50%, and 0.75%) were prepared from Sargassum cinerum, Ulva intestinalis, and Padina tetrastromatica, which were tested on Sorghum bicolor and Pennisetum glaucum. Growth parameters like root length, shoot height, flowering-fruiting, and biochemical analysis like proteins were analyzed by using seaweed liquid fertilizer and chemical fertilizer. Plants treated with 0.50% concentration of seaweed liquid fertilizer showed higher protein content. Total chlorophyll was found to be higher in treated plants than in control plants. Flowering and fruiting were observed earlier in seaweed liquid fertilizer treated plants. This concludes that seaweed extracts can be used as a promising alternative to chemical fertilizers, which plays a significant role in the holistic growth enhancement of plants.


Introduction
Around half of the population of India is employed in agriculture and has the second-largest cultivable land area in the world.After the onset of the 20 th century, India's population grew significantly and hence to feed the ever-increasing population, excessive use of chemical fertilizers and pesticides has done to expedite crop development.Crops grow rapidly as a result, but excessive use affects human health and degrades soil fertility. 1Consequently, there is LAD et al., Curr.Agri.Res., Vol.11(3) 915-927 (2023)   an alarming need to choose and utilize organic fertilizers, which are usually administered in the form of manures and bio-stimulants.Microorganisms, organic compost, animal dung, chicken droppings, and domestic sewage are just few examples of the sources that organic fertilizers comprise of.Moreover, macro and microscopic algae have been used to make organic fertilizers. 2 alga plays an important role in agriculture since it is utilized as a biofertilizer and as a soil stabilizer.It increases the uptake of phosphorus and nitrogen.They contain high levels of iodine, calcium, phosphorus, magnesium, iron, sodium, and potassium.Several major trace elements, including chromium, manganese, selenium, zinc and copper are additionally present. 3They contain a variety of bioactive components including various minerals, lipids, proteins, carbohydrates, amino acids, and phytohormones. 4Similar characteristics are witnessed in seaweeds, which are readily available in the coastal region.Seaweeds are used as biofertilizers and bio-stimulants.It investigated the effect of seaweed liquid fertilizer (SLF), specifically Sargassum wightii and Ulva lactuca, without the use of chemical fertilizers on seed germination, growth, and biochemical characteristics of the legume plant Glycine max (Soybean).This led to the use of seaweed liquid fertilizer at low concentrations to improve the germination of seeds and the growth of seedlings in farmed crops.Furthermore, increased pigment levels were seen in crops treated with low-concentration seaweed liquid fertilizer. 5Additionally, it has been noted that applying seaweed liquid fertilizers to rice crops can reduce NPK doses while promoting plant development on an agronomic level. 6Application of seaweed liquid fertilizer on Cucumis sativus (Cucumber) showed enhancement of chemical and physical characteristics associated with immunity, productivity, stress resistance and increased yield. 7Different combinations of seaweed liquid fertilizers are also tested with equal concentrations.This result suggests that with lower concentrations of seaweed liquid fertilizers, vegetative plant growth, and germination bioassays both considerably increased.Furthermore, the combination of seaweed liquid fertilizer (SLF) enhanced photosynthetic pigments, total soluble sugar, and total soluble protein and its agronomic growth compared with single seaweed liquid fertilizer treatment. 8Ascophyllum nodosum, Fucus spp., Sargassum spp., Laminaria spp., and Turbinaria spp.(Phaeophyceae) are utilized as agricultural biofertilizers and are among the most researched brown algae species.According to studies, seaweed protects plants from bacterial, viral, and fungal diseases. 9Seaweed like Sargassum cinereum, Padina tetrastromatica, Ulva intestinalis, Ulva rigida, etc. are primarily found in our nation; therefore, they are primarily used as biofertilizers and biostimulants for growth promotion as they are comparatively less expensive than the seaweeds that are exported from European Countries such as Ascophyllum nodusum, a brown alga.Due to its high price, it suffers from lower demand and popularity. 10Because of this, different concentrations of seaweed liquid fertilizer (0.25%, 0.50%, and 0.75%) were made from Ulva intestinalis, Padina tetrastromatica, their combination, and Sargassum cinerum were used as foliar and fertilizers to study how it improved agronomic growth on Sorghum bicolor and Pennisetum glaucum.The chosen crops are given NPK and Control treatments to compare their agronomic and biochemical characteristics.
The use of seaweeds also makes a significant contribution to economic growth because they are prepared using inexpensive, naturally occurring resources.The local villagers and fishermen can take advantage of the numerous employment opportunities it offers.They prepare seaweed liquid fertilizer as their prosperous start-up by gathering seaweeds.

Collection of Seaweed
Samples of seaweed were taken from Maharashtra Coastline.Ulva intestinalis and Padina tetrastomatica belonging to class Chlorophyceae and Phaeophyceae were collected from rocky areas in Bandra and Devgad, respectively.The seaweeds were properly cleaned to eliminate any salt or sand contents, and large contaminants such as sea snails and shrimp were separated.They were cleaned with regular tap water and dried under shade at room temperature.To preserve the biochemical properties, they were sealed up in collection bags and kept in an incubator at 37 °C.For the further study, samples were used in dried form.

Preparation of Seaweed Liquid Fertilizer
1gm of dried seaweed powder was dice in which 100 ml Distilled water was added.It was autoclaved for 1 hour at 15 psi.The autoclaved extract was cooled at room temperature and was filtered through a muslin cloth/ cheese cloth.The cooled extracts were centrifuged at 3000 rpm for 10 minutes at 30 °C.Supernatant was transferred into a clean conical flask, cotton capped, and placed in the refrigerator for further use.The prepared 1% Seaweed liquid fertilizer was made into different concentrations using sterile distilled water, as 0.25%, 0.50% and 0.75% respectively.As a result, various concentrations of each extract were obtained and used to treat seeds.Using this technique, two seaweeds extracted along with a combination of both were each converted into seaweed liquid fertilizers and was then diluted to test the impact of changing concentrations.

Procedure of Seed Preparation
The seeds were rinsed twice with distilled water, followed by a 70% alcohol and D/W wash.They were cleaned again with Mercuric chloride and rinsed 2-3 times with sterile distilled water to prevent infection.Seeds were first dried on filter paper before being added to various seaweed liquid fertilizer concentrations.For 48 hours, 10 seeds were soaked in each seaweed liquid fertilizer concentration.10 seeds were steeped in normal D/W as a control.The seed was inspected after 48 hours and then placed in distinct, labelled pots with various seaweed liquid fertilizer concentrations and control.

Maintenance of Seeds
After the seeds were planted in pots, they were kept in a greenhouse to maintain temperature and humidity.Seaweed liquid fertilizer was applied to the plants every week.25 ml of each concentration was applied directly into the soil as well as foliar spray.Control plants were also tested in the same way i.e., 25 ml of 1% NPK was added to the control treated plant every week.Rest of the days the plants were watered with tap water.Plants were thus maintained and observed daily.Plants were carefully grown until the fruiting was observed and matured fruits were further monitored in the study.

Agronomic Characters
After seeds being sown for three days, leaves were visible.After 7 days, observation of each shoot length was recorded.Triplicates of each set of seaweed liquid fertilizer concentrations, along with controls and water were formed, to study the effect of seaweed liquid fertilizer on the germination rate of the seeds.Germination rates were recorded for 24, 48, and 72 hours.Observations of panicle growth were made 90 days after planting.
Weight and length of the panicle as well as number of seeds it bore were also studied.

Biochemical Parameter Chlorophyll Estimation
Plants grown for 40 days were selected and chlorophyll content was estimated.Chlorophyll estimation was carried out by Aaron's method.Further, measurements of absorbance were made at 645, 652, and 663 nm using UV-Vis spectrophotometer / colorimeter.Formula-20.2(A675) + 8.02(A663) x V/100 x W

Procedure for Protein Estimation
Lowry's method of estimation of protein was carried out.Seeds collected from selected fully matured plants were extracted into aqueous extracts.1 g of seeds and 3 ml of D/W were ground in a mortar and pestle.For preparation of standard, 0.1% Bovine Serum Albumin was used.Further procedure was conducted as per Lowry's method (11) and analysed at 660 nm.

Bio-active Compounds Test Qualitative Phytoconstituents Analysis
A qualitative phytochemical analysis of crude powder was done to isolate various phytoconstituents.For these tests, the solvent with the highest extractive value i.e. aqueous extract was utilized.Among the phytoconstituents examined were alkaloids, flavonoids, phenols, saponins, tannins, and polysaccharides.

Alkaloids
Wagner's test 1 ml of seaweed liquid fertilizer extract + 5-6 drops of Wagner's reagent.(Formation of brown/reddish precipitate showed the presence of alkaloids.)Mayer's Test 1 ml of seaweed liquid fertilizer extract + 5-6 drops of Mayer's reagent.(Formation of creamish yellow precipitate indicated the presence of alkaloids).

Flavonoids (Alkaline Reagent Test)
Seaweed liquid fertilizer Extract + a few drops of NaOH solution.(Intense yellow colour is formed, which becomes colourless in addition to diluting HCl, confirming the presence of flavonoids).
Tannins (Using Lead Acetate) 1 ml of the seaweed liquid fertilizer extract + 1 ml of lead acetate.(Formation of white precipitate indicated the presence of Tannins).

Saponins (Foam Test)
2 ml of seaweed liquid fertilizer extract + 2 ml of D/W were shaken well.(Formation of persistent foam indicated the presence of saponins).

Phenols (Using lead acetate)
1 ml of seaweed liquid fertilizer extract + 1 ml of lead acetate.(Formation of white precipitate indicated the presence of Phenols).

Elemental Analysis using the ICP-AES Method
Prepared seaweed extracts were tested for presence of heavy metals as well as micro and macro elements.It was analysed using Induced coupled plasma atomic emission spectroscopy (ICP-AES) method from SAIF lab -IIT Bombay, Mumbai.

Shelf-life of seaweed liquid Fertilizer
It has been noted that seaweed liquid fertilizer becomes contaminated with bacteria and fungi when exposed to the environment.Therefore, keeping it in the refrigerator is necessary.Using natural preservatives like clove oil, the storage of seaweed liquid fertilizers at room temperature was also put to the test.To prevent contamination and extend the shelf life of seaweed liquid fertilizer, clove oil was employed as a preservative.

Germination Rate
Rates of germination were observed for 24 hrs, 48 hrs and 72 hrs.Tables 1

Discussion
The use of seaweed as fertilizers have been studied throughout the last few decades.Seaweed liquid fertilizer (SLF) is utilized as a bio-stimulant.Europe has the world's largest supply of seaweed, and Ascophyllum nodosum is one of the seaweed species found there. 12These seaweeds are utilized as bio-stimulants for enhancing plant development characteristics such as panicle growth, chlorophyll content, and protein content.They are applied as foliar sprays and added to the soil at regular intervals to act as fertilizer.
Two of the three seaweeds employed in the current experimental study showed maximum growth enhancement.On Pennisetum glaucum and Sorghum bicolor, seaweed liquid fertilizer of Ulva intestinalis and a combination of Ulva intestinalis and Padina tetrastromatica were administered respectively.As described by Bhosale et.al. 13eaweeds were autoclaved with distilled water to prepare seaweed liquid fertilizer, which is a relatively inexpensive and efficient process.Adding distilled water produced additional, different concentrations of 0.25%, 0.50%, and 0.75% of the corresponding seaweed liquid fertilizer concentrations and were applied to the plants as treatment, with NPK while water served as the control.
By treating the seeds with Ulva intestinalis, Padina tetrastromatica, a combination of it, and Sargassum cinerum the rate of germination on both plants were examined.The seeds were soaked and later examined after 48 hrs.Among them, the early and highest germination rate was seen in 0.50% of Ulva intestinalis and a combination of U. intestinalis + P. tetrastromatica as compared to the control which showed the beneficial results.Brinjal, Tomato, and Chilly treated with seaweed liquid fertilizers of Gracilaria textorii and Hypnea musciformis showed that maximum seed germination was observed in 1:4 and 1:6 concentration of Gracilaria textorii and 1:6 concentration of Hypnea musciformis. 14Capsicum longum seeds treated with seaweed liquid fertilizer Caulerpa taxifolia exhibited greatest germination percentages at 2%, 3%, and 4% concentrations, whereas seeds treated with seaweed liquid fertilizer Caulerpa racemosa exhibited highest growth at 5% concentration. 15 a result, they were later planted and grown in soil.The plants after 2 weeks of growth were treated with a foliar spray made up of Ulva intestinalis, Padina tetrastromatica, and a combination of it with their different concentrations respectively.It was observed that after 8 weeks the seaweed liquid fertilizer-treated plants with 0.50% showed longer shoot lengths as compared to control plants.
Likewise, the application of 20% of seaweed liquid fertilizer Sargassum wightii on Triticum aestivum shows better results in agronomic growth where it shows a 6.7% increase in shoot length over control. 16omato plants treated with 0.4% and 1.0% seaweed liquid fertilizer Ecklonia maxima were significantly bigger than control plants. 17Further, the pigment estimation of leaves of both the seaweed liquid fertilizer treated plants and control was carried out among which both seaweed liquid fertilizer treated plants showed good results in both chlorophyll a and b, carotenoid, treated with Ulva intestinalis, Padina tetrastromatica, a combination of it.Solanum lycopersicum Mill.leaf chlorophyll content and photosynthetic rates positively increased with the application of seaweed extract of Sargassum horneri. 18Solanum lycopersicum treated with the fresh and dried combination of Ulva reticulata showed high ascorbic acid, beta-carotene, and lycopene contents. 19ter 13 weeks, flowering and fruiting were noticed, Pennisetum glaucum and Sorghum bicolor were treated with U.intestinalis and a combination of U. intestinalis and P. tetrastromatica respectively, both displayed early and maximum fruiting.Even in the panicle study, the greatest panicle length and the total number of seeds in the 0.50% seaweed liquid fertilizer have been observed.The protein content in seeds of Pennisetum glaucum fruiting treated with 0.50% of U. intestinalis was 0.253 mg/g, which was higher than NPK, which is 0.229 mg/g, and the control is 0.222 mg/g.The protein content in seeds of Sorghum bicolor fruiting treated with 0.50% of P. tetrastromatica + U. intestinalis was 0.247 mg/g, which was higher than NPK, which is 0.224 mg/g, and for control is 0.226 mg/g.Similar research was done in the Effect of Seaweed Liquid Fertilizer of Colpomenia sinuosa (Mert.ex-Roth) Derbes & Solier, where Pennisetum glaucum was treated with Colpomenia sinuosa (brown algae) as seaweed liquid fertilizer.Greater agronomic growth with a 10% concentration was the result of the studies. 20ing ICP-AES, the presence of micronutrients and heavy metals have been detected.Calcium, Magnesium, Potassium, and Phosphorus are the micronutrients essential for plants among which Calcium and Magnesium are present in seaweed liquid fertilizer of U. intestinalis and also in its combination with P. tetrastromatica.In both stressful and non-stressful environments, Ca2+ ions act as secondary messengers for plant growth and development. 21Furthermore, it promotes other nutrient absorption by roots and translocation throughout the plant.Magnesium is the second-most common cation in plants and is essential for the formation of nucleic acids, proteins, and enzymes as well as for photosynthesis . 22hus, from this study, it can be concluded that seaweed liquid fertilizers play a significant role in the holistic growth and enhancement of plants as compared to chemical fertilizers.

Conclusion
According to the results of the current study, U. intestinalis, P. tetrastromatica, its combination, and S. cinerum exhibited beneficial effects on Pennisetum glaucum and Sorghum bicolor, observations were carried out from the treatment of the seeds to its fruiting stage.Germination rate was reported after 48hrs in seeds treated with 0.50% concentration seaweed liquid fertilizer when compared to control seeds.85% germination in Pennisetum glaucum was observed in only 0.50% concentration U. intestinalis treated seeds which is 18 -20% greater than the control crop.On the other hand, 96% germination rate in Sorghum bicolor was observed in 0.50% concentration combination of U. intestinalis and P.tetrastromatica treated seeds only which is 15-16% greater than the control crop.The investigation of subsequent growth in crops with increased germination was done following the findings of the germination study.Crops treated with 0.50% seaweed liquid fertilizers demonstrated general agronomic growth, including growth in shoot and root length, leaf size, stem diameter, and fruiting.According to the panicle study, crops treated with 0.50% seaweed liquid fertilizer had panicles longer, heavier and with more seeds overall.The protein and Chlorophyll content of these crops were also in favour of 0.50% treated crops.
According to the qualitative phytochemical analysis performed, Alkaloids, Tannins, Saponins, Phenols, and Polysaccharides are found in seaweed liquid fertilizers to enhance the morpho-agronomic and bioactive characteristics of crops. 23The existence of bioactive components is confirmed by ICP-AES results, confirming the enhancement carried on by seaweed liquid fertilizer.Hence, seaweed liquid fertilizer is proved to be the most efficient and affordable bio-stimulant for agronomic growth and enhancement of crops.

Acknowledgement
Authors would like to acknowledge Department of Botany, Ramnarain Ruia Autonomous College for providing necessary research facilities and technical support.Authors also thank SAIF lab IIT-Bombay for ICP-AES analysis.

1: Germination Rate of Pennisetum glaucum treated with different Seaweed liquid fertilizer. Shoot lengths
.1 and 1.2 show results for germination rate after 72 hrs of treatment with seaweed liquid fertilizer as well as water as control.In table 1.1, SLF of Ulva intestinalis treated showed gradual increase in rate of germination.While in Table 1.2, Combination of Seaweed extracts showed maximum germination rate.Based on this data, further Pennisetum glaucum were treated with Ulva intestinalis SLF and Sorghum bicolor was treated with a combination of Seaweed liquid fertilizer, respectively.
Shoot lengths of the plants were recorded after every 7 days.As observed from table no 1, initial growth was observed in plants treated with 0.50% concentration and control.According to further observations, maximum growth was observed in 0.50% concentration of Ulva intestinalis treated Pennisetum glaucum crop also in combination of Ulva intestinalis and Padina tetrastromatica treated Sorghum bicolor crop.Graph 2: