Bio Fertilizer

American-Eurasian J. Agric. & Environ. Sci., 6 (4): 392-399, 2009 ISSN 1818-6769 © IDOSI Publications, 2009

Studies on the Effect of Sargassum polycystum (C.Agardh, 1824) Extract on the Growth and Biochemical Composition of Cajanus cajan (L.) Mill sp V. Erulan, P. Soundarapandian, G. Thirumaran and G. Ananthan Centre of Advanced Study in Marine Biology, Annamalai University. Parangipettai, Tamilnadu, India Abstract: In the present study, an attempt has been made to investigate the effect of seaweed liquid fertilizer of S. polycystum on the seed germination growth, yield, biochemical and pigment characteristics of C. cajan. The 0.50% concentration of both water and alcohol extract showed better results of seed germination, growth parameters, biochemical and pigments constitutions. The lowest concentration (0.50%) showed higher seed germination (99.12±6.03%), shoot length (18.51±2.52 cm), root length (6.96±0.19 cm), fresh weight (0.326±0.037 mg gG1 fr.wt) and dry weight (0.093±0.005 mg gG1 fr.wt). The highest concentrations (1.5%) exhibit the lower seed germination (81.19±4.83%), shoot length (9.63±1.08 cm), root length (3.21±0.173 cm), fresh weight (0.148±0.031 mg gG1 fr.wt) and dry weight (0.042±0.005 mg gG1 fr.wt). The biochemical composition such as protein (4.48%±0.138%), lipid (5.96%±0.039%) and carbohydrate (9.21%±0.482%) showed the maximum at 0.50% concentration. The minimum biochemical composition such as protein (1.187%±0.047%), lipid (2.573%±0.050%) and carbohydrate (4.987%±0.411%) was recorded at 1.5% concentration. Pigment content such as chlorophyll ‘a’(0.615±0.032 mg gG1 fr.wt), chlorophyll ‘b’ (0.445±0.13 mg gG1 fr.wt), total chlorophyll (1.107±0.041 mg gG1 fr.wt) and carotenoids (0.712±0.52 mg gG1 fr.wt) shows the maximum at lower concentration (0.50%) than that of higher concentration (1.5%). Key words:Seaweed Liquid Fertilizer % Sargassum polycystum % Cajanus cajan % Growth % Yield % Proximate Composition and Pigment Analyses INTRODUCTION

hormones, regulators, promoters carbohydrates, amino acids, antibiotics, auxins, gibberellins and vitamins consequently which enhance the yield and quality which are induce the yield of crops, seed germination, resistance to frost, fungal and insect attacks. Seaweed cast continued to be so valuable to farmers, even in the early 1990s [1] in many countries seaweed and beach cast are still used in both agriculture and horticulture [2, 3]. Many studies in the past three decades have found wide application in modern agriculture for using marine macro algae as fertilizer. They are used as whole or finely chopped powdered algal manure or aqueous extracts. The effect of seaweed liquid fertilizers (SLF) of Caulerpa recemosa and Gracilaria edulis on growth and biochemical constituents of Vigna catajung has been studied. The lowest concentration (10%) of aqueous extract promoted the seedling growth, fresh and dry weight, chlorophyll content, protein, amino acid and deducing for total sugar content in Vigna catajung [4].

Seaweeds are marine macro algae, which form an important component of the marine living resources of the world. They are available largely in shallow coastal waters of sea, estuaries and backwaters. Seaweeds are marine non-flowering plant which lack flowers, true shoot, root, stem and leaf system. Seaweeds usually grow vertically away from the substratum which brings them closure to light; major environmental factors such as light, temperature, salinity, water motion and nutrient availability are related to seaweeds growth. Water (Nutrients) uptake and photosynthesis take place over entire surface of the thallus in contrast to vascular plants. Seaweeds are classified into three groups namely green (Chlorophyceae), brown (Phaeophyceae) and red (Rhodophyceae) based on their pigments like chlorophylls, carotenoids and phycobiliproteins. The growing agricultural practices need more fertilizers for higher yield to satisfy food for human beings. The seaweed extracts contain plant growth

Corresponding Author: P. Soundarapandian, CAS in Marine Biology, Annamalai University, Parangipettai,Tamilnadu, India-608502 Tel: +91-4144-243223

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The extracts (SLF) from Enteromorpha intestinalis showed the maximum activity in terms of increase in seed germination, length of main root and shoot system and also increased chlorophyll content [5]. The levels of endogenous free and total Auxin (IAA), Gibberellic acid (GA3), Abscisic acid (ABA) and Cytokinin (zeatin), were examined in some species of mosses and lichens [6]. The effect of seaweed liquid fertilizer (SLF) prepared from Sargassum wightii on biochemical constituents of V. radiata has been studied by Sivasankari et al. [7]. The lowest (10%) concentration increased the pigment content, protein, amino acid, total sugar content, catalase, peroxidase and polyphenol oxidase activities. The effect of Chaetomorpha antennina and Rosenvingea intricata on seed germination, fruit settling and weight of vegetable of Abelmoschus esculenus were investigated by Thirumaran et al [8]. An attempt was made to investigate the effect of seaweed liquid fertilizer of Rosenvigea intricata with or without chemical fertilizer on seed germination, growth, yield, pigment content and soil profile of Abelmoschus esculentus [9, 10]. In recent years, the use of these marine macro algae in modern agriculture has been investigated by Rama Rao [11, 12], Manimala and Rengasamy [13], Whapham et al. [14], Lopez-Musquera and Pazas [15] and Rajarajan [16]. Apart from the effect of SLF on the growth of higher plant there are also few studies on the effect of SLF on microorganisms [17]. Seaweed extract is definitely capable of promoting growth in both higher plants and prokaryotic organisms [18]. Also, there is a need to study the use of macro algal preparations on the growth and dynamics of the soil microbial community, especially those concerned with biological nitrogen fixation and their host legume. The present study intends to investigate the effect of seaweed liquid fertilizer (SLF) prepared from the brown algae, Sargassum polycystum on the growth,

biochemical and pigment characteristics of red gram (C. cajan). MATERIALS AND METHODS Study Area: Gulf of Mannar Biosphere Reserve is actually situated in the southern part of the peninsular India; this extends from Adams Bridge to Cape Comorin (Fig. 1). This area is remarkable for its diversity of flora and fauna and it also sustains a good fishery. The species of marine macro alga S. polycystum (C.Agardh) were collected from shallow waters of the Gulf of Mannar region (Lat.9° 17’N Long.79° 17’E) at Mandapam, southeast coast of India. Site was chosen in this region based on the abundance and distribution of different species of algae. The substrate comprised of sand with boulders and platforms of compressed sand stones with rough uneven surfaces at different lends. In the intertidal region, the algae are completely submerged during high tide and are exposed during the low tide. This species was available on mud covered coralline rocks and they were fully covered with this algae. Edna (1981) reported this as an edible one. S. polycystum was obtained in sufficient quantity for subsequent extraction. Collection of Seaweeds: The seaweed S. polycystum was collected from Gulf of Mannar, south east coast of India near Mandapam. The collected seaweeds were washed with salt water to remove all the epiphytes and then allowed in distilled water to remove the salts. Subsequently the seaweeds were air dried for few days, then cut into small pieces and made into powder. The powdered seaweed was extracted with water and alcohol and both extracts were used for different analyses like growth parameters, biochemical and pigment composition in C cajan.

Fig. 1: Map Showing the Study Area 393

Am-Euras. J. Agric. & Environ. Sci., 6 (4): 392-399, 2009

Flow chart for the Preparation of Seaweed Liquid Fertilizer

Details of Treatment: Different concentrations of seaweed liquid fertilizer (water and alcohol extract) C

Seaweed liquid fertilizer preparation [11] 9 Fresh seaweed 9 Washed thoroughly to remove all epiphytes and sand 9 Particles with tap water 9 Shade dried for five days 9 Hand crushed 9 The sample was ground using mixer-grinder 9 The coarse powder was collected 9 The coarse powder was mixed with distilled water in ratio of 1:20 (W/V) 9 Autoclaved at 121°C, 20 1bs for 60 minutes 9 Filtered through cheese cloth 9 The filtrate was collected at 4°C 9 The supernatant was centrifuged and dried on an oven at 60°C for 48 hours 9 The 100% seaweed extract was collected using distilled water 9 Prepare different concentrations of SLF were prepared from 100% seaweed

C

Water Extract-Different Concentrations (0.1, 0.25, 0.50, 0.75, 1.0 and 1. 5%) Alcohol Extract-Different Concentrations (0.1, 0.25, 0.50, 0.75, 1.0 and 1. 5%).

Experiment-I: One forty numbers of healthy and uniform sized seeds of red gram (C. cajan) were divided into 7 batches (Each petriplate/20 seeds). Then they were treated with different concentrations of water extracted seaweed (0.1, 0.25, 0.50, 0.75, 1.0 and 1. 5%) for every 12 hrs. One batch of seeds was kept as control and treated with water. Experiment-II: One forty numbers of healthy and uniform sized seeds of red gram were divided into 7 batches (Each petriplate/20 seeds). Then they were treated with different concentration of alcohol extracted seaweed (0.1, 0.25, 0.50, 0.75, 1.0 and 1. 5%) for every 12 hrs. One batch of seeds was considered as control and treated with water. Germination Test (Inclined plate Method): Germination test of 100 with four sub replicates of 20 seeds each was carried out in paper medium with modification using inclined plate method in a germination room. It was maintained at 25±1.5°C and RH 96±2 percent with diffuse light (approximately 10 hours) during the day. The normal seedling was recorded on 7th day of the culture period and the percentage of germination was computed.

Preparation of SLF using distilled water: Chopped seaweed (1 Kg) was boiled with 1 liter of distilled water for 1 hour and then filtered. The filtrate was taken as 100% concentration; then that was made up into different concentrations (0.1, 0.25, 0.50, 0.75, 1.0 and 1. 5%) by using distilled water.

Root and Shoot length: All the normal seedlings from each replication were used for growth measurements. The shoot length was measured from the collar region to tip of the shoot and the root length was calculated from the collar region to tip of the primary root. The mean values were expressed in Cm.

Preparation of SLF using Ethanol: Powdered seaweed (100g) was mixed with 100 ml of alcohol and stirred well and then filtered. The filtrate was considered as 100% concentration of SLF and then made up to different concentrations (0.1, 0.25, 0.50, 0.75, 1.0 and 1. 5%).

Fresh and Dry weight: The uprooted plants were washed and separated into root and shoot; they were blotted in blotting paper and weighed. They were dried in a hot air oven at 80°C for 24 hours and then dry weight was taken by using an electrical single pan balance. The mean values were expressed in mg gG1.fr.wt.

Crop Plant Selection: The crop plant selected for the present study was red gram (C. cajan). It is a Leguminoseae family, cultivated throughout Indian subcontinent. The seeds for the study purpose were collected from the regional pulses research station, Agricultural University, Department of Seed Science Technology, Madurai, Tamil Nadu, India. Healthy seeds free from visible infection, with uniform size, colour and weight were segregated; then the seeds were stored in metal tin container as suggested by Rao [19].

Pigment Analyses: The amount of chlorophyll present in the leaf was estimated by method of Arnon [20]. Carotenoids were estimated by Kirk and Allen [21].

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Biochemical Analyses: The total carbohydrate was estimated by Phenol-sulphuric acid method of Dubois et al. [22]. The total protein was estimated using the Biuret method of Raymont et al. [23]. The extraction of lipid was done by the chloroform-methanol method [24]. RESULTS Growth Parameters: The effect of different concentrations of water and alcohol extracts of seaweed (S. polycystum) treated on the growth of C. cajan was studied. The parameters such as seed germination, shoot length, root length, fresh and dry weights were recorded.

Fig. 2: Seed Germination of C. Cajan

Seed germination: The percentage of seed germination of C. cajan was varied from 81.19±4.83 to 99.12±6.03. The highest germination percentage (99.12±6.03) was observed at 0.50% concentration of alcohol extracts in red gram. The lowest germination percentage (81.19±4.83) was recorded from 1.5% concentration of same alcohol extract. The germination percentage was increased upto 0.50% concentration and thereafter declined. The value was expressed in percentage/seedlings (Fig.2). In C.cajan, shoot length ranged from 9.63±1.08 to 18.51±2.52 cm/seedlings. The highest shoot length (18.51±2.52) occurred at 0.50% concentration of alcohol extracted seaweed and the lowest value of shoot length (9.63±1.08) was recorded at 1.5% concentration of the similar alcohol extract (Fig.3). The root length of red gram varied from 3.21±0.173 to 6.96±0.198 cm/seedlings and the maximum value (6.24±0.187; 6.96±0.198) was obtained at 0.50% concentration of both water and alcohol extracts, respectively. The minimum value (3.21±0.173) occurred at 1.5% concentration of alcohol extract (Fig.4). Fresh weight of C.cajan ranged from 0.148±0.031 to 0.326±0.037 mg gG1. fr.wt. Seedlings and the greatest values (0.326±0.037; 0.293±0.041) occurred at 0.50% concentration of both alcohol and water extracts. The lowest value (0.148±0.031) obtained at 1.5% concentration of alcohol extract. The fresh weight was increased up to 0.50% concentration and there after it showed a declining manner (Fig.5). The dry weight of C.cajan values varied from 0.093±0.005 to 0.042±0.005 mg gG1. fr.wt seedlings and the maximum value (0.093±0.005 to 0.083±0.006) was found at 0.50% concentration of both alcohol and water extracts. The minimum value (0.042±0.005) was observed at 1.5% concentration of water extracts (Fig.6).

Fig. 3: Shoot Length of C. Cajan

Fig. 4: Root length of C. cajan

Fig. 5: Fresh Weight of C. Cajan Photosynthetic pigments: The Chlorophyll ‘a’ composition ranged between 0.615±0.032 to 1 0.331±0.014 mg gG fr.wt. The maximum (0.615±0.032) was observed at 0.50% concentration of alcohol extracts and the minimum (0.331±0.014) chlorophyll ‘a’ content was recorded at 1.5% concentration of the same alcohol extract (Fig.7).

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Fig. 6: Dry Weight of C. Cajan

Fig. 9: Total Chlorophyll Content of C. Cajan

Fig. 7: Chlorophyll ‘a’ Content of C. Cajan

Fig. 10: Carotenoid Content of C. Cajan 7 Protein %

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Fig. 8: Chlorophyll ‘b’ Content of C. Cajan

Fig. 11: Protein Content of C. Cajan The Chlorophyll ‘b’ in C. cajan ranged from 0.455±0.013 to 0.243±0.021 mg gG1 fr.wt and the highest value (0.455±0.013) was obtained at 0.50% concentration of alcohol extract. The minimum (0.243±0.021) chlorophyll ‘b’ content was record at 1.5% concentration of alcohol extract (Fig.8). The total chlorophyll content ranged between 1.107±0.041 to 0.586±0.041 mg gG1 fr.wt and the highest value (1.107±0.041; 0.904±0.062) of total chlorophyll was obtained from 0.50% of both alcohol and water extracts respectively. The lowest (0.586±0.041) concentration of total chlorophyll content was obtained at 1.5% concentration of alcohol extract (Fig.9). The carotenoids content ranged from 0.712±0.052 to 0.415±0.021 mg gG1 fr. wt and highest value (0.712±0.052; 0.682±0.045) were found at 0.50% concentration of both alcohol and water extracts. The lowest value (0.415±0.021) was obtained at 1.5% concentration of alcohol extracts.

The experimental plant significantly increased pigment concentration upto 0.50% and thereafter decreasing trend when compared to control (Fig.10). Biochemical parameters: The biochemical parameters like protein, lipid and carbohydrate contents were analyzed at different concentrations of water and alcohol extracts. The protein content ranged between 4.482%±0.138% to 1.187%±0.047% and the maximum (4.482%±0.138%, 4.163%±0.126%) was recorded at 0.50% of both alcohol and water extracts respectively. The minimum (1.187%±0.047%) was observed at 1.5% concentration of alcohol extract. The protein value was increased upto 0.50% concentration of both alcohol and water extracts and thereafter it showed a diminishing trend when compared to control (Fig.11).

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Dhargalkar and Untawale [25] reported that seaweed liquid fertilizer (SLF) treatment enhanced the rate of seed germination in green chilies and turnip and found that lower concentration of seaweed liquid fertilizer increased the germination percentage than higher concentration. Similar observation was made in C. cajan seaweed extract of the present study seaweed extracts treated on the vegetable crops of Abelmoschus esculentus and Lycopersicon lycopericum. Different concentrations (10, 15, 20, 25 and 30%) of SLF were used and better results were obtained in lower doses (10, 15, 20) reported by Selvaraj et al. [26]. Studies on the effect of Chaetomorpha antennina and Rosenvingea intricata on seed germination, fruit settling and weight of vegetable of Abelmoschus esculenus. Were carried out by Thirumaran et al. [8]. They got better results at lower concentration than that of higher concentration In the present study, lower concentration (0.50%) exhibited higher seed germination (99.12±6.03%) shoot length (18.51±2.52 cm), root length (6.96±0.19 cm), fresh weight (0.326±0.037 mg gG1 fr.wt) and dry weight (0.093±0.005 mg gG1 fr. wt) than higher concentration. The growth rate was increased up to 0.50% concentration and thereafter it showed a decreasing trend. This result was coinciding with study of Bhosle et al. [27]. They showed better growth when S. tenerrimum extract was used especially in lower concentrations. Seaweed liquid fertilizer (SLF) at low concentrations enhanced the growth parameters viz., shoot length, root length, leaf area, fresh weight, dry weight and moisture content. Biochemical parameters like chlorophyll ‘a’ and ‘b’, protein, sugars, starch, ascorbic acid and in vivo nitrate reductase activity were also found higher at 1.5%. The higher concentrations like 2, 2.5 and 5% appeared to be the inhibitory levels for Sorghum reported by Ashok et al. [28]. The investigation on the effect of crude extract was performed using the seaweed S. plagiophyllum (SLF) and the commercial seaweed extract-SM3 on the seed germination and seedling growth in green gram and black gram. Both SLF and SM3 promoted seedling growth upto a concentration of 0.75% in black gram. Green gram showed the maximum seedling growth at 1.5% concentration of SLF and 1.0% concentration of SM3 [29]. The earlier investigation was in contrast to the present study, as the concentrations varied from 0.1 to 1.5% and the maximum growth rate, biochemical and pigment concentration was observed at 0.50% concentration.

5 4.5 4 Lipid %

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Fig. 12: Lipid Content of C. cajan 12 10 Carbohydrate %

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Fig. 13: Carbohydrate Content of C. cajan The lipid content of C. cajan varied from 5.96%±0.039% to 2.573%±0.050% and the higher value (5.96%±0.039%, 4.562%±0.028%) obtained at 0.50% concentration of both alcohol and water extracts. The minimum value (2.573±0.050) was observed at 1.5% concentration of alcohol extract (Fig.12). The carbohydrate content of C. cajan ranges between 9.210%±0.482% to 4.987%±0.411% and the maximum value (9.210±0.482; 8.673±0.436) was obtained at 0.50% concentration of both alcohol and water extracts. The minimum value (4.987±0.411) was observed at 1.5% concentration of alcohol extract. The protein, lipid and carbohydrate values were increased up to 0.50% concentration of both alcohol and water extracts and thereafter it showed a decreasing manner when compared to control plant (Fig. 13). DISCUSSION The effect of seaweed Liquid fertilizers (SLF) of Caulerpa recemosa and G.edulis on growth and biochemical constituents of V. catajung has been studied by Anandharaj and Venkatesalu [4]. The lowest concentration (10%) of aqueous extract promoted the seedling growth, fresh and dry weight in V. catajung.

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Studies on water stress in the black gram V. mungo (L) Hepper with SLF and PEG (Polyethylene Glycol) revealed that the seedling drenched with 1% SLF did not wilt up to 4 days after stress imposition and appeared like control plants. Whereas, plants treated with 1% PEG and plants grown without any treatment wilted on the 3rd day after subjecting to waters deficiency. The seedlings grown in 1% SLF accumulated total soluble proteins at significant levels during the stress period [30]. The lowest concentrations of SLF (0.025 and 0.05%) from Ulva lactuca to be responsible for enhancing the growth characteristics of the alga (Pirulina flatensis) was reported by Sridhar and Rengasamy [31]. The effect of seaweed liquid fertilizer (SLF) prepared from S. wightii on biochemical constituents of V.radiata was studied by Sivasankari et al. [7], where the lowest (10%) concentration increased the pigment content, protein, amino acid, total sugar content, catalase, peroxidase and polyphenol oxidase activities. The seaweed liquid fertilizer (SLF) and seagrass liquid fertilizer (SGLF) not only promoted the seedling growth but also increased the chlorophyll ‘a’ and ‘b’ contents in the leaf up to 12 days of germination in Zea mays which was reported by Asir Selin Kumar et al. [32]. The lowest concentration of SLF (10%) has shown remarkable effect on plants than higher concentrations. The results of the present study the coincides with that of the above study. The biochemical composition such as protein (4.48%±0.138%), lipid (5.96%±0.039%), carbohydrate (9.21%±0.482%) and pigment contents such as chlorophyll ‘a’ (0.615%±0.032%) chlorophyll ‘b’ (0.445%±0.13%). Total chlorophyll (1.107%±0.041%) and carotenoids (0.712%±0.52%) showed a maximum at lower concentration (0.50%) and thereafter it showed a reducing manner at a higher concentration (1.5%). The investigation on the effect of seaweed liquid fertilizer of Rosenvigea intricata with or without chemical fertilizer on seed germination, growth, yield, pigment content and soil profile of Abelmoschus esculentus was carried by Thirumaran et al. [9, 10].The Seed germination, shoot length, root length, number of lateral roots, number of leaves, number of vegetables, length of vegetables, weight of vegetables, chlorophyll ‘a’, chlorophyll ‘b’, total chlorophyll and carotenoids were found to be the maximum at 20% SLF with or without chemical fertilizer. So it may be concluded from their study that, lower dose (20%) of SLF with or without chemical fertilizer leads to higher growth, yield, chlorophyll pigment and soil profile compared to other concentrations.

CONCLUSION Approximately 70% of the population thrives in rural areas, engaged in agriculture, making the backbone of the economy. The fast growing population is mounting tremendous pressure in food production. To meet out this increasing demand, farmers use chemical fertilizers to enhance the crop production. The toxic chemicals (arsenic and cadmium) from the chemical fertilizers accumulate in plant products and ultimately reach to dinning tables causing health problems in human beings by biomagnifications in recent years. The use of natural seaweed products as substitutes to the conventional synthetic fertilizers has assumed importance. In agriculture, the application of seaweeds are so many, as soil conditioners, fertilizers and green manure, due to the presence of high amount of potassium salts, micronutrients and growth substances. The present study is an additional information for an alternate to synthetic fertilizers and further study is necessary to recompense the constraints. REFERENCES 1.

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