Effect of aqueous extract of Sargassum johnstoniiSetchell & Gardner on growth, yield and qualityof Lycopersicon esculentum Mill.

Reeta Kumari & Inderdeep Kaur & A. K. Bhatnagar

Received: 26 March 2010 /Revised and accepted: 13 December 2010 /Published online: 19 January 2011# Springer Science+Business Media B.V. 2011

Abstract Experiments were conducted on tomato to studythe potential of brown alga Sargassum johnstonii as abiofertilizer. Seaweed extract was applied as a foliar spray,soil drench, and soil drench + foliar spray to assess its effecton plant growth, yield, and concentration of lycopene andvitamin C. The main objective of the study was to enhancethe biochemical constituents with neutraceutical and anti-oxidant values in tomato fruit. Different concentrations(0.1%, 0.4%, 0.8%, 2%, 6%, 8%, and 10%; v/v) of seaweedextract were used and growth was observed over a period of7 months. A total of 14 sprays/drenches were applied at 15-day intervals during the entire vegetative and reproductivephase. A statistically significant increase in vegetativegrowth (plant height, shoot length, root length, and numberof branches), reproductive parameters (flower number, fruitnumber, and fresh weight), and biochemical constituents(photosynthetic pigments, proteins, total soluble sugars,reducing sugars, starch, phenols, lycopene, and vitamin C)was recorded following all three methods of treatment athigher concentrations of seaweed extract. The study alsoreports auxin- and cytokinin-like activity, and the presenceof macro- (Ca, Mg, Na, and K) and micronutrients (Fe, Cu,Zn, and Mn), in seaweed extract of S. johnstonii, whichmakes it a potential biofertilizer.

Keywords Biofertilizer . Nutrients . Lycopene .

Lycopersicon esculentum . Seaweed extract . Vitamin C

Introduction

Extensive application of chemical fertilizers to increasecrop productivity has caused considerable damage to theecology of agricultural systems and has even reduced thenutritional quality of crops. For health considerations, andalso to meet the stringent consumer standards, agriculturalpractices are increasingly being modified with a focus onorganic farming (Kramer et al. 2006). Organic farming is asystem of ecological soil management that relies, in part orin full, on building organic matter through crop rotation,organic waste amendments, balanced mineral nutrientmanagement, and mechanical and biological controls withminimum adverse effects on soil health. The use ofbiofertilizers to enhance plant growth and yield has gainedmomentum due to hazardous effects of chemical fertilizers.Biofertilizers enhance crop productivity through processessuch as nitrogen fixation, phosphate solubilization, andplant hormone production (Pereira and Verlecar 2005).Biological resources, namely symbiotic micro-organisms(e.g., Rhizobium), free-living soil bacteria (Azotobacter spp.and Bacillus megatherium), fungi (arbuscular mycorrhiza),algae (blue green algae and seaweeds), and soil faunasuch as earthworms (Mahfouz and Sharaf-Eldin 2007;Karthikeyan et al. 2008), have been investigated aspotential biofertilizers. Seaweeds need to be evaluated notonly as rich sources of mineral nutrients that are oftendeficient in farm soils but also for the growth promotingsubstance they possess (Crouch and van Staden 1992;Sivasankari et al. 2006).

The use of seaweeds as biofertilizers in horticulture andagriculture has increased in the recent years (Dhargalkar

Electronic supplementary material The online version of this article(doi:10.1007/s10811-011-9651-x) contains supplementary material,which is available to authorized users.

R. Kumari : I. Kaur :A. K. Bhatnagar (*)Environmental Biology Laboratory, Department of Botany,University of Delhi,Delhi 110 007, Indiae-mail: akb_du@rediffmail.com

R. Kumarie-mail: gautam.rita@gmail.com

I. Kaure-mail: inderdeep_botany@rediffmail.com

J Appl Phycol (2011) 23:623–633DOI 10.1007/s10811-011-9651-x

and Pereira 2005). Seaweeds can be used in liquid extractform as foliar spray or soil drench (Blunden et al. 1997;Lingakumar et al. 2004; Thirumaran et al. 2009) or ingranular/powder form as soil conditioner and manure.Seaweed extracts from Sargassum wightii and Kappaphy-cus alvarezii have been found to increase the yield of Vignasinensis and Phaseolus radiata, respectively (Sivasankari etal. 2006; Zodape et al. 2010). For seaweeds to be utilized asa biofertilizer, availability of biomass in plenty is one of themajor criteria. Seaweeds are abundantly available in warmtemperate and tropical waters of the world. Fresh plants canbe easily collected during low tide periods and “driftseaweed” during high tide periods on the sea coast. To meetthe agricultural demand, seaweed biomass is raised throughmass cultivation in many countries, including France,Spain, Egypt, Vietnam, Thailand, Indonesia, Bangladesh,Malaysia, Philippines, Korea, Japan, and China (Tseng1993). Sargassum johnstonii Setchell & Gardner is widelydistributed in west coast of India where it shows activevegetative growth from July to September before enteringreproductive phase in mid October. After an active sexualphase, the aerial plant parts senesce and die during Marchto May. These have a great potential to regenerate fromholdfast in the months of May–July (Vijayaraghavan andKaur 1991). The present study investigates the potential useof aqueous seaweed extract of S. johnstonii as a biofertilizer.

Tomato is one of the most important vegetables in theworld, ranking second after potato, with an annual productionof 122.9 million t of fresh fruit (FAO 2005). It is a rich sourceof minerals, carbohydrates, fats, proteins, vitamins, lycopene,α and β carotene, lutein, zeaxanthin, and β-crytoxanthin.Lycopene constitutes 80–90% of the total carotenoid contentof red ripe tomato (Shi and Le Maguer 2000). Vitamin C(ascorbic acid), while being an effective antioxidant in plants,is an important phytochemical constituent of tomato fruit.Low caloric value, high fiber content, and high concentrationof minerals, vitamins, and phenols such as flavonoids maketomato an excellent fruit vegetable providing physiologicaland nutritional benefits (Dorais et al. 2008).

In the present study, efforts have been made to enhancegrowth and yield of tomato plants and to improve lycopeneand vitamin C content of fruits, by treatments with aqueousextract of S. johnstonii.

Material and methods

Preparation of aqueous seaweed extract

Sargassum johnstonii (Phaeophyceae, Fucales) was collect-ed during low tide periods, in January 2008, from PortOkha, (22°28.528′ N, 69°04.322′ E) located along thenorthwest coast of India. Plants were handpicked and

washed in seawater to remove debris, shells, and sand.These were brought to the laboratory in air tight plasticbags. Samples were washed with tap water to remove saltfrom the surface, sun-dried for 24 h, oven-dried for 48 h at60°C, and then ground in pestle and mortar to obtainpowder form. To 500 g of powdered seaweed 5,000 mL ofwater was added and contents were heated for 45 min at60°C in a plugged conical flask (modified after Rathore etal. 2009). After cooling, the contents were filtered throughfour muslin cloth layers. The filtrate recovered (3,075 mL)was used as 10% (w/v) aqueous seaweed extract, fromwhich seven different concentrations, 0% (control), 0.1%,0.4%, 0.8%, 2%, 6%, and 8% (v/v) were prepared usingdouble distilled water.

Experimental design and treatments

Seeds of tomato (Lycopersicon esculentum Mill.) cultivarPusa Ruby were procured from the National Seeds Corpo-ration, Pusa Campus, New Delhi (India). Three sets ofexperiments were conducted in 38-cm pots during December2008, at the Department of Botany, University of Delhi,Delhi (India). Eight seeds per pot were planted and werelater thinned to five plants, with three replicates (3×5) pertreatment. Seedlings were raised under natural conditions.

Three treatments were given to the potted plants, namelyfoliar spray, soil drench, and soil drench + foliar spray. Foreach treatment, a series of concentrations (0, 0.1, 0.4, 0.8,2, 6, 8, and 10%) of aqueous seaweed extract was used. Ineach of the foliar and drench treatments, 100 mL aqueousextract was applied. However, in soil drench + foliar spraytreatment, 50 mL each of drench and spray was appliedconcurrently. The first spray/drench treatments were givento 15-day-old seedlings. Thereafter, eight sprays/drenchesat intervals of 15 days each were given during vegetativephase of growth, and six sprays/drenches were given duringthe reproductive phase. Spraying was done in the earlymorning and in the evening when the ambient temperaturewas cooler. All plants were watered as required throughout theexperiment, except after foliar application, when the plantswere not watered for 24 h. Handweeding was done frequently.

Hormone-like activity in aqueous seaweed extract

Cytokinin-like activity was determined for aqueous sea-weed extract using cucumber cotyledon bioassay (Fletcheret al. 1982). The experiments were conducted twice withtwo replicates per treatment and five cotyledons per Petridish. The cotyledons were placed in 3 mL test solution(40 mM KCl, 10 mM CaCl2, and H2O) and after keeping at28°C for 12, 16, 20, and 24 h were exposed to fluorescentlight for 3.5 h with 12.9 W m−2 light intensity. Thecotyledons were homogenized in 8 mL 80% acetone to

624 J Appl Phycol (2011) 23:623–633

extract chlorophyll. Volume was made to 10 mL withacetone and centrifuged at 2,500×g for 10 min. Theabsorbance of the supernatant was read at 663 nm (Chl a)and 645 nm (Chl b). Total chlorophyll was calculated usingArnon formula (1949). A kinetin standard over a range of100–10−5 mg L−1 was used to detect the concentration ofcytokinin-like activity in the test solutions.

Mung bean rooting bioassay (Hess 1964) was used wherethe number of roots formed was recorded as a measure ofauxin-like activity. Vigna mungo hypocotyl cuttings (12 cm)after 10 days were transferred to vials containing seaweedextract (20 mL) and incubated in biological oxygen demandincubator (BOD) at 26°C for 8 h. Experiments were repeatedtwice with five cuttings in each vial and two vials pertreatment. After pulse treatment, cuttings were rinsed indeionized water and incubated at 24°C in low light (9 μmolphotons m−2 s−1) with a 16 h light : 8 h dark cycle. Thenumber of roots was recorded after 8 days. IBA over aconcentration range of 10−8–10−4 M was used as a standard.

Nutrients in aqueous seaweed extract

Nutrient analysis of the aqueous extract was carried outfollowing the method described by Allen (1989). Ten mL of10% seaweed extract was filtered through Whatman filterpaper No. 44, heated in a digester at 200°C for 80 h withconc. HNO3, 60% HClO4, and conc. H2SO4 upon which aclear solution was obtained. After adding 10 mL distilledwater, it was filtered through Whatman filter paper No. 44.The volume of filtrate was made to 50 mL with deionizedwater. Concentration of macro- (Na, K, Ca, and Mg) andmicronutrients (Fe, Cu, Mn, and Zn) was determined usingatomic absorption spectrophotometry (Shimadzu AA-130,AAS, lamp current 7 ma, slit width of 1.9 cm, and burnerheight of 7 cm) at wavelengths 589.0, 766.5, 285.2, 433.7,248.3, 324.8, 213.9, and 279.5 nm, respectively. Workingstandards were prepared for each of the nutrients.

Growth parameters

Out of five seedlings that were retained per pot uponthinning, two were uprooted at 80 days for study ofvegetative parameters, i.e., plant height, plant fresh weight,shoot and root length, and number of branches. Remainingthree seedlings were maintained until harvest. Among thereproductive parameters, for each treatment, the number offlowers was recorded at 90 days, and the number of fruitsand their fresh weight were recorded at 130 days.

Photosynthetic pigments

In a test tube 0.1 g of 120-day-old freshly harvested leaf tissuewas placed and 7 mL of dimethyl sulfoxide (DMSO) was

added. The tubes were incubated for 1 h at 65°C in dark andvolume was made to 10 mL with DMSO. Absorbance wasmeasured at 480, 510, 645, and 663 nm (Hiscox and Israelstam1979). The amount of chlorophyll a, b, and carotenoids wascalculated using the formula given by Arnon (1949).

Proteins

Fresh leaves (0.5 g) at 90 days of growth were homogenizedin 5 mL phosphate buffer (pH 7.0). The homogenate wasfiltered through four muslin cloth layers and the filtrate wascentrifuged at 6,440×g for 10 min at 4°C. To the aliquot,5 mL Bradford reagent was added and vortexed. Absorbancewas read at 595 nm (Bradford 1976). Concentration ofsoluble proteins was calculated and quantified against astandard curve prepared using bovine serum albumin.

Total soluble sugars

Fresh leaf tissue (1 g) at 90 days was hydrolyzed in 5 mL2.5 N HCl for 3 h. The hydrolyzed extract was cooled andneutralized with sodium carbonate until effervescencestopped. Volume was made to 100 mL with deionized waterand centrifuged at 4,472×g for 10 min. The supernatant(0.5 mL) was collected and mixed with equal volume ofdistilled water and 4 mL anthrone reagent was added. Testtubes were placed in boiling water for 8 min followed bycooling. Optical density was measured at 490 nm. Amountof total soluble sugars was estimated from standard curveprepared with D-glucose (Yemm and Willis 1954).

Reducing sugars

Fresh leaf (1 g) samples at 90 days were homogenized with10 mL hot 80% ethanol. The extract was filtered and thefiltrate evaporated in boiling water. To dissolve the sugarsin samples, 10 mL distilled water was added to the extract.To 0.5 mL of the above prepared extract, 2.5 mL distilledwater and 3 mL dinitrosalicyclic acid reagent were added.Test tubes were placed in boiling water for 5 min, uponwhich the color developed. Following this, 1 mL 40%Rochelle salt solution (sodium potassium tartrate solution)was added. Test tubes were cooled and absorbance was readat 510 nm. Amount of reducing sugars was estimated froma standard curve prepared with D-glucose (Miller 1959).

Starch

Estimation of starch was done in 90-day-old leaves by themethod described by Thimmaiah (1999). Fresh leaf tissue(0.1 g) was homogenized in 10 mL hot 80% ethanol andcentrifuged at 4,472×g for 10 min. The resulting pellet waswashed repeatedly with ethanol and dried in a water bath.

J Appl Phycol (2011) 23:623–633 625

The dried pellet was re-dissolved by adding 5.0 mL H2Oand 6.5 mL 52% perchloric acid and centrifuged at 0°C for20 min at 4,472×g. The pellet formed was repeatedlywashed with perchloric acid. The supernatants obtainedfrom repeated washings were then pooled and volume madeup to 100 mL using distilled water. To 0.1 mL of the aboveprepared solution, 0.9 mL water and 4 mL anthrone reagentwere added. The contents were heated for 20 min in boilingwater and cooled. The absorbance was read at 630 nm.Amount of glucose content was estimated from standardcurve prepared with D-glucose. The concentration obtainedwas multiplied by a factor 0.9 to calculate starch content(Hassid and Neufeld 1964).

Total phenols

Freshly harvested leaves (0.5 g) at 90 days were homog-enized with 5 mL 80% ethanol and centrifuged at 4,472×gfor 20 min at 4°C. The supernatant was re-extracted withethanol, centrifuged, pooled, and evaporated to dryness.The residue was dissolved in 5 mL distilled water and usedfor quantification. To the aliquot, 0.5 mL Folin &Ciocalteu’s reagent was added. After 3 min, 2 mL 20%Na2CO3 was added. The tubes were placed in boiling waterfor 1 min, cooled, and the absorbance read at 650 nm. Totalphenolic content was calculated based on standardsprepared with catechol (100 μg mL−1) and expressed asmg phenols/100 g material (Malick and Singh 1980).

Lycopene

The extraction of lycopene pigment was based on modifiedmethod of Fish et al. (2002). Three or four freshlyharvested tomato fruits (140 days) were taken and pulpedwell to a smooth consistency in a Waring Blender. Tomatopulp (5 g) was weighed and extracted repeatedly withacetone in a pestle and mortar until residue was colorless.The acetone extract was pooled and transferred to aseparating funnel containing 20 mL petroleum ether, andthe contents were mixed gently. To this, 5% sodium sulfatesolution was added. To reduce the loss due to evaporation,20 mL petroleum ether was added. The two solvent phaseswere separated and the lower aqueous phase was re-extractedwith additional petroleum ether until it was colorless. Theabove extracts containing carotenoids were pooled into abrown bottle containing anhydrous sodium sulfate and allowedto stand for 30 min. The extracts were transferred to avolumetric flask through a funnel containing cotton/glass wool.The sodium sulfate slurry was washed with petroleum etheruntil it was colorless, transferred to a volumetric flask, andmade up the volume to 100 mL with deionized water. Usingpetroleum ether as a blank, absorbance of supernatant contain-ing lycopene was read at 503 nm. One mole of lycopene when

dissolved in 1 L petroleum ether (40–60°C) and measured in aspectrophotometer at 503 nm in 1 cm light path gives anabsorbance of 17.2×104. Therefore, a concentration of3.1206 μg lycopene mL−1 gives unit absorbance. Amount oflycopene in tissues was estimated by the following formula:lycopene mg g�1ð Þ ¼ x=yð Þ � A503 � 3:12, where x is theamount of petroleum ether (milliliters), y the weight of fruittissue (grams), A503 the absorbance at 503 nm, and 3.12 is theextinction coefficient.

Vitamin C (ascorbic acid)

To prepare a standard solution (ascorbic acid 100 μg mL−1),4% oxalic acid was added and titrated against 2,6-dichlorophenolindophenol dye (V1). End point was detectedby the appearance of a pink color which persisted for 5 min.Amount of dye consumed is equivalent to the amount ofascorbic acid. Tomato pulp (0.5 g) was taken from 140-day-old plant and extracted in 10 mL 4% oxalic acid. Thevolume was made up to 100 mL with deionized water andcentrifuged at 5,411×g for 10 min. To the above-preparedextract 4% oxalic acid was added and titrated against thedye (V2) (Thimmaiah 1999). The ascorbic acid concentra-tion was calculated by:

Ascorbic Acid mg 100 g�1� � ¼ 0:5 mg� V2 � 100 mL

V1 � 15 mL�W:

where W = weight of the sample (grams)

Statistical analysis

All experiments were repeated at least twice with aminimum of five replicates per treatment. All data wereanalyzed for significant differences by analysis of variancewith means separation using the least significant difference.One-way analysis of variance was carried out for eachparameter studied to assess significant differences at 5%level in treatments and their interaction for all parameters.All statistical analyses were performed with SPSS version10 (SPSS Inc., USA).

Results and discussion

Hormone-like activity in aqueous seaweed extract

In all incubation periods, there was a linear increase inchlorophyll concentration of cucumber cotyledons onapplication of 0%, 0.1%, 0.4%, and 0.8% seaweed extract,whereas a drop in chlorophyll was observed at higherconcentrations (2%, 6%, 8%, and 10%) of seaweed extract(Fig. 1a). This is correlated to the highest amount of

626 J Appl Phycol (2011) 23:623–633

cytokinin-like activity observed in 0.8% seaweed extractafter 24-h dark incubation which is equivalent to 10−2–10−1 mg L−1 kinetin (Fig. 1b).

Auxin-like activity was observed in aqueous seaweedextract. All concentrations of seaweed extract showed a linearincrease in rooting of V. mungo seedlings up to 0.8%, afterwhich there was a decrease in rooting with no rooting at 10%(Fig. 1c). The highest activity of root count at 0.8% seaweedextract was comparable to IBA at 10−6–10−5 M (Fig. 1d).Higher concentrations of seaweed extract caused detrimentaleffects such as browning of roots and disintegration ofhypocotyl cuttings (also refer Supplementary Figure).

Stirk and van Staden (1997) reported auxin- andcytokinin-like activity in commercially available seaweedextracts such as Kelpak, Marinure, Maxicrop, Seamac, andSM3. Cytokinins present in seaweed extracts stimulateplant growth by mobilizing nutrients in leaves, providingprotection from marginal frost, retarding senescence, andregulating physiological processes that include apicaldominance, chloroplast development, and cell division.Cytokinins stimulate the conversion of proplastids toplastids in cotyledons (greening process) and intensify theformation of chlorophyll (Mok 1994). In the present work,the bioassay revealed enhanced greening and expansion of

cucumber cotyledons with increasing seaweed concentrationup to 0.8% (refer Supplementary Figure). Higher concen-trations of seaweed extract caused a drop in biological activityleading to sickly, brown cotyledons, probably either due toloss of chloroplast integrity (Wu and Lin 2000) or because ofinterplay of other compounds in the seaweed extract.

A commercial seaweed concentrate prepared from thebrown alga Ecklonia maxima, exhibited a remarkable root

Fig. 1 a–d Hormone-like activity detected in aqueous extracts madefrom S. johnstonii. a Cytokinin-like activity detected using cucumbercotyledon bioassay after 12 h,□ 16 h, 20 h, and 24 h ofincubation. b Kinetin standard for cytokinins. c Auxin-like activity

detected using the mung bean bioassay. d IBA (M) standard forauxins. Histograms with different letters are significantly different.Error bars represent the standard error (n=20; P=0.05)

Table 1 Nutrient analysis of 10% aqueous extract of Sargassumjohnstonii

Constituents Concentration in seaweedextract (μg mL−1)

Sodium 34,536.7±3.58

Potassium 2,363.9±0.48

Calcium 97,247.5±3.78

Magnesium 4,225.6±2.23

Iron 4,258.2±0.211

Copper 5,750.1±0.575

Manganese 924.8±0.310

Zinc 277.3±0.220

Values are mean ± standard error (n=9)

J Appl Phycol (2011) 23:623–633 627

Tab

le2

Effectof

seaw

eedextractmadefrom

Sargassum

john

ston

iion

vegetativ

eandreprod

uctiv

egrow

thparametersof

Lycopersicon

esculentum

Treatments

Aqu

eous

seaw

eedextract

concentrations

(%)

Growth

parameters

Num

berof

branches

Roo

tleng

th(cm)

Sho

otleng

th(cm)

Plant

fresh

weigh

t(g)

Total

plant

height

(cm)

Flower

number

Fruitnu

mber

Fruitfresh

weigh

t(g)

Foliarspray

02.3±0.33

a2.7±0.05

a11.2±0.72

a1.32

±0.08

0a

13.9±0.75

a0.0±0.00

a0.3±0.33

a17

.3±1.02

ab

0.1

3.6±0.33

b3.9±0.17

b10

.7±1.56

a2.74

±0.36

2b

14.6±1.72

a1.3±0.33

ab1.3±0.33

ab17

.7±0.75

a

0.4

4.6±0.33

b4.0±0.28

b15

.9±1.51

b2.97

±0.39

1b

19.9±1.27

b2.6±0.43

bc2.0±0.57

b16

.5±2.19

ab

0.8

6.6±0.33

cd4.1±0.30

b15

.3±1.73

b3.18

±0.29

9b

19.4±1.64

b3.3±0.33

c3.3±0.33

cd16

.3±3.59

ab

26.0±0.00

c4.6±0.15

bcd

24.3±0.66

c4.16

±0.05

1c

28.9±0.51

cd3.3±0.20

c2.3±0.33

bc14

.1±1.94

a

67.6±0.66

de5.0±0.13

cd22

.1±1.46

c5.50

±0.15

2d

27.0±1.36

c4.0±0.25

c4.3±0.33

d15

.9±1.18

a

88.6±0.33

ef4.3±0.13

bc24

.3±0.08

c5.71

±0.28

0d

28.6±0.00

cd4.1±0.43

c4.0±0.57

d21

.2±1.00

ab

109.0±0.57

f5.2±0.56

d25

.0±0.20

c5.21

±0.33

5d

30.1±0.76

d4.3±0.38

c4.0±0.57

d23

.3±1.70

b

Soildrench

04.3±0.33

a2.7±0.08

a10

.5±0.02

a2.30

±0.27

1a

13.2±0.63

a2.0±0.15

a0.3±0.33

a18

.4±0.80

a

0.1

7.0±0.57

bcd

4.0±0.20

b13

.3±0.02

a2.89

±0.10

1ab

17.3±0.54

a2.3±0.33

ab2.3±0.33

bc16

.9±1.78

ab

0.4

6.6±0.33

bc3.6±0.40

b15

.7±0.02

bc3.54

±0.16

7c

19.3±0.70

b2.6±0.33

ab2.6±0.33

bc28

.5±1.45

c

0.8

7.6±0.33

cd3.4±0.23

ab16

.2±0.02

bc3.30

±0.21

7bc

19.7±1.69

b3.3±0.28

abc

3.0±0.57

bc17

.9±2.10

ab

26.3±0.33

b3.7±0.29

b16

.7±0.02

c3.35

±0.26

7bc

20.1±1.61

b4.0±0.12

bc2.0±0.57

b19

.5±2.49

b

68.0±0.57

d3.3±0.23

ab21

.5±0.02

d3.82

±0.22

2cd

24.8±1.23

c2.3±0.13

ab0.6±0.33

a11.8±2.49

a

89.3±0.33

e3.4±0.11

ab28

.4±0.02

e4.25

±0.37

7d

31.8±2.13

d4.3±0.13

bc3.3±0.33

c18

.0±2.42

ab

1010

.6±0.66

f4.2±0.32

b29

.1±0.02

e5.82

±0.311e

33.3±0.60

d5.0±0.37

c3.0±0.57

bc31

.7±0.18

c

Soildrench

+foliarspray

04.3±0.33

a2.2±0.08

a12

.6±0.02

ab2.52

±0.18

3a

14.9±0.32

ab0.0±0.00

a0.0±0.00

a19

.7±1.00

a

0.1

5.3±0.33

ab3.1±0.26

ab13

.4±0.02

abc

2.27

±0.12

1a

16.5±1.06

ab1.0±0.00

ab2.3±0.33

c20

.7±1.62

a

0.4

6.3±0.33

bc2.7±0.23

b14

.4±0.02

abc

2.62

±0.12

2a

17.2±1.33

bc1.0±0.00

ab0.6±0.33

a21

.3±1.80

a

0.8

6.0±0.40

bc3.2±0.29

b11.7±0.02

a2.67

±0.117a

15.0±1.06

ab0.66

±0.33

ab1.0±0.00

ab18

.3±0.60

a

26.6±0.33

c3.2±0.20

b14

.1±0.02

abc

3.31

±0.31

8a

17.3±1.08

bc2.3±0.16

bc2.0±0.57

bc21

.3±2.30

a

67.0±0.57

cd3.5±0.08

b16

.7±0.02

c3.00

±0.44

1a

20.2±0.96

c2.6±0.26

bc2.6±0.33

c17

.0±2.87

a

87.3±0.33

cd3.2±0.06

b20

.3±0.02

d3.61

±0.115b

23.5±0.26

c2.7±0.26

bc2.3±0.33

c22

.6±2.10

ab

1011.0±0.0e

4.6±0.37

c25

.4±0.02

e5.44

±0.96

8b

30.0±0.65

d4.0±0.15

c2.6±0.33

c29

.1±2.10

b

Valuesaremean±standard

error.Reading

swith

different

letters

aresign

ificantly

different(n=27

;P=0.05

)as

analyzed

byTuk

eypo

stho

ctest

628 J Appl Phycol (2011) 23:623–633

promoting ability in cuttings. This rooting response wasattributed to endogenous indoles which were positivelyidentified in the seaweed concentrate (Stirk et al. 2004).Initiation and development of roots probably requires lowconcentration of the active compounds and therefore adecline in rooting was observed at higher (10%) seaweedextract concentrations. This coincided with the observedfall in cytokinin-like activity of the extract. Such a responseis common with hormones, often promoting physiologicalprocesses at low concentrations and inhibiting at highconcentrations (Crouch and van Staden 1991).

Nutrient analysis of aqueous seaweed extract

Aqueous seaweed extract from S. johnstonii when analyzedfor nutrients showed the presence of both macro- (Na, K,Ca, and Mg) and micronutrients (Fe, Cu, Mn, and Zn;Table 1). The concentration of calcium was maximum andthat of manganese minimum in the extract (Table 1). Theliquid extract prepared from S. wightii contains high levelsof Mg, Na, K, P, Fe, Cl, Zn, Cu, and N (Sivasankari et al.2006). Seaweeds are a good source of nutrients for theplants when added as fertilizer. The liquid sprays providenutrients to the plant through stomata and enhance theyield, vigor, and pest resistance of plant.

Effect of seaweed extract on growth and yield parametersof tomato

All three treatments enhanced vegetative and reproductivegrowth of the tomato plants as compared to the control(Table 2). Application of aqueous seaweed extract to plantsas foliar spray resulted in maximum increase in root lengthand number of fruits, while number of branches wasmaximum in soil drench + foliar spray at 10%. The soildrench at 10% resulted in a statistically significant increasein shoot length, plant fresh weight and height, number offlowers and fruit fresh weight (Table 2). The increasedsupply of nutrients provided by the aqueous seaweedextract resulted in healthy root and shoot, improved heightand higher plant fresh weight.

As flower initiation and development are related not only tothe physiological age of the plants but also to their vegetativegrowth, superior reproductive performance was observed inplants treated with higher concentrations of seaweed extractsresulting in improved total fruit production. Such a correlationbetween vegetative and reproductive parameters is wellestablished (Crouch and van Staden 1992). The reproductivephase has a higher nutrient requirement which is fulfilled byapplication of higher concentrations of aqueous seaweedextract containing both macro- and micronutrients (Table 1).Enhanced vegetative growth in tomato plants at higherconcentrations of seaweed extract could be due to mineral

nutrients present in seaweed extract. The beneficial effect ofseaweed extract application can be attributed to its manycomponents working synergistically at different concentrations(Fornes et al. 2002). The present study clearly indicates thataqueous seaweed extract is able to enhance overall growth intomato plants as compared to the control plants.

Effect of seaweed extract on biochemical parametersof tomato

Photosynthetic pigments

The plants treated with seaweed extract in all three modesof treatment showed a significant increase in photosynthetic

Fig. 2 a–c Photosynthetic pigments in 120-day-old tomato leavestreated with aqueous extracts made from S. johnstonii applied as afoliar spray; b soil drench; and c soil drench + foliar spray.

□ Chlorophyll a, Chlorophyll b, and Carotenoids. Histogramswith different letters are significantly different. Error bars representthe standard error (n=27; P=0.05)

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pigments as compared to control (Fig. 2a–c). The foliarspray and soil drench + foliar spray treatments causedmaximum increase in levels of chl a, chl b, and carotenoidsat 10% seaweed extract (Fig. 2a, c), whereas soil drenchtreatment at same concentration led to a significant riseonly in chl a and carotenoids (Fig. 2b). From these results,it can be concluded that foliar spray is more effective inincreasing the photosynthetic pigments of tomato leaves.Enhanced levels of photosynthetic pigments in leaf tissueswith the application of seaweed extracts from Ascophyllumnodosum, either as a soil drench or as foliar spray, havebeen reported earlier in tomato (Whapham et al. 1993). Inanother study, Sargassum seaweed extract was found

effective in enhancing chlorophyll synthesis in Zea maysand Phaseolus mungo (Lingakumar et al. 2004).

Proteins

The application of aqueous seaweed extract in all theconcentrations and treatments resulted in a significantincrease in protein concentration. The highest proteinconcentration was measured with 10% seaweed extractapplication. Soil drench + foliar spray treated plants gavethe best response, followed by soil drench and foliar spray(Fig. 3a). Such a rise in protein content may be attributed tothe increased availability and absorption of necessary

Fig. 3 a Protein concentration, b soluble sugar concentration, creducing sugar concentration, d starch concentration, and e phenolconcentration in 90-day-old tomato leaves treated with aqueous extracts

made from S. johnstonii.□ Foliar spray, Soil drench, and Soildrench + foliar spray. Histograms with different letters are significantlydifferent. Error bars represent the standard error (n=27; P=0.05)

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elements (Ca, Na, Mg, Cu, and Zn) present in the aqueousextract that enhance the source efficiency of leaves. Z. maysand P. mungo responded well in maintaining high level ofsoluble proteins at 1% and 0.5% Sargassum extractapplication (Lingakumar et al. 2004). Significant increasein the levels of protein content of Sorghum vulgare wasrecorded with 1.5% seaweed liquid extract prepared fromHydroclathrus clathratus (Ashok et al. 2004). The increasein protein content at lower concentrations of liquid extractmay be due to absorption of most of the necessary elementsby the seedling (Anantharaj and Venkatesalu 2001).

Total soluble sugars and reducing sugars

In comparison to control, plants treated with aqueousseaweed extracts in all three treatments showed a signifi-cant increase in total soluble sugars and reducing sugars atall concentrations above 0.1%. For foliar spray, statisticallysignificant increase was recorded at 8% and 10% ofseaweed extract. With soil drench concentrations, statisticallysignificant increase was recorded for concentrations above0.8% as compared to the other treatments (Fig. 3b). Highestconcentration of reducing sugars was measured at 10%seaweed extract applied as a foliar spray. This wasstatistically similar to 10% of soil drench + foliar spray(Fig. 3c). The concentration of total soluble sugars andreducing sugars was maximum at higher concentrations ofspray and drench treatments. This may be consideredindicative of the fact that seaweed extracts stimulate variousbiological processes that increase the carbohydrate levels intomato plants. Similar observations were recorded for Vignacatajung using 10% Caulerpa racemosa extracts (Anantharajand Venkatesalu 2001). In V. sinensis, the sugar contentincreased up to 20% with S. wightii liquid extract but showeda decline at higher concentrations (Sivasankari et al. 2006).

Starch

The effect of various treatments on starch concentration didnot display any specific trend. Highest starch concentrationwas measured with application of 0.8% and 2% seaweedextracts when applied as a foliar spray. In the soil drench +foliar treatment, significant increase over control wasobserved with 0.1% and 0.8%, followed by 2% seaweedextract. In comparison to control, 6%, 8%, and 10%extracts showed a significant linear decrease in all thetreatments. In the soil drench treatment, starch concentra-tion declined with all seaweed extract applications(Fig. 3d). Thus, in the present study, maximum concentra-tion of starch was recorded in plants treated with loweraqueous seaweed extract concentrations. High starch con-centrations were measured in Z. mays seedlings treated with1% Sargassum seaweed extract (Lingakumar et al. 2004)

and in S. vulgare treated with 1.5% seaweed extractprepared from H. clathratus (Ashok et al. 2004).

Phenols

The tomato plants treated with soil drench + foliar sprayhad a linear increase in phenols compared to control, with amaximum phenol concentration observed at 10%. Soildrench-treated plants also resulted in a similar linearincrease except at 8%. Foliar application of the seaweedextract had the least effect on phenol concentration(Fig. 3e). Phenolic compounds play an important role inthe protection of the plant tissue against grazers, pathogens,and epiphytes (Pavia and Toth 2000). Similar increase inthe phenols of S. vulgare was recorded when treated withseaweed extract (Ashok et al. 2004).

Lycopene

There was a statistically significant linear increase in lycopenecontent in fruits with all three seaweed extract applications. Themaximum lycopene concentration was measured at 10%seaweed extract. Although not significant, variation wasrecorded between the three modes of treatment, with the soildrench application givingmaximum lycopene content (Fig. 4a).

Fig. 4 a, b Chemical constituents of 140-day-old fruits treated withaqueous seaweed extract. a Lycopene and b Vitamin C. □ Foliarspray, Soil drench, and Soil drench + foliar spray. Histogramswith different letters are significantly different. Error bars representthe standard error (n=27; P=0.05)

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The increase in lycopene content of the fruit can help tocounteract harmful effects of various substances that contrib-ute to age-related macular degeneration, cardiovasculardisorders and cancer in humans (Dorais et al. 2008). Due tothe increasing popularity of lycopene for use in food andnutritional supplements, there is a demand for developinglycopene-rich products and ingredients (Choudhary et al.2009). Lycopene concentration in fresh tomatoes may varyfrom 5 to 50 mg kg−1, depending upon cultivar, ripeningstage, and temperature during crop growth (Hadley et al.2002). In the present study, lycopene concentration in tomatofruits increased up to 9.4 mg g−1 in plants treated with higherconcentrations of seaweed extract. This provides evidencethat seaweed extract may be responsible for the translocationof cytokinin from the roots to the developing fruit orpossibly even for increasing the level or synthesis ofcytokinins within the fruit (Hahn et al. 1974). Withcytokinin-like substances present in aqueous extract of S.johnstonii, mobilization of nutrients to the fruit maycontribute to improving the lycopene concentration.

Vitamin C

The effect of various seaweed extract treatments on thevitamin C content of tomato fruit showed a linear increase,with the highest vitamin C content observed at 10%seaweed extract. The application of seaweed extract as asoil drench resulted in the maximum increase in vitamin Cas compared to the other two treatments (Fig. 4b). VitaminC, one of the most important of all vitamins, is an indicatorof fruit ripening. It plays an important role in variousbiochemical processes, such as collagen formation, ironabsorption and is involved in neurotransmission andimmune response in humans (Peng et al. 2008). Levelsof vitamin C generally increase as the fruit ripens frommature-green stage onward. In our investigation, vitaminC was maximum in tomato fruits when the plants weretreated with the highest concentration of seaweed extractas a soil drench.

Conclusions

The present study strongly suggests the presence of variousnutrients and hormone-like activity (auxin and cytokinin) inaqueous seaweed extract of S. johnstonii. An overallassessment of the effect of this extract on L. esculentumin terms of shoot length, root length, plant height, andconcentration of photosynthetic pigments, proteins, totalsoluble sugars, starch, phenols, lycopene, and vitamin Crevealed that this brown seaweed has the potential to beused as a biofertilizer. Since most of the parametersincreased linearly with application of higher concentrations

of the extract up to 10%, it would be interesting to test stillhigher concentrations of aqueous seaweed extract. Theextract can be recommended in agriculture as soil drenchand foliar spray to increase yield and/or quality of tomatoplants. This work further suggests that organic farmingusing such seaweed extracts can lead to the production ofhigh-quality fruits. Further, seaweed extracts can berecommended as biofertilizer to be used alone or incombinations with other biofertilizers and applied to eithersoil or foliage for improved growth. With abundantdistribution, great regeneration potential and easy masscultivation, the seaweed biofertilizer seems a feasiblesubstitute to synthetic fertilizers. If such seaweeds extractsare used for organic farming, our dependence on chemicalfertilizers can be reduced.

Acknowledgements Reeta Kumari is thankful to the UniversityGrants Commission, New Delhi (India) for the Rajiv Gandhi NationalFellowship award for 2008–2009. Inderdeep Kaur gratefully acknowl-edges the financial support provided by the Department of Scienceand Technology under their Women Scientist Scheme (WOS-A). Wealso thank Dr P.V. Subha Rao, Scientist, Central Salt and MarineChemical Research Institute, Bhavnagar, Gujarat (India) for his help incollection of seaweed material from the Port Okha, Gujarat. We thankDr. Rupam Kapoor, Associate Professor, Department of Botany,University of Delhi for her comments on the manuscript.

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