E-ISSN 2087-3956

| Nus Biosci | vol. 4 | no. 3 | pp. 97-137 | November 2012 || ISSN 2087-3948 | E-ISSN 2087-3956 |

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EDITORIAL BOARD:

Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia ([email protected])Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia([email protected])

Editorial Advisory Boards:Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia ([email protected])Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia([email protected])Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India ([email protected])Biomedical Sciences, Afiono Agung Prasetyo, Sebelas Maret University, Surakarta, Indonesia ([email protected])Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia ([email protected])Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia([email protected])Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia ([email protected])Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia ([email protected])Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia([email protected])Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt ([email protected])Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia ([email protected])Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia ([email protected])Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China ([email protected])Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia ([email protected])

Management Boards:Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta ([email protected])Associated Editor (English Editor), Wiryono, State University of Bengkulu ([email protected])Associated Editor (English Editor), Suranto, Sebelas Maret University SurakartaTechnical Editor, Ari Pitoyo, Sebelas Maret University Surakarta ([email protected])Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok,Indonesia ([email protected])

PUBLISHER:Society for Indonesian Biodiversity

CO-PUBLISHER:School of Graduates, Sebelas Maret University Surakarta

FIRST PUBLISHED: 2009

ADDRESS:Bioscience Program, School of Graduates, Sebelas Maret UniversityJl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: [email protected]

ONLINE:biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity Sebelas Maret University Surakarta

| Nus Biosci | vol. 4 | no. 3 | pp. 97-137 | November 2012 || ISSN 2087-3948 | E-ISSN 2087-3956 |

I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

N U S A N T A R A B I O S C I E N C E ISSN: 2087-3948Vol. 4, No. 3, pp. 97-100 E-ISSN: 2087-3956November 2012 DOI: 10.13057/nusbiosci/n040301

Determination of ethanol in acetic acid-containing samples by abiosensor based on immobilized Gluconobacter cells

ANATOLY N. RESHETILOV1,♥, ANNA E. KITOVA1, ALENA V. ARKHIPOVA2, VALENTINA A. KRATASYUK2,MAHENDRA K. RAI3

1Laboratory of Biosensors, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 5 Prospect Nauki,Pushchino, 142290, Russia. Tel.: +7-4967-318600, Fax: +7-495-9563370, email: [email protected]

2Department of Biophysics, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, 79 Svobodny Prospect, 660041, Russia3Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra, India

Manuscript received: 3 November 2012. Revision accepted: 28 November 2012.

Abstract. Reshetilov AN, Kitova AE, Arkhipova AV, Kratasyuk VA, Rai MK. 2012. Determination of ethanol in acetic acid containingsamples by a biosensor based on immobilized Gluconobacter cells. Nusantara Bioscience 4: 97-100. A biosensor based onGluconobacter oxydans VKM B-1280 bacteria was used for detection of ethanol in the presence of acetic acid. It was assumed that thisassay could be useful for controlling acetic acid production from ethanol and determining the final stage of the fermentation process.Measurements were made using a Clark electrode-based amperometric biosensor. The effect of pH of the medium on the sensor signaland the analytical parameters of the sensor (detection range, sensitivity) were investigated. The residual content of ethanol in acetic acidsamples was analyzed. The results of the study are important for monitoring the acetic acid production process, as they represent amethod of tracking its stages.

Key words: Gluconobacter, biosensor, ethanol, acetic acid

Abstrak. Reshetilov AN, Kitova AE, Arkhipova AV,. Kratasyuk VA, Rai MK. 2012. Penentuan etanol dalam sampel yang mengandungasam asetat dengan biosensor sel Gluconobacter yang diimobilisasi. Nusantara Bioscience 4: 97-100. Sebuah biosensor berdasarkanbakteri Gluconobacter oxydans digunakan untuk mendeteksi etanol pada sampel yang mengandung asam asetat. Pengukuran dilakukandengan elektroda Clark berdasarkan biosensor amperometrik. Uji ini diharapkan berguna untuk mengendalikan produksi asam asetatdari etanol dan menentukan tahap akhir proses fermentasi. Pengaruh pH pada stabilitas pengukuran dipelajari. Berbagai jenis larutanbufer (sitrat, Tris maleat, natrium fosfat) diuji untuk memilih varian optimal, yang merupakan bufer fosfat dengan pH dalam kisaran 6sampai 7 unit. Sampel yang dianalisis dengan asam asetat pada konsentrasi sesuai dengan fermentasi selesai (9%) diencerkan 80 kali.Sensor tes etanol diaktifkan dalam kisaran 0,0125-2,00 mM (0,0006-0,0092%). Kandungan etanol dalam sampel komersial asam asetatdari berbagai produksi dinilai. Hasil dari penelitian ini penting untuk memantau proses produksi asam asetat, karena mereka mewakilimetode pelacakan tahapannya.

Kata kunci: Gluconobacter, biosensor, etanol, asam asetat

INTRODUCTION

Bacteria of the genus Gluconobacter are widely used invarious biotechnological processes, in particular, inproduction of vinegar and acetic acid from alcohol-containing products. At the initial stage of the fermentationprocess, the bioreactor contains a maximum amount ofethanol, which decreases as the content of acetic acid goesup. The decrease of ethanol concentration down to a certainlevel indicates the completion of the process. A realfermentation process (modeling, measurement and control)of acetic acid production by the repeated batch methodusing Acetobacter strains is given in Hekmat andVortmeyer (1992). The fermentation was controlled bysuch parameters as acetic acid concentration, ethanolconcentration and optical density of cell suspension.According to the data of the work, the uptake of ethanoland accumulation of acetic acid had close-to-linear

dependences. Accumulation of acetic acid and uptake ofethanol were mutually dependent. Thus, the initial ethanolconcentration of 30±5 g/L was totally utilized within 25 h,and the initial level of acetic acid increased from 10±5 g/Lup to 95±5 g/L. The content of ethanol was determined bya gas sensor, and acetic acid was assayed by a gel-based pHelectrode.

There are various biosensor approaches to detection ofethanol and acetic acid, based on the use of enzymes ormicrobial cells (Tkac et al. 2002; Wang et al. 2006). Theyenable monitoring the formation of acetic acid by thecontent of ethanol in the fermentation medium. Still,approaches based on microbial biosensors making possibleethanol assays in the presence of acetic acid have not beendescribed.

The aim of the work was to assess the possibility ofassaying ethanol in acetic acid-containing samples by abiosensor based on G. oxydans bacteria and, for

N U S A N T A R A B I O S C I E N C E 4 (3): 97-100, November 201298

comparison, on alcohol oxidase. We assumed that thisassay could be useful for controlling acetic acid productionfrom ethanol and determining the final stage of the process.In spite of a significant dilution of the analyzed initialsample containing acetic acid, pH of the basic solutioninevitably changes, which can affect the biosensor signal.A comprehensive theoretical calculation of the model,including pH changes and biosensor responses in thisprocess, appears to be complex, due to which anexperimental verification was used. Special attention waspaid to the case simulating the final stage of acetic acidproduction, when the content of ethanol in the sample islow (from 1 down to 0.1%), and the concentration of aceticacid is high (of the order of 10%). The purpose of the workwas also to choose the type of buffer solution and its effecton the stability of the bioreceptor, as well as to study theparameters of the sensor when the optimal type of bufferwas used. Commercial solutions of acetic acid were takenas an approximation to the applied aspect of the problem.Publications on the subject have not investigated the issuein this statement of the problem.

MATERIALS AND METHODS

A microbial biosensor based on cells of Gluconobacteroxydans VKM B-1280 (purchased from All-RussianCollection of Microorganisms) and an enzyme sensorbased on alcohol oxidase (isolated from Hansenulapolymorpha NCYC 495 ln, activity 14 units/mg) (Ashin etal. 2004) were used for the ethanol assay.

The strain G. oxydans VKM B-1280 was grown on anutrient medium containing (g/L): sorbitol, 100; yeastextract, 10. The cells were grown for 18 h on a shaker (200rpm, 28°С) in 750 mL Erlenmeyer flasks containing 100mL growth medium. Biomass was separated bycentrifugation at 10,000 g for 5 min and washed twice withsodium phosphate buffer (30 mM, рН 6.6).

In formation of the microbial biosensor, cells wereimmobilized by their physical sorption on Whatman GF/Aglass fibre filters. For this, 1 mg biomass was applied on afilter and dried for 20 min at room temperature (frozenbiomass was used; during the preparation toimmobilization, cells were preliminarily unfrozen).

Alcohol oxidase was immobilized in a layer of DEAEdextran on nitrocellulose membranes activated withbenzoquinone (Zaitsev et al. 2007).

The bioreceptor (enzymes or cells immobilized onmembranes) 33 mm2 in size was fixed on the measuringsurface of a Clark oxygen electrode (Kronas Ltd., Russia).The rate of stirring the solutions by a magnetic stirrer was400 rpm. The measurements were carried out in an opencuvette (volume, 2 mL) by an IPC2L galvanostat/potentiostat (Kronas Ltd, Russia) connected to a computer.A 100-µL sample of a required concentration wasintroduced into the cuvette. The sample was diluted 1:20.The measurements were done at room temperature. A 30mM sodium phosphate buffer, pH 6.6, was used as a basicsolution. The registered parameter was the maximum rateof signal change (nA/s).

The pH dependences were studied using the followingbuffer solutions: sodium phosphate buffer, MacIlvaine’scitrate phosphate buffer and a buffer containing Tris(hydroxy-methyl) aminomethane maleate (Tris maleate).The molarity of the buffer solutions was 30 mM.

Samples simulating the main stages of the fermentationprocess were used for the analysis: 10% ethanol, the onsetof the process; 5% ethanol in 5% acetic acid, the midpointof the process; 1% ethanol in 9% acetic acid and 0.1%ethanol in 9% acetic acid, the completion of the process.

RESULTS AND DISCUSSION

Choice of the type of buffer solutionVarious types of buffers (sodium phosphate,

MacIlvaine’s citrate phosphate, Tris-maleate) were used. Itwas assumed that buffer solution components could affectin different ways the stability of cells to pH changes. Theconcentrations of ethanol and acetic acid in the initialsample were, respectively, 0.1% (22 mM) and 9% (1.5mM). This content of acetic acid corresponds to aminimum concentration of acetic acid in the fermentationmedium, at which the process is terminated. Formeasurements, the sample was diluted 80-fold.

The effect of the type of buffer on the stability of cellsis given in Figure 1. The range of investigated pH valuesfor MacIlvaine’s citrate phosphate buffer solution was 3.2-7 (curve 1). The bioreceptor enabled measurements withouta loss of activity within the pH range of 3.2 to 6.4. At pH 7,the measurement error exceeded 10%.

рН

3 4 5 6 7

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or re

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se, n

A/s

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

1

2a

3

2b

Figure 1. Dependences of the responses of a G. oxydans-basedbiosensor on pH of the buffer solution (1, citrate phosphatebuffer; 2 (a,b), sodium phosphate buffer; 3, Tris-maleate buffer).

When using sodium phosphate buffer, the bioreceptorwas stable within the pH range 5-7 in assaying ethanolsamples (curve 2a). When, assaying samples containingethanol and acetic acid (curve 2b), the sensor responseswere stable within the pH range of 6-6.6. When using abuffer solution containing Tris-maleate, the range of

RESHETILOV et al. – Biosensor for determination of ethanol in acetic acid 99

investigated pH values was 5-6.1 (curve 3). The responseswere observed to be decreased at a pH rise.

The type of buffer solution affected the magnitude ofsensor response. Thus, the highest response was obtainedusing a citrate buffer solution. However, in studies ofsensor stability in citrate buffer the responses decreased inthe first 6 h, which was not characteristic of other buffertypes. Sodium phosphate buffer was chosen as an optimalbasic solution and was used in further experiments.

Study of the major parameters of the sensorThe main analytical parameter of a biosensor is its

calibration dependence. It was plotted as a function ofethanol concentrations in the measuring cuvette. Figure 2(curve 1) presents the calibration dependence of abiosensor based on cells of the strain G. oxydans VKM B-1280. The range of assayed concentrations was 0.0125-2.00mM (0.0006-0.0092%) (the final concentration of ethanolin the measuring cuvette is given). Sensor responses wererecorded in a 30 mM sodium phosphate buffer solutionwith pH 6.6. The maximum sensitivity in the linear rangewas 1.2 (nA/s)/mM; the linear range of detection, 0.0125-0.5 mM (0.0006-0.023%). Curve 2 is a calibration curvefor ethanol samples containing acetic acid as a backgroundconcentration (0.11% (18.7 mM), concentration in thecuvette; in the initial sample, the concentration was 9%).The linear range was 0.0125-1.25 mM (0.0006-0.06%) ofethanol; the sensitivity in the linear range, 1.2 (nA/s)/mM.

Sens

or re

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se, n

A/s

0.0

0.5

1.0

1.5

2.0

2.5

Ethanol, mM

0.0 0.5 1.0 1.5 2.0 2.5

Time, s0 200 400 600 800 1000120014001600

Sign

al a

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itude

, nA

-42-40-38-36-34-32-30-28-26-24

1

2

Figure 2. Calibration dependences of a biosensor based on thestrain G. oxydans VKM B-1280 for detection of ethanol (1) andethanol in the presence of acetic acid (2); the dashed line showsthe maximum tangent slope at the initial segment of the curve.Insert, a typical shape of sensor response.

A bioreceptor based on G. oxydans cells was evaluatedfor the reproducibility of responses to samples containingethanol and acetic acid (a sample contained 0.025% (22mM) ethanol and 2.4% (375 mM) acetic acid, in thecuvette, the sample was diluted 20-fold). The coefficient of

variation was 6%. The measurements were done in asodium phosphate buffer solution of a 30 mMconcentration. The response time was 120 s. The sensorenabled 6 measurements per hour.

Assay of samples simulating the acetic acid productionprocess with various contents of ethanol in acetic acidsolution

Information on the ethanol content is vital for the aceticacid production process to be optimal, as its decrease to acertain final level (0.1%) indicates the completion of theprocess. We analyzed samples simulating acetic acidproduction at various stages of the process.

The values of sensor responses to samples simulatingvarious fermentation stages are given in Table 1. In theanalysis of a sample containing 9% (1.5 M) acetic acid(concentration in the cuvette, 0.11% (18.7 mM)) and 0.1%(22 mM) ethanol (0.00125% (0.26 mM)), the pH value ofthe initial buffer solution equal to 6.6 decreased by unity.For subsequent assays, the dilution was increased by anorder of magnitude, which in practice had no effect on pHof the buffer solution.

Table 1. Dependence of sensor responses on ethanolconcentrations in a model sample.

Sample рН ofsamples

Dilution ofsamples

with buffer

pH ofsamples inmeasuring

cuvette

Sensorresponse,

nA/s

0.1% ethanol in9% acetic acid

2.4 80-fold 5.5 0.200±0.005

1% ethanol in9% acetic acid

2.4 800-fold 6.6 0.206±0.004

5% ethanol in5% acetic acid

2.5 4000-fold 6.6 0.202±0.009

10% ethanol 5.0 8000-fold 6.6 0.200±0.019

Thus, to assess ethanol in assayed samples they shouldbe diluted 80 times (final dilution in a cuvette) and more.With this dilution, pH of the basic buffer solution changesinsignificantly.

Assay of ethanol in real samplesWe estimated the residual ethanol in samples of vinegar

produced by a microbiological method (Table 2). Thecorrelation coefficient of the data obtained using amicrobial sensor and enzyme (alcohol oxidase-based)sensor was 0.98.

Table 2. Content of residual ethanol in acetic acid.

Ethanol (mM)SampleG. oxydans Alcohol oxidase

Apple vinegar (Egorye) 12.0±0.7 11.9±1.1Apple vinegar (Abriko) 10.9±1.0 11.7±0.8Wine vinegar (Baltimor) 16.6±1.2 14.9±1.1

High dilutions of the initial sample simulating thecomposition of the fermentation medium in acetic acidproduction were shown to practically buffer low levels of


pH. At these dilutions with the basic buffer solution,ethanol concentrations corresponded to the linear range ofthe biosensor based on G. oxydans cells. Insignificant pHchanges caused by the presence of acetic acid did not affectthe measurement results when using this type of biosensor.The buffer systems studied had different effects on thebiosensor parameters, due to which an optimal (sodiumphosphate) buffer solution was chosen.

CONCLUSION

The optimal conditions for operating a Gluconobacteroxydans-based biosensor in an acetic acid-containingmedium were determined. Under these conditions, thebiosensor was shown to be capable of assaying the contentof ethanol in acetic acid-containing samples within thelinear segment (0.0125-0.5 mM) of the calibrationdependence. The data obtained indicate a possibility ofmonitoring the acetic acid production process by bothmicrobial and enzyme biosensors. The enzyme biosensorhaving a higher selectivity as compared with the microbial

biosensor can be used as a control for estimating the totalcontent of alcohols.

REFERENCES

Hekmat D, Vortmeyer D. 1992. Measurement, control, and modeling ofsubmerged acetic acid fermentation. J Ferment Bioeng 73 (1): 26-30

Tkac J, Vostiar I, Gemeiner P, Sturdik E. 2002. Monitoring of ethanolduring fermentation using a microbial biosensor with enhancedselectivity. Bioelectrochem 56: 127-129

Wang YF, Cheng SS, Tsujimura S, Ikeda T, Kano K. 2006. Escherichiacoli-catalyzed bioelectrochemical oxidation of acetate in the presenceof mediators. Bioelectrochem 69 (1): 74-81

Ashin VV, Toropova IA, Kuvichkina TN, Reshetilov AN. 2004. Acomparative characteristic of alcohol oxidase from methylotrophicyeasts Pichia methanolica and Hansenula polymorpha. Abstracts ofpapers of the 3rd Congress of Russian Biophysicists. Voronezh,Russia

Zaitsev MG, Ashin VV, Reshetilov AN. 2007. A novel method ofimmobilization of methylotrophic cells and alcohol oxidase isolatedfrom them for biosensor detection of lower alcohols. Book ofabstracts of the 3rd International School-Conference of YoungScientists “Important aspects of modern microbiology”. Moscow


Eco-friendly synthesis and potent antifungal activity of 2-substituted coumaran-3-ones

PRABHA SOLANKI1,♥, PRACHI SHEKHAWAT2

1Department of Chemistry, Vidyabharti Mahavidyalaya, C.K. Naidu Road, Amravati 444602, Maharashtra, India. Tel. +91-721-2662740, Fax. +91-7212662740, email: [email protected]

2Department of Pharmaceutics, Vidyabharati College of Pharmacy, Amravati, Maharashtra, India

Manuscript received: 16 September 2012. Revision accepted: 19 november 2012.

Abstract. Solanki P, Shekhawat P. 2012. Eco-friendly synthesis and potent antifungal activity of 2-substituted coumaran-3-ones.Nusantara Bioscience 4: 101-104. 3-halochromones (IIa-c and IIIa-c) have been synthesized by treating 1- (2-hydroxyphenyl)-3-methyl-1,3-propanediones (Ia-c) with bromine or sulphuryl chloride in dioxane respectively. These chromones were employed in the synthesisof 2-acetyl-coumaran-3-ones (IVa-f). These were subjected to Knoevenagel condensation to give 2-cinnamoyl coumaran-3-ones. Invitro assay and field trials of these compounds against Fusarium oxysporum were carried out to study the antifungal effect of targetcompounds. Compound Va was the most effective growth inhibitor of the pathogen, whereas Vc showed a little tendency and Vb, Vd,Ve and Vf hardly inhibits the growth.

Key words: microwave, cyclodehydration, Knoevenagel condensation

Abstrak. Solanki P, Shekhawat P. 2012. Sintesis ramah lingkungan dan potensi aktivitas antifungi dari 2-tersubstitusi coumaran-3-on.Nusantara Bioscience 4: 101-104. 3-halokromon (IIa-c dan IIIa-c) telah disintesis dengan memperlakukan 1- (2-hidroksifenil)-3-metil-1,3-propanedion (Ia-c) dengan brom atau belerang klorida dalam dioksan secara berturut-turut. Kromon ini digunakan dalam sintesis 2-asetil-coumaran-3-ona (IVa-f). Lalu, dilakukan kondensasi Knoevenagel untuk menghasilkan 2-cinnamoyl coumaran-3-on. Uji in vitrodan uji coba lapangan dari senyawa-senyawa ini terhadap Fusarium oxysporum dilakukan untuk mempelajari efek antifungi senyawatarget. Senyawa Va adalah inhibitor pertumbuhan patogen yang paling efektif, sedangkan Vc menunjukkan kecenderungan sedikit danVb, Vd, Ve dan Vf tidak menghambat pertumbuhan.

Kata kunci: microwave, siklodehidrasi, kondensasi Knoevenagel

INTRODUCTION

Synthons having chromone moiety are associated withvarious biological activities such as antibacterial (Tanaka etal. 2009), antifungal, antiallergic and diuretic (Abrahamand Rotella 2010). Substitution of halogen in thesemolecules enhanced the above activities, likewise 2-coumaranones i.e. 3H-2-benzofuranones are also proved tobe potential synthons for various products extended foragriculture or having physiological effects. Therefore,processes are continuously being sought which allow it tobe obtained rapidly and cheaply from inexpensivecommercially available products. Researchers have alsosynthesized coumaranone from cyclohexanone andglyoxalic acid in presence of dehydration catalyst (Vallejoset al. 1997). Process has been also described forpreparation of enol lactone 2-oxocyclihexidine acetic acidand to its application to preparation of 2-coumaranone by(Carmona et al. 1998). Benzofuranone derivatives found topossess potential antipsychotic (Aranda et al. 2008),anticancer (Charrier et al. 2009; Mishra et al. 2011),peroxidase activity (Ghadami et al. 2012), cytotoxicityactivity (Terasawa et al. 2001), antibacterial activity (Hadj-

Esfandiari et al. 2007) and other biological activities (Liand Chen et al. 2008; Adadiran et al. 2001). With referenceto observation and versatility of chromones andcoumaranones, attempts have been made to synthesize thecompound under microwave irradiation (Goncalo et al.1999) with a rapid environment benign, cleaner andcheaper work up.

MATERIAL AND METHODS

Eco-friendly synthesisAll chemicals and solvents were purchased from Sigma

Aldrich. Melting points were determined by open capillarymethods on a ‘Veego’ VMP-D apparatus and areuncorrected. TLC was done using silica gel G plates using3x8 cm (Sigma-Aldrich) and visualized in an iodinechamber. The IR spectra (KBr) were determined on "PerkinElmer 577 spectrometer and the values are expressed incm-1 and H1NMR (chemical shift in δ ppm) were recordedon Perkin Elmer R-32 and Varian XL-100A high NMRspectrophotometer using TMS as reference either in CDCl3

or DMSO-d6 as solvent. C, H and N analyses were carried


out on Carlo Erba 1106 Analyser (Italy). Physicalparameters and schematic diagram of this eco-friendlysynthesis can be shown in Table 1 and Figure 1.

3-bromo-2-methylchromones (IIa-c): 1- (2-hydroxy-phenyl)-3-methyl-1-propane diones (Ia-c) (10 mmol) wasdissolved in dioxane (0.5 mL) and solution of pure bromine(0.5 mL) in dioxane (15 mL) was added with constantstirring. The reaction mixture was warmed and kept for 30minutes. After cooling, the reaction mixture was dilutedwith water and then the crude product was filtered andcrystallized using ethanol to get 75-80% yield. Ia-IR in cm-

1-1630 (C=O), 1490 (C=C), 1340 (-yrone). PMR-δ 2.44 (s,3H, Ar-CH3), 2.62 (s, 3H, heteroaromatic), 7.25-8.00 (m,3H, Ar-H). UV λmax 310 mm.

3-Chloro-2-methylchromones (IIIa-c): A mixture of1- (2-Hydroxyphenyl)-3-methyl-1,3-propanediones (Ia-c)(10 mmol) and sulfuryl chloride (10 mmol) in dioxane (25mL) was irradiated under microwave at 450 W for 45 secwith intermittent heating. It was then diluted and crudeproduct thus obtained was crystallized from ethanol to get70-75% yield. IIIa-IR in cm-1-1640 (C=O), 1490 (C=C),1340 (-pyrone). PMR-δ 2.45 (s, 3H, Ar-CH3), 2.62 (s, 3H,heteroaromatic), 7.25-8.00 (m, 3H, Ar-H). UV λmax 305 mm.

2-Acetyl coumaran-3-ones (IVa-c): A solution of 3-halochromones (IIa-c) or (IIIa-c) (1 g) in ethanol (20 mL)was treated with aq. KOH solution separately and thereaction mixture was exposed to MW for 1 minute. Cooland diluted product was acidified with HCl. The crudeproduct thus obtained was crystallized from ethanol to getcompound (IVa-c) in 50-60% yield. IVa-IR in cm-1-3340 (-OH), 1625 (C=O), 1490 (C=C), 2945 (C-H). PMR - δ 2.44(s, 3H, Ar-CH3), 2.46 (s, 3H, COCH3), 6.25 (s, 1H,CO-CH), 7.21-8.00 (m, 3H, Ar-H). UV λmax 345 mm.

2-Cinnamoyl coumaran-3-ones (Va-f): A mixture of2-acetylcoumaran-3-one (IVa-c) (10 mmol) and aromaticaldehyde (20 mmol) in ethanol (20 mL) and few drops ofpiperidine (0.5 mL) was exposed to MW for 1 minute withintermittent heating. After cooling the reaction mixture wasdiluted, filtered and crystallized from ethanol to get 65-70% yield. Va-IR in cm-1-2950 (-OH),1600 (C=O). PMR-δ2.41 (s, 3H, Ar-CH3), 7.21-7.82 (m, 8H, Ar-H), 6.5-7.1 (dd,2H,-CH=CH).

Table 1. Physical parameters of the eco-friendly synthesis

Sr.No. Entry M.F. R R1 R2 M.W.

SecM.P.(°C)

1. IIa C11H9O2Br CH3 H - - 1322. IIb C10H7O2Br H H - - 1323. IIc C11H9O2Br H CH3 - - 934. IIIa C11H9O2Cl CH3 H - 45 1245. IIIb C10H7O2Cl H H - 40 1286. IIIc C11H10O2Cl H CH3 - 45 1217. IVa C11H10O3 CH3 H - 60 1268. IVb C10H8O3 H H - 55 1389. IVc C11H10O3 H CH3 - 60 14210. Va C18H13O3 CH3 H H 60 11911. Vb C19H15O4 H H OCH3 60 21112. Vc C17H11O4 H H H 60 15413. Vd C18H13O4 H H OCH3 65 13414. Ve C18H13O3 H CH3 H 60 14015. Vf C19H15O4 H CH3 OCH3 65 149

Figure 1. Scheme of the eco-friendly synthesis

In vitro assay of target compounds against Fusariumoxysporum

Application and utility of heterocycles in agriculturecrop to eradicate the alarming diseases has drawn theattention of research scientist. The following samples weretested at the concentration noted against Fusariumoxysporum by using poisoned food technique (Schmitz,1930). One week old culture of F. oxysporum was grownon potato dextrose agar medium (PDA) in petri plates forassessing efficiency of newly synthesized samples.Solutions of 100 ppm concentration were taken in 250 mLconical flask containing 100 mL of sterilized and meltedpotato dextrose agar medium, mixed thoroughly by gentleswirling the flask and poured into a sterile petri disc andallowed to solidify. A 8 mm culture disc pathogen is F.oxysporum was inoculated and the plates were incubated inan inverted position at room temperature. Inoculated PDAmedium without sample served as control. Threereplications were maintained. The mean radial growth ofthe colony was measured at 48, 72 and 96 hrs afterinoculation respectively. The results were expressed aspercent inhibition over control (Table 2). The percentinhibition of the growth was calculated by the formula ofVincent (1927).

I = (C-T)/C X 100

I: Inhibition of mycelia growth, C: Growth in control,T: Growth in treatment

SOLANKI & SHEKHAWAT – Synthesis and antifungal activity of 2-substituted coumaran-3-ones 103

Table 2. Radial growth of F. oxysporum on PDA medium at 100ppm concentration of samples at 48, 72 and 96 hrs of incubation;and percent growth inhibition of F. oxysporum at 100 ppm after96 hours.

Mean radii growth (mm)

Sample48 hrs 72 hrs 96 hrs

% Growthinhibition

after 96 hrs

Va 0.00 0.00 0.00 100.00Vb 24.33 44.66 68.83 0.72Vc 15.83 26.50 41.83 39.66Vd 25.33 45.16 69.50 -0.24Ve 24.33 46.16 69.16 0.28Vf 24.66 45.83 69.33 0.00

Control 25.66 46.33 69.33 -

Among the six samples tested, sample Va was the mosteffective recorded percent growth inhibition over controlafter 96 hrs of incubation. Compound Vc also showed39.66% of inhibition where as samples Vb, Vd, Ve and Vfdid not inhibit the growth of F. oxysporum.

Control of fungal diseases in agricultural cropsThe most general means of controlling plants diseases

is in field, green house and sometimes in storage, throughthe use of chemical compounds that are toxic to the pathogens,such chemical either inhibits germination, growth andmultiplication of the pathogen or outright lethal to thepathogen. In this part the synthesized compounds Va andVc had been tested on Bengal grams (Chickpea or Gram;Cicer arietinum L.) whose growth is generally retarded dueto vascular cause by F. Oxysporum (Table 3-4).

Gram is mostly affected by wilt F. oxysporum. It ismost important rabi crop grown on large scale in India. Soin the present work, in vitro assay of compound I and IVthat have shown to possess a remarkable fungicidalactivity, had been selected to evaluate the antifungalactivity against F. oxysporum, a pathogen of Bengal gramcrop. Post culture method is adopted in this experiment.Studies have been have carried out in triplicate to select therequisite concentration of newly synthesized fungicide forplant growth.

Design of experimentSeven pots of the size 30x20 cm were taken for the

three replication to increase the precision of theexperiment. Approximately 1.5 kg soil for each pot wasautoclaved for complete sterilization. A control pot C wasfilled up with sick soil 100 g of fungal culture had beenmixed with soil in 100: 1 proportion. Three pots for eachsample were filled with sick soil and labeled as P1-P3 andQ1-Q3. Pregerminated seeds of Bengal gram were procuredfrom Krishi Vigyan Kendra Durgapur, Amravati for thesetrials. Aqueous solution of 100 pp, of the sample P and Qwere prepared and ten seeds for each replication, soaked inthe sample solution and allowed to dry. Treated seedssowed in pot P1-P3 and Q1-Q3. Seeds soaked in distil waterwere sown in control pot C. Some physical parameters like(a) percent germination (b) number of leaves per plant (c)plant height and (d) mortality had been observed and notedperiodically.

Table 3. Fungicide effect of newly synthesized compound Va onCicer arietinum

ReplicationsObservationsfor controlexperiment A B C

Perio-dicitydays

a b c d a b c d a b c d a b c d8 20 8 4 80 70 7 5 30 80 8 5 20 75 8 5 2515 - 30 10 - 80 75 20 - 85 74 20 - 88 72 25 -30 - 25 10 10 90 25 - - 95 25 - - 75 29 -45 - 25 10 5 102 27 - - 105 29 - - 98 32 -75 - 26 10 - 125 30 - - 115 22 - - 112 35 -120 - - - - 130 35 - - 120 32 - - 118 35 -

Table 4. Fungicide effect of newly synthesized compound Vc onCicer arietinum.

ReplicationsObservationsfor controlexperiment A B C

Perio-dicitydays

a b c d a b c d a b c d a b c d8 20 8 4 80 70 6 4 30 92 8 5 28 65 6 5 -15 - 30 10 - 75 62 20 25 68 65 22 32 70 70 25 -30 - 25 10 10 - 85 26 - - 80 25 - - 80 30 -45 - 25 10 5 - 98 26 - - 95 27 - - 92 33 -75 - 26 10 - - 115 24 - - 120 29 - - 118 35 -120 - - - - - 133 23 - - 130 30 - - 125 35 -Note: a-% Germination, b-No. of leaves per plant, c-Plant height,d-Mortality


Cyclodehydration of substituted 1, 3-propanedione (Ia-c)with bromine in dioxane gave 3-bromo-2-methyl chromones(IIa-c), which is characterized by pyrone nucleus. IR ofIIa shows characteristic peak at 1340 cm-1 (-pyrone) anddisappearance of phenolic-OH singlet of 1,3-propane-diones. PMR spectra also shows prominent signals at 2.44(s, Ar-CH3), 2.63 (s, Ar-CH3 heteroaromatic) and 7.25-8.00(m, 3H, Ar-H) whereas signals of keto-enol tautomers of 1,3-dione get disappeared due to cyclodehydration. Similarly,1,3-propanedione (Ia-c) reacts with sulfuryl chloride indioxane under microwave at 450 W for 45 sec gave 3-chloro-2-methylchromones (IIIa-c) in 80% yield. IR andPMR showd presence of -pyrone nucleus and absence ofsignals of phenolic-OH and keto-enol tautomerism.

2-Acetylcoumaranones (IVa-c) have been synthesizedfrom a solution of 3-halochromones (IIa-c) or (IIIa-c) inethanol in aqueous alkaline medium under microwaveexposure for one minute. PMR signal at 6.25 δ is identifiedas (-COCH) coumaran proton and a acetyl signal at 2.46 δ.In IR spectra frequency for -pyrone (1340 cm-1) getdisappeared, a signal at 3340 cm-1 (-OH) also confirm thestructure (IVa-c). These compounds (IVa-c) are subjectedto condensation with aromatic aldehyde (0.02 mol) inethanolic condition in presence of piperidine undermicrowave radiation. The absence of acetyl signal andappearance of-COCH=CH-dd at 7.19-7.25 range confirmsthe presence of cinnamoyl group and a singlet at 9.18 δ for-OH group. IR frequency at 2950 cm-1 (-OH) and 1600


(>C=O) also correlates with the structure as 2-cinnamoylcoumaran-3-ones (Va-f).

A remarkable antifungal activity was shown by compoundVa and Vc. Fungicidal activity of these compounds wasfound to be excellent in different trials in vitro assayagainst. F. oxysporum. Compound Va had shown to havebetter fungicidal effect as compared to compound Vc. Incontrol experiments rate of mortality is found to be veryhigh due to sick soil, which diminished in remarkableextent in three trials where the seeds were treated withsynthesized compounds. Thus synthesized heterocycles arefound to possess potential antifungal activity

CONCLUSION

Microwave assisted organic synthesis has attractedattention in recent years due to enhanced reaction rates,higher yields, improved purity, ease of work up afterreaction and eco-friendly reaction conditions compared tothe conventional methods. Some of the synthesizedcompounds possess potent antifungal activities againstFusarium oxysporum. Compound Va was the mosteffective growth inhibitor of the pathogen, whereas Vcshowed a little tendency and Vb, Vd, Ve and Vf hardlyinhibits the growth. Candidly, these are few prototype trialstaken to root out the overwhelming situation prevailing inbiosphere. This work will definitely provide an access toorganic chemists synthesizing ever lasting number ofcompounds of high potent which may serve mankind.

ACKNOWLEDGEMENTS

The author expresses their sincere thanks to thePrincipal of Vidya Bharti Mahavidyalaya, Amravati,Maharashtra, India and the Director of Krishi VigyanKendra, Durgapur Badnera, Maharashtra, India forproviding necessary laboratory facilities.

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In vitro rapid multiplication of Stevia rebaudiana: an important naturalsweetener herb

SHIVAJI DESHMUKH♥, RAVINDRA ADEDepartment of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India. Tel: +91-721-2662206 to 8, Fax: +91-721-

2662135, 2660949, email: [email protected]

Manuscript received: 20 April 2011. Revision accepted: 5 November 2012.

Abstract. Deshmukh S, Ade R. 2012. In vitro rapid multiplication of Stevia rebaudiana: an important natural sweetener herb. NusantaraBioscience 4: 105-108. Stevia rebaudiana Bertoni, belonging to family Asteraceae and natural sweet plant, but due to poor seedviability, fertility and vigor, Stevia cultivation is a challenging task. In the present study in vitro rapid multiplication method wasestablished for S. rebaudiana by inoculating explants on M.S. medium, supplemented with different combination of phytoharmone. Themaximum number of shoots (18.3±0.8) was obtained on M.S. medium supplemented with BAP + KIN (1.5 + 0.5 mg/L). The highestrooting percentage (95.25) was observed with (IAA 0.1 mg/L). The rooted plants were successfully established firstly in soil with cocopeat (1:1) and then directly in ordinary soil.

Key words: Stevia rebaudiana, in vitro culture, multiplication, sweetener, micropropagation.Abbreviations: IAA: Indole-3-acetic acid, BAP: 6-Benzyl amino purine, KIN: Kinetin, GA: Gibberellic acid. NAA: 1 Naphthaleneacetic acid

Abstrak. Deshmukh S, Ade R. 2012. Perbanyakan cepat secara in vitro Stevia rebaudiana: herbal pemanis alami yang penting.Nusantara Bioscience 4: 105-108. Stevia rebaudiana Bertoni, anggota suku Asteraceae merupakan tanaman pemanis alami, namunkarena viabilitas, kesuburan dan kekuatan benih yang buruk budidaya Stevia menjadi tugas yang menantang. Dalam penelitian inimetode perbanyakan cepat secara in vitro dilakukan pada S. rebaudiana dari eksplan inokulasi pada media MS, dilengkapi dengankombinasi fitoharmon yang berbeda. Jumlah maksimum tunas (18,3±0,8) diperoleh pada media MS dengan BAP + KIN (1,5 + 0,5mg/L). Persentase perakaran tertinggi (95,25) diamati dengan (IAA 0,1 mg/L). Tanaman berakar berhasil ditanam pertama kali padatanah dengan coco peat (1:1) dan kemudian langsung di tanah biasa.

Kata kunci: Stevia rebaudiana, kultur in vitro, perbanyakan, pemanis, mikropropagasi.

INTRODUCTION

Stevia rebaudiana Bertoni, the member of the familyAsteraceae, is a perennial herb which can be growing up to1 meter (Kinghorn et al. 1985; Handro et al. 1989; Tadhaniet al. 2005). It is natural sweetener plant called as “sweetweed”, “sweet leaf” and “honey leaf” (Ahmed et al. 2007).The leaves of Stevia are source of glycoside, viz steviosideand rebaudioside which are 100-300 time sweeter thansucrose (0.4% solution) but zero calories (Mousumi 2008),hence it is important medicinal plant and it has beentraditionally used for hundreds of year in Paraguay andBrazil in South America continent to sweeten tea andmedicine and also used as a sweet treat (Tiwari 2010). It isrecommended for diabetes and has been extensively testedon animal and human with no side effects (Megeji et al.2005). The crude extracts from leaves have been used fewdecades to sweeten soft drinks and other foods(Komissarenko et al. 1994).

The stevioside, can be used in tea and coffee, cooked orbaked goods, processed foods and beverages, fruit juices,tobacco products, pastries, chewing gum and coldrink etc.Stevioside have zero calories and can be used wherever

sugar is used, including in bakery. Stevia has generatedmuch attention with the rise in demand for lowcarbohydrate, low sugar food alternative. Stevia also hasshown promise in medical research for treating obesity andhigh blood pressure, due to these important medicinalproperties the Stevia is being cultivated in Japan, Taiwan,Philippines, Hawaii, Malaysia and overall South Americaand used in several food and pharmaceutical products (Daset al. 2011).

The main problem in cultivation of Stevia is that theplant is heterozygous. Self incompatible nature of flowersleads to lack of fertilization, poor seed viability and vigor,due to this plant propagation by seed is not efficient.(Tadhani and Rema 2006; Rathi and Arya 2009).Propagation by seeds does not allow the production ofhomogeneous population, resulting in great variability inimportant features like sweetening level and composition(Tamura et al. 1984). Due to such difficulties in cultivationof Stevia, tissue culture is the only rapid process for themass propagation. The present study was carried out tooptimize a suitable and efficient protocol for In vitro rapidmultiplication of S. rebaudiana Bertoni.



Collection and surface sterilization of explantsStevia rebaudiana plants were collected from the

Government Nursery, Pune University, Pune, Maharashtra,India. The twigs about 5-6 cm were taken and the leaves,auxiliary buds and apical buds were first washed in runningtap water, then treated with 0.1% (w/v) Bavistin solutionfor 5 min to remove superficial dust particle as well asfungal spores. After that the explants were treated with70% alcohol for 2 min followed by 4-5 time wash bydouble distilled water to remove bacterial contaminants.Sodium hypochlordte (0.3%) also used for thedecontamination. It was again treated with 0.1% HgCl2

aseptically for 3-4 min and washed with sterile distilledwater 4-5 times.

Inoculation of explants on the multiplication mediumThe surface sterilized auxiliary bud were aseptically

inoculated vertically on MS medium (pH 5.7)supplemented with specific concentration of growthregulators (BAP, KIN and NAA) singly as well as incombination, 0.7 % agar was used as a gelling agent and 30gm/lit sucrose was used as a carbon source.

Culture conditionsAll the standard conditions were provided such as

photoperiod was 16 hrs light (2000 lux) to 8hrs darkness.The cultures were maintained at 26±1°C and 70%humidity. Subculturing was done for every fortnight andwell-grown sub-cultured shoots were further inoculated onmultiple shoot formation medium. Regenerated multipleshoots were cut and individual shoots were placed in MSmedium containing different concentrations of IBA, NAAand IAA for root induction.

Figure 1. In vitro multiplication of S. rebaudiana from nodal explants on MS medium supplemented with BAP + Kin (1.5+1.0). A.After 15 days, B. After 30 days, C. After 45 days of culture. D. Adventitious root formation from micro-cuttings on MS supplementedwith IAA (0.1 mg/L). E. Growth of Stevia in plastic pot containing coco peat and soil 1:1. F. In ordinary soil.

A

FD E

B C

DESHMUKH & ADE – In vitro rapid multiplication of Stevia rebaudiana 107

Hardening of plantsIn vitro rooted shoots were kept under normal growth

room condition for 7-8 days until the induced roots becomepartially brown. The shoots were taken out from culturebottle carefully and medium attached to the roots weregently washed out with running tap water. The rootedplants treated with 0.1% bavistin (antifungal) for 1-2 minthen transferred on soil: coco peat (1:1) for hardening thendirectly in ordinary soil (Figure 1).


Shoot-apex were inoculated on MS medium supple-mented with different concentration of BAP (0.5, 1.0, 1.5,2.0 mg/L) and KIN (0.5, 1.0, 1.5, 2.0 mg/L) alone or BAPwith KIN or with NAA (0.5, 1.0mg/L) as shown in Table 1.This shows different response during the primaryestablishment period. After 15 days, multiple shootsemerged directly from auxiliary node of cultured explants.The response of explants to the treatment is presented inTable1. The maximum number of shoots was observed onMS medium containing BAP (1.5 mg/L) among singlehormonal set. Similar findings were recorded by (Sivaramand Mukundan 2003). BAP + KIN (1.0 + 0.5 mg/L) andBAP + KIN (1.0 + 1.0 mg/L) also shows significantmultiplication. The response was best at BAP + KIN (1.5 +0.5 mg/L) combination, where highest percentage ofexplants showing shoot proliferation was found to be89.25%,whereas highest average number of total shoot wasfound to be 18.3 + 0.8, with average length 4.8 + 0.8 cmwere recorded (Table 1). Similar results have already beenreported in Fragaria indica (Bhatt et al. 2000). Patil et al.(1996) reported that shoot tips and auxiliary buds producemultiple shoots. When the explants were inoculated on MSmedium with IAA + BAP (0.5 + 5.0 mg/L) supplementedwith 10 mg/L GA. The numbers of multiple shoots werearound 20-25 per plant.

Multiple shoot formation requires the presence ofcytokinins in the culture medium (Tadhani and Rema2006). In present study the BAP containing medium isbetter for the shoot formation at lower concentration ascompared to KIN. However Tamura et al. (1984) reportedthat high concentration of KIN (10 mg/L) was required formultiple shoot production in S. rebaudiana. Progressivelyhigher concentration of BAP resulted in decreasingmultiple shoot formation in all the explants of Stevia.(Table 1)

Although the process of in vitro rooting is a laborintensive in the micropropagation studies of Stevia, but itseems to be an essential step for plant survival. Theaddition of auxin at certain level enhances the rootformation. Micro cuttings taken from in vitro proliferatedshoots were implanted on MS medium containing differentconcentrations 0.1mg/L, 0.3 mg/L, 0.5 mg/L, 0.8 mg/L, 1.0mg/L, of IAA, NAA and IBA for rooting. Here eachtreatment consists of four replications and in eachreplication 10 explants were used.

Within 6-12 days root initiation starts in the MSmedium with IAA 0.1 mg/L and it shows a best response

for rooting with highest length of root (4-5cm), number ofroots 12-13 and maximum root induction (95.25%) asshown in Table 2. It was observed that the root inductiongradually decreased with increased concentration of auxin.There was not any satisfactory root induction in anothercase except IAA. Similar findings were recorded inChrysanthemum morifolium (Hoque et al. 1995), Pigeon pea(Sivaprakash et al. 1994), Vitex negundo (Thiruvengadamet al. 2000) and Psoralea corylifolia (Jeyakumar et al.2002). However Tadhani et al. (2005) reported 0.1mg/LIBA was the best concentration for rooting.

In vitro rooted shoots were kept under normal growthroom condition for 2 weeks until the induced roots becomepartially brown. The shoots were taken out from growthroom and from the culture bottle carefully and gentlywashed with running tap water. The rooted plant wastreated with 0.1% Bavistin for 1 minute and transferred tosoil and coco peat (1:1) for primary hardening followed byordinary soil in natural environment.

Table 1. Effect of auxin and cytokinin concentration on in vitromultiplication.

Growth regulator(mg/L)

Explantsproliferation

(%)

Average no.of shoots

Averagelength of

shoots (cm)BAP 0.5 49.50 5.1+ 1.7 2.6 + 0.5

BAP 1.0 55.25 4.2 + 1.3 2.3 + 0.4

BAP 1.5 68.00 4.1 + 1.4 3.3 + 0.6

BAP 2.0 57.50 4.5 + 1.5 2.4 + 0.5

KIN 0.5 42.00 3.6 + 0.9 2.3 + 0.4

KIN 1.0 60.25 3.2 + 0.9 3.4 + 0.5

KIN 1.5 58.50 3.2 + 0.9 2.4 + 1.0

KIN 2.0 52.25 3.6 + 0.9 1.6 + 0.6

BAP+KIN (1.0+0.5) 67.25 13.4 + 1.2 3.5 + 0.5

BAP+KIN (1.0+1.0) 76.50 14.4 + 1.7 3.4 + 0.5

BAP+KIN (1.5+0.5) 89.25 18.3 + 0.8 4.8 + 0.4

BAP+KIN (1.5+1.0) 57.50 12.8 + 0.9 4.4 + 0.5

BAP+KIN (2.0+1.0) 56.25 11.6 + 0.8 3.4 + 0.5

BAP+KIN (2.0+0.5) 59.50 11.3 + 0.6 1.6 + 0.6

BAP+NAA (1.0+0.5) 35.25 7.4 + 0.5 1.6 + 0.5

BAP+NAA (1.0+1.0) 34.50 4.3 + 0.4 3.3 + 0.4

BAP+NAA (1.5+0.5) 37.40 12.8 + 0.9 2.6 + 0.5

BAP+NAA (1.5+1.0) 44.50 11.3 + 0.6 2.7 + 0.4

BAP+NAA (2.0+0.5) 42.00 11.5 + 0.5 3.5 + 0.5

BAP+NAA (2.0+1.0) 52.50 13.9 + 0.8 1.6 + 0.5

NAA +KIN (1.0+0.5) 26.00 5.7 + 0.8 1.7 + 0.4

NAA +KIN (1.0+1.0) 45.25 5.0 + 0.8 1.5 + 0.5

NAA +KIN (1.5+0.5) 56.50 5.7 + 0.8 1.4 + 0.5

NAA +KIN (1.5+1.0) 35.25 2.5 + 0.5 3.6 + 0.5

NAA+KIN (2.0.+0.5) 42.00 3.7 + 0.8 2.8 + 0.6

NAA +KIN (2.0+1.0) 48.25 12.8 + 0.9 1.4 + 0.5

Note: * Each treatment consists of three replications and in eachreplication 10 explants were used.


Table 2. Effect of different type of auxin on adventitious rootformation.

Auxin Conc.(mg/L)

Rootinitiation

(days)

Rootinitiation

(%)

Averageno. ofroots

Averagelength of

roots (cm)0.1 6-8 95.25 12.8 + 0.6 4.7 + 0.40.3 10-11 92.0 10.7 + 0.8 2.4 + 1.00.5 7-8 85.0 9.5 + 0.5 1.4 + 0.50.8 10-12 65.50 7.4 + 0.5 3.5 + 0.5

IAA

1.0 9-10 67.75 5.1 + 0.9 2.4 + 0.50.1 7-8 70.35 5.2 + 0.4 2.3 + 0.40.3 7-8 65.25 4.6 + 0.5 2.7 + 0.40.5 7-8 58.50 4.7 + 0.4 1.4 + 0.50.8 7-8 62.00 4.5 + 0.5 2.4 + 1.0

NAA

1.0 7-8 51.50 3.5 + 0.5 2.7+ 0.60.1 8-10 65.75 7.4 + 0.5 1.9 + 0.80.3 10-12 60.25 6.5 + 0.5 2.4 + 1.00.5 10-12 45.50 4.5 + 0.5 1.5 + 0.50.8 8-9 48.00 4.3 + 0.4 2.4 + 0.5

IBA

1.0 8-9 42.75 4.6 + 0.5 3.6 + 0.5Note: *Each treatment consisted of three replications and in eachreplication 10 explants were used.

CONCLUSION

Stevia is an important sweetener herb. In normalpropagation it is very difficult to regenerate due to variousreasons like heterozygous nature, self incompatibility offlowers, lack of efficient fertilization and most importantlypoor seed viability, hence there is urgent need to developefficient protocol for rapid multiplication and thusconservation, because till date no appropriate and efficientmethod is available for its regeneration. Since tissue culturetechnology is the only process for the mass propagation,this rapid and efficient regeneration protocol provides agood platform for the multiplication and effectiveconservation of this important plant.

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Tagetes erecta mediated phytosynthesis of silver nanoparticles: an eco-friendly approach

UMESH P. DHULDHAJ1,2, SHIVAJI D. DESHMUKH1, ANIKET K. GADE1, MADHU YASHPAL2,MAHENDRA K. RAI1,♥

1Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India. Tel: +91-721-2662206 to 8, Fax: +91-721-2662135, 2660949, email: [email protected]

2 Department of Botany, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India.

Manuscript received: 27 September 2012. Revision accepted: 18 November 2012.

Abstract. Dhuldhaj UP, Deshmukh SD, Gade AK, Yashpal M, Rai MK. 2012. Tagetes erecta mediated phytosynthesis of silver nanoparticles:an eco-friendly approach. Nusantara Bioscience 4: 109-112. Nanotechnology is a multidisciplinary field having applications in thevarious fields like medicine, pharmacy, engineering and biotechnology. An important step in nanotechnology is to develop simple andeco-friendly method for the nanomaterial synthesis. Here we describe simple and eco-friendly method for synthesis of silvernanoparticles by extract of Tagetes erecta plant leaves. The phytosynthesis (synthesis by plant) of silver nanoparticles was detected bycolor change from light-green to dark-brown. Synthesis of silver nanoparticles was confirmed by UV-Vis spectrophotometry, furthercharacterization includes nanoparticle tracking analysis system (NTA) (LM20) and transmission electron microscopy (TEM). TEManalysis confirms the synthesis of the polydispersed spherical silver nanoparticles of 20-50 nm, with the average size of 30 nm.

Key words: Tagetes erecta, eco-friendly, nanoparticles, phytosynthesis, TEM.

Abstrak. Dhuldhaj UP, Deshmukh SD, Gade AK, Yashpal M, Rai MK. 2012. Fotosintesis nanopartikel perak yang diperantarai olehTagetes erecta: pendekatan ramah lingkungan. Nusantara Bioscience 4: 109-112. Nanoteknologi merupakan bidang multidisiplin yangdapat diaplikasikan pada berbagai bidang seperti kedokteran, farmasi, teknik, dan bioteknologi. Sebuah tahap penting padananoteknologi adalah mengembangkan metode yang sederhana dan ramah lingkungan untuk menyintesis nanomaterial. Dalampenelitian ini, digambarkan metode sederhana dan ramah lingkungan untuk sintesis nanopartikel perak oleh ekstrak daun tanamankenikir Tagetes erecta. Fotosintesis (sintesis oleh tanaman) dari nanopartikel perak terdeteksi oleh perubahan warna dari hijau-terang kegelap-coklat. Sintesis nanopartikel perak dikonfirmasi dengan spektrofotometer UV-Vis, lebih lanjut karakterisasi meliputi sistemanalisis pelacakan nanopartikel (NTA) (LM20) dan mikroskop elektron transmisi (TEM). Analisis TEM menegaskan terjadinya sintesisbola nanopartikel perak dari 20-50 nm yang multidispersi, dengan ukuran rata-rata 30 nm.

Kata kunci: Tagetes erecta, ramah lingkungan, nanopartikel, fotosintesis, TEM.

INTRODUCTION

Nanotechnology is the branch of science dealing withthe synthesis of nanomaterials and nanoparticles of size 1-100 nm and their technological applications in variousfields (Tenover 2006). The combination of thenanotechnology and biology presents the new branch ofnanotechnology called Nanobiotechnology. It deals withthe application of biotechnological principle for thedevelopment of devices, and systems at nano level(Hassellov et al 2008; Kholoud et al. 2010).

There are different methods for nanoparticles synthesisas chemical, physical and a recently developed biologicalmethod. Biological method is cost-effective and eco-friendly than chemical and physical methods (Rai et al.2008; Raveendran et al. 2003). In biological method, fungi,bacteria and plants are used for the synthesis ofnanoparticles. Biosynthesis of inorganic materials,especially metal nanoparticles using microorganisms(Mandal et al. 2006, Gade et al. 2010) and plants (Gardea-Torresdey et al. 2003) were carried out. Various plants and

fungi having the potential of metal nanoparticle synthesis,proving that biological synthesis of metal nanoparticles issimple and cost effective (Rai et al. 2008). Among thebiological agent Fusarium sp. (Ingle et al. 2008, 2009;Bawaskar et al. 2010), Aspergillus niger (Gade et al. 2008),was proved to be a novel agent for synthesis of silvernanoparticles.

Use of plant extracts for the nanoparticles synthesis israpid, eco-friendly and simple alternative to bacterial andfungal system as leaf extract is easily prepared and there isno need of isolation, culture and maintenance of bacterialand fungal culture. Plant extracts have more potential ofreducing metal ions than microbes (Rai et al. 2008). Thesuccessful examples of synthesis of nanoparticles eitherintracellularly or extracellularly include geranium leaf-extract (Shivshankar et al. 2003, 2004, 2005) sun driedleaves of Cinnamomum (Huang et al. 2007), Azadirachtaindica (neem) (Shankar et al. 2004), Aloe vera (Chandranet al. 2006), Capsicum annuum (Li et al. 2007), Caricapapaya (Mude et al. 2009), Opuntia ficus-indica (Gade etal. 2010b), and Murraya koenigii (Bonde et al. 2012).


The present study focuses on the development of thesimple, novel and eco-friendly method for phytosynthesisof silver nanoparticles from Tagetes erecta.


Synthesis of silver nanoparticlesLeaves of the Tagetes erecta were collected from the

garden of S.G.B. Amravati University, Amravati,Maharashtra, India. After several washes with tap water,leaves were surface sterilized with HgCl2 (0.1%) for 1-2min to remove microbial contamination, if any. Theseleaves were cut into small pieces and crushed with mortarand pestle by adding little amount of double sterilizeddistilled water. The extract obtained was filtered withWhatman filter paper No. 42, the volume was adjusted to100 ml by double sterilized distilled water. The extract waschallenged with the silver nitrate (1mM) solution, andincubated at room temperature. Only leaf extract withoutsilver nitrate (1 mM) treatment was used as the control andthe experiments were carried out in triplicate.

Detection and characterization of silver nanoparticlesVisual observation

Synthesis of silver nanoparticles was detected bychange of color from light-green to dark-brown of thefiltrate challenged with silver nitrate.

By UV-visible spectrophotometerThe synthesis was confirmed with the help of UV-Vis

spectrophotometer (Perkin-Elmer, Lambda 25) by scanningthe absorbance spectra in the range of 200-800 nmwavelengths.

Nanoparticle tracking and analysis system (NTA) (LM 20)The nanoparticles tracking analysis was carried out by

using the liquid sample of silver nanoparticles prepared bydiluting with the nuclease free water and 0.5 ml of dilutedsample was injected onto the sample chamber and observedthrough LM 20. Nanoparticles present within the laserbeam path were observed by optical instrument (LM-20,NanoSight Pvt. Ltd., UK) having CCD camera and size ofthe nanoparticles was measured on the basis of Brownianmotion of the particles.

TEM analysisThe synthesized silver nanoparticles were also

characterized by TEM (Philips, CM 12), on conventionalcarbon coated copper grids (400 meshes, Plano Gmbh,Germany). For TEM analysis, 5 µL of silver nanoparticlessample was taken, and three image of each sample wereselected for the clarification of the composition.

RESULTS AND DISCUSSIONS

The indication of the silver nanoparticles synthesis wasmarked by the rapid change in the color of plant extractfrom light-green to dark-brown after treatment with silver

nitrate (1 mM AgNO3) at room temperature. The colorchange was due to the reduction of silver ions to silvernanoparticles i.e. Ag+ to Ag0 and appearance of browncolor indicates Silver nanoparticle synthesis (Figure 1). Theconformation of the silver nanoparticle synthesis was doneby UV-visible spectrophotometer analysis, which showsthe absorbance peak at 438 nm (Figure 2). Theseobservations are similar to those reported by manyresearchers (Ingle et al. 2008; Raheman et al. 2011).

The dispersion characteristics i.e. average size andparticle size distribution were measured with NTA byNanoSight LM-20. NTA allows individual nanoparticles ina suspension to be microscopically visualized and on thebasis of the Brownian motion the size distribution andaverage size of the particle was obtained. It shows that theaverage size of nanoparticles are 40 nm most repeatednanoparticles was of 37 nm, the concentration of thenanoparticles was 1.47x 108 particles/mL-1 The particle sizedistribution histogram and 3-D plot of particle sizedistribution were shown in Figure 3 and 4, respectively.Particle size and intensity distribution of nanoparticles wasshown in Figure 4 which was found to be similar to theresults obtained by Montes-Burgos et al. (2010) andRaheman et al. (2011).

Finally, the synthesis of spherical and polydispersivesilver nanoparticles by the leaf extract was confirmed byTEM analysis and it was found that the particles are in therange of 20-50 nm having 30 nm as the average diameter(Figure 5). Even the size predicted by TEM analysis wassmaller than predicted by NTA analysis. Generally, NTAanalysis resulted in larger particle sizes measurementscompared to TEM, which is in agreement with previouswork (Farkas et al. 2010, 2011). The differences can bemainly explained by the bias from each specific method(Hassellov et al. 2008). For example, TEM is a numberbased method has a bias towards the smaller particle sizescompared to NTA which is a number based method butfails to probe the weakly scattering particles (below 10-20nm) and is consequently mainly measuring the NPaggregates which may explain some of the differences insize measurements.

Figure 1. Synthesis of silver nanoparticles from leaf extract ofTagetes erecta (A) Leaf extract before treatment with AgNO3

(control) and (B) Leaf extract after treatment with AgNO3

(experimental)

A B

DHULHAJ et al. – Phytosynthesis of silver nanoparticles from Tagetes erecta 111

Figure 2. UV-visible spectra of leaf extract (showing no peak)and silver nanoparticles showing peak at 438 nm

Figure 3. NTA (Nanosight-LM 20) nanoparticle size distributionhistogram showing the average size of 37 nm

Figure 4. NTA (Nanosight-LM 20) 3-D plot of nanoparticle sizedistribution

Figure 5. TEM micrograph showing synthesis of polydispersedand spherical silver nanoparticle having average size of 30 nm(scale bar 200 nm)

For the development of green synthesis, Raveendran etal. (2003) suggested three main factors in nanoparticlesynthesis should be considered i.e. solvent choice, the useof an environmentally benign reducing agent, and the useof a non-toxic material for nanoparticle stabilization. In thepresent study water is used as an environmentally benignsolvent, replacing toxic organic solvents from chemicalmethods and biomolecules from leaf extract of Tageteserecta was used as both reducing and stabilizing agents forgreen synthesis.

CONCLUSION

The leaf extract of Tagetes erecta has potential of silvernanoparticles synthesis. These synthesized silvernanoparticles are found to be stable. The green synthesismethod is not only cost effective but also eco-friendly,simple and efficient than the others , and has appreciablecontrol over size and shape of the nanoparticles.

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Seed viability of Jatropha curcas in different fruit maturity stagesafter storage

BAMBANG BUDI SANTOSO1,♥, ARIS BUDIANTO2, I.G.P. MULIARTA ARYANA1

1Energy Crops Centre, Faculty of Agriculture, University of Mataram, Jl. Majapahit No. 62 Mataram 83125, West Nusa Tenggara, Indonesia. Tel. +62-0370 621435, Fax. +62-0370 640189, email: [email protected]

2Faculty of Agriculture, University of Mataram, Mataram 83125, West Nusa Tenggara, Indonesia.

Manuscript received: 8 October 2012. Revision accepted: 10 November 2012.

Abstract. Santoso BB, Budianto A, Aryana IGPM. 2012. Seed viability of Jatropha curcas in different fruit maturity stages after storage.Nusantara Bioscience 4: 113-117. The effect of fruit maturity stages and seed storage period to seed viability was investigated. Seedsamples of West Lombok, West Nusa Tenggara genotype of Jatropha curcas were collected from a stand of two-year-old trees at anexperimental field. The seed samples obtained were in four different stages of fruit maturity involving early maturity (green fruit),physiological maturity (yellow fruit), over maturity (brownies fruit), and senescence (black-dry fruit). The results showed that fruitmaturity and storage period had an influence on the seed viability of J. curcas. The best fruit maturity stage for seed viability includingseed oil content was found in yellow fruit and brownies fruit. For germination to be preserved, seeds could be stored in the ambientroom storage for at least five months. For the purpose of oil extraction, seed should preferably be stored not more than four monthsunder ambient room conditions.

Key words: germination rate, Jatropha curcas, room condition, seed oil content, seed quality

Abstrak. Santoso BB, Budianto A, Aryana IGPM. 2012. Viabilitas biji Jatropha curcas pada tahapan kematangan berbeda setelahpenyimpanan. Nusantara Bioscience 4: 113-117. Pengaruh tahapan kematangan dan periode penyimpanan terhadap viabilitas biji telahditeliti. Sampel biji Jatropha curcas genotip Lombok barat diambil dari tegakan pohon berusia dua tahun di lapangan percobaan.Sampel biji yang diperoleh memiliki empat tahapan kematangan, yaitu awal kematangan (buah hijau), kematangan fisiologis (buahkuning), kematangan berlebih (buah kecoklatan) dan tua (buah kering hitam). Hasilnya menunjukkan bahwa kematangan dan periodepenyimpanan memiliki pengaruh pada viabilitas biji J. curcas. Tingkat kematangan terbaik untuk biabilitas biji ditemukan pada buahkuning dan kecoklatan. Untuk .mempertahankan perkecembahan, biji harus disimpan paling tidak lima bulan di kondisi ruanganpenyimpanan. Untuk ekstraksi minyak, biji sebaiknya disimpan tidak lebih dari empat bulan di kondisi ruangan penyimpanan.

Kata kunci: laju perkecambahan, Jatropha curcas, kondisi ruangan, kadar minyak biji, kualitas biji

INTRODUCTION

Jatropha curcas L. is a multipurpose plant with manyattributes and considerable potential. It is a tropical plantthat can be grown in low to high rainfall areas and can beplanted in reclaimed land as a fence or commercial crop.The seed of this plant produces oil. Because Jatropha oilcan be used in place of kerosene and diesel fuel, it has beenpromoted to make rural areas self sufficient in fuel forcooking, lighting, and motive power (Openshaw 2000).Then, J. curcas is expected to be a highly potential energycrop in Indonesia (Nazir and Setyaningsih 2010).

Despite these numerous benefits and potential, theproduction and development program of J. curcas inIndonesia has been faced with a number of challenges. Oneof the constraints is the lack of good seeds in quality andquantity owing to some problems in seed multiplication.Little information is available on quality seed productionand post harvest handling.

Seed quality is often interpreted in terms of genetictraits, germination capacity, purity and storage potential

(ISTA 1999). Simic et al. (2007) also viewed seed qualityas a multiple criterion that encompasses several importantseed attributes such genetic and chemical composition,germination and vigor, seed water content, and also thepresence of seed-borne pathogen. Moreover, poorgermination can be resulted from the use of immature seeds(Batin 2011) and storage duration and condition(Dharmaputra et al. 2009; Akowuah et al. 2012). Cropproductivity can be increased by increasing the germinationrate which is possible by optimizing important parametereswhich are crucial for germination (Cheema et al. 2010). Inthe same manner, successful plantation activities for J.curcas provide viable seeds for the production of qualityseedlings. Seed germination and seedling establishment arethe most critical stages for survival during the life cycle ofthe individual J. curcas plant. To date, the potential of thisplant is still constrained by the lack of technicalinformation particularly in selecting the best fruit maturitycolor that could give the most excellent seed germinationand seedling growth performance.


As Cheema et al. (2010) state, that seed should beproduced in proper condition as seed from harshenvironment is not good for further crop production. So,proper selection of fruit maturity of J. curcas based oncolor must be ascertained in order to produce qualityplanting stocks to meet the increasing demand for J. curcasas rehabilitation species and source of oil. Hence, thedocumentation of fruit color indicating maturation stages ofJ. curcas is necessary to address issues on poorgermination and growth. This article described the effect offruit maturity stages at different time of seed storage periodon seed viability.


Plant materialsSeed samples of West Lombok, West Nusa Tenggara,

Indonesia genotype of J. curcas were collected from astand of two-year-old trees at an experimental field. Theseed samples were obtained in four different stages ofmaturity involving early maturity (green fruit), physiologicalmaturity (yellow fruit), over maturity (brown fruit), andsenescence (black and dry fruit) as shown in Fig.1. Theharvested was done in February-March 2010 and the seedstorage was done during April-September 2010.

ProceduresCollection, packaging, and storing of seeds

Seeds of J. curcas were sun-dried for two days and themoisture content was determined using standard hot airoven method at 105± 1OC for 24 hours (Pradhan et al.2009). Then two kg of sun-dried J. curcas seed was placedin a sac of polypropilen (PP) plastic and then stored in theroom condition for duration of 6 months. Three replicateswere used for each seed maturity stages. The ambienttemperature and relative humidity of the storage room wererecorded using a thermohygrometer.

Samples handlingEach sample (100-150 g of seeds) derived from each

seed sack (replication) was taken monthly for thedeterminations of seed water content, seed oil content, seedweight, and seed viability (number of germinate seed andgermination rate).

Determination of seed viabilityViability (percentage of germination and germination

rate) of seeds from each sample was determined bygrowing 100 seeds in plastic container containing sandmedia under green house conditions. Daily germinationcounts were taken and recorded up to 15 days (time afterwhich no seed was observed to germinate). The resultswere calculated as percentage of normal seedlings.

Determination of seed water contentWater contents of seeds (based on wet basis) were

determined every month based on oven method(gravimetry method). Two samples were used for eachreplicate (sack).

Determination of seed oil (lipid) contentSeed (kernel) oil (lipid) contents were determined based

on Soxhlet extraction method (AOAC 1999) with hexaneas the solvent. The extracted lipid was obtained byfiltrating the solvent using a rotary evaporator apparatus at40 OC followed by heating in an oven at 105 OC for threehours to evaporate any remaining solvent and water.

Statistical analysisThe data were statistically analysed using mean and

standard deviation. Analysis of Variance was applied totest the variation between different stages of fruit maturatythrough seed viability, seed moisture content, seed oilcontent, and other characteristics. Least significantdifference (LSD at 5% level) was also subjected onsignificant findings.

Figure 1. Maturity stages of J. curcas fruit studied. A. early maturity (green fruit), B. physiological maturity (yellow fruit), C. overmaturity (brown fruit), and D. senescence (black and dry fruit).

SANTOSO et al. – Seed viability of Jatropha curcas 115


ResultsThe range of ambient temperature and relative humidity

of the storage room is presented in Table 1. Thetemperature ranged between 26.1 and 29.6 OC, whereas thehumidity ranged between 71.4 and 83.4 %. The conditionof storage room was relatively steady state during thestorage period.

Table 1. The range of temperature and relative humidity ofstorage room during storage.

Duration of storage(month)

Temperature( OC)

Relative humidity(%)

0 - 1 26.5 - 28.3 72.2 - 80.51 - 2 26.1 - 28.9 75.1 - 83.42 - 3 26.3 - 29.4 75.5 - 81.93 - 4 26.7 - 29.5 73.4 - 80.84 - 5 26.4 - 29.6 72.6 - 80.85 - 6 26.3 - 29.4 71.4 - 80.7

Variations and significantly difference of fresh fruit andseed characteristic among fruit maturity are given in Table2. Fruit weight was found maximum (14.7±1.37 g) in greenfruit and minimum (5.8±0.78 g) in black-dry fruit of fruitmaturity. Not only weight of fruit but also seed moisturecontent, weight of fruit shell, and weight of seed werefound maximum in green fruit and minimum in black-dryfruit.

Table 2. Characteristics of fresh fruit and seed after harvest atdifferent fruit maturity stages

Fruitmaturity(fruit color)

Seedmoisturecontent(%)

Weight offruit(g)

Weight offruit shell(g)

Weight ofseed (g)

Green fruit 40.6 ±1.64 14.7 ±1.37 11.8 ±1.02 2.7 ±0.33Yellow fruit 32.7 ±1.42 12.4 ±1.02 9.4 ±0.87 2.5 ±0.21Brown fruit 29.8 ±1.11 9.7 ±0.82 7.9 ±0.83 2.1 ±0.11Black-dry fruit 21.9 ±0.95 5.8 ±0.78 4.1 ±0.82 1.8 ±0.06LSD 5% 6.6 3.8 4.5 0.7Note: ±: value of standard deviation. Means differ significantly atP<0.05.

Percentage of seed germination and germination rate ofJ. curcas seeds in this study differ significantly amongfruits maturity stages in the storage period of six months(Table 3 and Table 4). Seed taken from yellow fruit andbrown fruit had the highest percentage of seed germinationduring six months of storage period. For germination rate,it was seed taken from yellow fruit, brown fruit, and black-dry fruit had higher rate that of green fruit.

Weight of 100 of J. curcas seeds in this study differsignificantly among fruit maturity stages in the storageperiod until six months (Table 5). Decrease of seed weightduring storage as consequence of decrease in their moisturecontent (Table 6). It was observed that there was significantdifference in the moisture content of the seeds at room

condition. However, there was no significant difference atthe 3 to 5 month of stored seed (Table 6.). Higher andlower seed water content was recorded at green fruit andyellow to black-dry fruit respectively. It could be said thatthere was a marginal decrease in seed water content of theseeds related to fruit maturity. Seed from green fruit hadhigh water content at the beginning of storage (12.4%),while the water content of seed from yellow to black-dryfruit ranged from 6.7-8.2%. Then, seed water content of J.curcas seeds decreased during seed storage.

Oil analysis of seeds was carried out after six months ofstorage. Seed oil content was influenced by storage periodand maturity stage of fruit (Table 7.). The oil content ofseeds varied from minimum of 32.3% (green fruit) tomaximum which ranged between 35.8 to 36.9% (yellow,brownies, and blck-dry fruit) at the beginning of storageperiod. After six months of storage, oil content of seedsvaried from minimum 8.9% (green fruit) to maximum23.3% (yellow fruit). Therefore, seed oil content of J.curcas seeds harvested at yellow maturity was not diffrentfrom that at brownies to black-dry maturityfruit. Thosephenomena existed during first three months of storageperiod. At the period of three to six month of storage theseed oil content harvested at yellow fruit was no differentfrom that of brownies fruit.

DiscussionSeed germination is affected by two factors, i.e. internal

and external factors. Internal factor consists of the level ofseed maturity, seed size, dormancy, and germinationinhibitor. In this study, fruit maturity stages had significanteffect on germination of seed, germination rate, and also onthe seed weight, water content, and oil content of storagedJ. curcas seeds (one to six months of storage). In presentstudy, the highest percentage of seed germination wasobserved in seeds taken from yellow fruit and browniesfruit from the beginning of storage until six months ofstorage.

Seed taken from young fruit (green fruit) producedimmature seed, therefore, resulting in low and delayedgermination. This proved the claim of Basra (2006) andBatin (2011) that seed viability includes J. curcas seed, ishigher at the mature stage and decreases at early or lateharvest or maturity. Harvesting J. curcas fruit too early(green fruit) results in more immature seeds with lowergermination of seed and germination rate.

The fresh weight of fruits, shells, and seeds changedduring maturation, ripening and senescence. Fruits, shells,and seeds fresh weight increased significantly when thefruits were ripe (fully yellow) but reduced when theystarted to senesce. Biomass of J. curcas fruits weresignificantly different according to their maturity stage dueto high water content at physiological maturity stage andlow water content at senescence stage (Gunaseelan 2009).Germination capacity as seed viability increases duringseed maturation. In this J. curcas, maximum seed viabilitycoincided with the attainment of maximum seed dry weightor physiological maturity (yellow to brownies fruit color)and decline thereafter. According to Adikadarsih and


Hastono (2007) , the lowest percentage ofJ. curcas seed germination (7%) wasfound in seeds derived from green fruitswhile the highest percentage (91.6%) wasfound in seeds derived from yellow fruit.Moreover, Wellbaum and Bradford (1990)found that germination capacity generallyincreased progressively and coordinatelyduring seed maturation. Probert and Hay(2000) state that maximum seed qualities,normally harvested as dry seeds, wereattained at or close to physiologicalmaturity eventhough for others continue toincrease well into the post-abscissionphase.

Hard seed coat prevents oxygen andmoisture entering the seed and preventsauto-oxidation of linoleic and linolenicacid which are responsible for degradationof cellular organelles (Cantliffe 1998).Therefore, as the time of seed storageincreases, so does cellular damage.Gingwal et al. (2004) also reported thatgermination of J. curcas seed fell below50% within 15 month of storage. In thesame manner, Ellis et al. (1990) andGhasemnezhad and Honemeier (2009) saythat melon and sunflower seeds aredifficult to store because germination andvigor deteriorate quickly in storage due tothe high oil content in the seed. This studyalso was in agreement with that ofKumari et al. (2011), that the Indias J.curcas seeds looses its viability uponstorage. In addition, Cheema et al. (2010)state, that there was a decrease in rate ofgermination of castor seeds due to lowavailability of moisture (seed watercontent). Therefore, the seeds of J. curcasfor seedling should be derived from theyellow and brown fruits with storageperiod not more than four month withinroom condition.

As storage period increased in presentstudy, oil content of seed decreased. Thenthis study is in agreement with that ofAkowuah et al. (2012) which showed thatpercentage of seed oil content of J. curcasgradually decreased with increasingstorage time. According to Ahmadhan andShahidi (2000) and Morello et al. (2004),it was due to the development of rancidityor deterioration of lipids in vegetable oilduring storage. As Akowuah et al. (2012)state that aging process naturally affectsthe quality of seeds during storage atvarious conditions, especially oil contentwhich is sensitive to deterioration as resultof the reaction between unsaturated fattyacid and oxygen. This might be a reason

Table 3. Seed germination at different fruit maturity stages after storage

Duration of storage (month)0 1 2 3 4 5 6

Fruit maturity(fruit color)

%Green fruit 90.3 a 86.6 a 57.3 a 41.5 a 29.6 a 11.9 a 8.6 aYellow fruit 97.5 bc 95.4 b 81.6 b 79.9 c 68.7 c 59.3 c 56.9 cBrown fruit 98.7 c 96.1 b 79.9 b 70.1 c 61.3 c 53.4 c 48.4 cBlack-dry fruit 92.1 ab 87.1 a 60.1 a 52.2 b 45.8 b 32.9 b 25.5 bLSD 5% 6.2 5.7 5.8 10.4 9.8 8.5 8.9Note: numbers in the column with the same letter did not differ significantly at P<0.05.

Table 4. Rate of seed germination at different fruit maturity stages after storage

Duration of storage (month)Fruit maturity(fruit color) 0 1 2 3 4 5 6

dayGreen fruit 9.3 b 7.8 10.6 b 14.2 b 20.5 b 26.1 b 33.2 bYellow fruit 6.6 a 6.9 7.4 a 7.6 a 7.9 a 9.6 a 9.9 aBrown fruit 6.7 a 6.8 7.4 a 7.5 a 7.8 a 9.8 a 10.2 aBlack-dry fruit 5.8 a 6.5 6.8 a 7.3 a 7.6 a 8.7 a 10.8 aLSD 5% 2.5 ns 2.6 3.1 4.5 4.7 5.2Note: numbers in the column with the same letter did not differ significantly atP<0.05, ns: not significant

Table 5. Weight of 100 seeds at different fruit maturity stages after storage



gGreen fruit 108.3 c 98.7 c 68.2 b 63.4 a 61.9 a 52.1 a 47.7 aYellow fruit 90.7 b 87.8 b 85.6 b 84.3 b 83.1 b 82.7 c 80.3 cBrown fruit 81.6 b 78.4 b 76.7 b 75.2 b 74.8 b 74.4 c 74.1 cBlack-dry fruit 69.8 a 67.2 a 66.5 a 65.1 a 64.7 a 63.2 b 62.8 bLSD 5% 11.5 10.2 9.8 9.4 10.3 10.1 12.2Note: numbers in the column with the same letter did not differ significantly at P<0.05.

Table 6. Seed water content at different fruit maturity stages after storage



%Green fruit 12.4 b 10.2 b 8.9 b 7.8 7.2 5.6 4.4 aYellow fruit 8.2 a 7.9 a 7.5 ab 7.3 7.1 6.8 6.4 bBrown fruit 7.4 a 7.2 a 7.1 ab 7.1 6.7 6.6 6.3 bBlack-dry fruit 6.7 a 6.5 a 6.4 a 6.1 5.8 5.2 4.9 aLSD 5% 2.1 1.9 2.2 ns ns ns 1.7Note: numbers in the column with the same letter did not differ significantly atP<0.05, ns: not significant

Table 7. Seed oil content at different fruit maturity stages after storage


Fruitmaturity

(fruit color) %Green fruit 32.3 a 31.9 a 26.6 a 23.7 a 18.3 a 14.2 a 8.9 aYellow fruit 36.9 b 37.2 b 36.5 b 34.6 c 31.1 b 29.7 c 23.3 cBrown fruit 36.2 b 36.8 b 36.1 b 34.2 bc 30.8 b 29.1 bc 22.8 bcBlack-dry fruit 35.8 b 36.1 b 35.3 b 32.1 b 29.7 b 27.5 b 20.1 bLSD 5% 3.3. 3.6 4.2 2.1 2.6 2.0 2.7Note: numbers in the column with the same letter did not differ significantly at P<0.05.

SANTOSO et al. – Seed viability of Jatropha curcas 117

why the percentage of oil of stored J. curcas seeds tends toreduce during storage. In addition Taiz and Zeiger (2002and Basra (2006) state that, the metabolism of seed duringstorage to provide energy for its physiological activitiescould be another reason of seed oil decrease during storage.

CONCLUSION

Fruit maturity and storage period had an influence onthe seed viability of J. curcas. The best fruit maturity stagefor good seed viability including seed oil content wasfound in yellow fruit and brown fruit. Germinationpercentage and germination rate were statistically the samewhen the fruits were harvested at yellow and brown ripe.For germination to be or preserved, seeds could be storedin the ambient room storage for at least five months. Inaddition, for the purpose of oil extraction, seed shouldpreferably be stored not more than four months underambient room conditions.

REFERENCES

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Akowuah JO, Addo A, Kemausuor F. 2012. Influence of storage durationof Jatropha curcas seed on oil yield and free fatty acid content.ARPN J Agric Biol Sci 7: 41-45.

AOAC [Association of Official Analytical Chemists]. 1999. Officialmethods of the Association of Agricultural Analytical Chemist.Association of Analytical Chemist, Inc., Arlington

Basra AS. 2006. Handbook of seed science and technology. HaworthPress, New York.

Batin CB. 2011. Seed germination and seedling performance of Jatrophacurcas L. fruit based on color at two different seasons in northern

Philippines. International Conference on Environment andBioScience IPCBEE 21: 94-100.

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Cheema NM, Malik MA, Qadir G, Rafique MZ, Nawaz N. 2010.Influence of temperature and osmotic stress on germination inductionof different castor bean cultivars. Pakistan J Bot 42: 4035-4041.

Dharmaputra OS, Worang RL, Syarief R, Miftahudin. 2009. The qualityof physic nut (Jatropha curcas) seeds affected by water activity andduration of storage. Microbiol Indon 3: 139-145.

Ellis RH, Hong TD, Robert EH. 1990. Effect of moisture content andmethode of rehydration on the susceptibility of C. melo seeds toimbibitional damage. Seed Sci Technol 18: 131-137.

Ghasemnezhad A, Honemeier B. 2009. Influence of storage conditions onquality and viability of high and low oleic sunflower seeds. Intl JPlant Prod 3: 39-48.

Gingwal SH, Phartyal SS, Rawat PS, Srivastava RL. 2004. Seed sourcevariation in morphology, germination and seedling growth of(Jatropha curcas) Linn. in Central India. Silvae Genetica 54: 76-80.

Gunaseelan NV. 2009. Biomass estimates, characteristics, biochemicalmethane potential, kinetics and energy flow Jatropha curcas on drylands. Biomass Bioen 33: 589-596.

ISTA [International Seed Testing Association]. 1999. International rulesfor seed testing. Seed Science and Technology. International SeedTesting Association, Bassersdorf, Switzerland

Kumari A, Joshi PK, Arya MC, Ahmed Z. 2011. Enhancing seedgermination of Jatropha curcas L. under Central-Western Himalayasof Uttrakhand, India. Plant Arch 11: 871-874.

Morello JR, Motilva MJ, Tovar MJ, Romero MP. 2004. Changes incommercial virgin olive oil (CV Arbequina) during storage withspecial emphasis on the phenolic fraction. J Food Chem 85: 357-364.

Nazir N, Setyaningsih D. 2010. Life cycle assessment of biodieselproduction from palm oil and jatropha oil in Indonesia. 7th BiomassAsia Workshop. Jakarta, Indonesia.

Openshaw K. 2000. A review of Jatropha curcas: An oil plant ofunfulfilled promise. Biomass Bioeng 19: 1-15.

Pradhan RC, Naik SN, Bhatnagar N, Vijay VK. 2009. Moisture-dependentphysical properties of jatropha fruit. Ind Crop Prod 29: 341-347.

Probert RJ, Hay FR. 2000. Keeping seeds alive. In: Black M, Bewley JD(eds). Seed technology and its biological basis. Sheffield AcademicPress, Sheffield, UK.

Simic B, Popovic R, Sudaric A, Rozman V, Kalinovic I, Cosic J. 2007.Influence of storage condition on seed oil content of maize, soybeanand sunflower. CCS Agriculturae Conspectus Scienticus 72: 211-213.

Taiz L, E Zeiger. 2002. Plant physiology. 3rd ed. Sinauer, Sunderland,MA.

Wellbaum GE, Bradford KJ. 1990. Water relation of seed developmentand germination in muskmelon (C. melo L.). V. Water relation ofimbibition and germination. Plant Physiol 92: 1046-1052.


Physiological response of Moringa oleifera to stigmasterol and chelated zinc

ABDALLA EL-MOURSI, IMAN MAHMOUD TALAAT♥, MOHAMED ABDEL-GHANY BEKHETA,KARIMA GAMAL EL-DIN

Department of Botany, National Research Centre, Dokki, Cairo 12622, Egypt. Tel. +202-3366-9948, +202-3366-9955, Fax: +202-3337-0931, e-mail:[email protected]

Manuscript received: 11 September 2012. Revision accepted: 12 November 2012.

Abstract. El-Moursi A, Talaat IM, Bekheta MA, Gamal El-Din K. 2012. Physiological response of Moringa oleifera to stigmasterol andchelated zinc. Nusantara Bioscience 4: 118-123. Two pot experiments were carried out in the screen of the National Research Centre,Dokki, Giza, Egypt, during two successive seasons (2009/2010 and 2010/2011), respectively to study the effect of foliar spray withchelated zinc (100, 200 and 300 mg/L) and stigmasterol (50, 100 and 150 mg/L) on growth and chemical constituents of moringa plants(Moringa oleifera). The results indicated that treatment of plants with 300 mg/L chelated zinc or 150 mg/L stigmasterol significantlyinfluenced the vegetative growth of moringa plants. The same treatments also significantly increased total sugars%, total protein%, totalphosphorous and microelements contents in the leaves. The changes in the pattern of protein electrophoresis (SDS-PAGE) extractedfrom the newly formed leaves of moringa plants treated with different concentrations of chelated Zinc (Zn) or stigmasterol showedbeneficial influences for improving plant growth, leaves quality and quantity.

Key words: Moringa oleifera, stigmasterol, chelated zinc

Abstrak. El-Moursi A, Talaat IM, Bekheta MA, Gamal El-Din K. 2012. Tanggapan fisiologis Moringa oleifera terhadap stigmasteroldan kelat seng. Nusantara Bioscience 4: 118-123. Dua pot percobaan dibuat di kebun percobaan Pusat Riset Nasional, Dokki, Giza,Mesir, selama dua musim secara berturut-turut (2009/2010 dan 2010/2011), masing-masing untuk mempelajari pengaruh penyemprotandaun dengan kelat seng (100, 200, 300 mg/L) dan stigmasterol (50, 100 , 150 mg/L) terhadap pertumbuhan dan kandungan kimiatanaman kelor (Moringa oleifera). Hasil penelitian menunjukkan bahwa perlakuan tanaman kelor dengan 300 mg/L kelat seng atau 150mg/L stigmasterol berpengaruh signifikan terhadap pertumbuhan vegetatif tanaman. Perlakuan yang sama juga secara signifikanmeningkat persentase gula total, protein total, fosfor total dan kandungan unsur mikro dalam daun. Perubahan pola elektroforesis protein(SDS-PAGE) yang diekstrak dari daun yang baru terbentuk dari tanaman kelor yang diperlakukan dengan konsentrasi kelat seng (Zn)atau stigmasterol yang berbeda menunjukkan pengaruh yang menguntungkan untuk meningkatkan pertumbuhan tanaman, kualitas dankuantitas daun.

Kata kunci: Moringa oleifera, stigmasterol, kelat seng

INTRODUCTION

The search for new drugs of plant origin has yieldedfruitful result in the past. Today it is possible to use molecularbiology techniques for detection of genetic variability andtagged desired traits as well as culling out duplicates inaccessions. The availability of high throughout screen hasmade the possibility of covering ‘hits’ into lead compoundsin comparatively short time. Drug development from plantsources using gene/molecular techniques is becomingincreasingly important. Although few people have everheard of Moringa oleifera tree today, Moringa could soonbecome one of the world's most valuable plants, at least inhumanitarian terms. Perhaps the fastest-growing of alltrees, it commonly reaches three meters in height just 10months after the seed is planted. Furthermore, it has morethan a dozen important uses, yielding, among other things,several types of food as well as oil, wood, paper, shade,beautification and liquid fuel (Morton 1991).

Moringa oleifera Lam. leaves on ethanolic extractionyielded a number of amino acids viz, aspartic acid,glutamic acid, serine, glycine, threonine, β-alanine, valine,leucine, iso-leucine, histidine, lysine, arginine, phenyl-alanine, tryptophan, cystine and methionine. The etherextract of leaves yielded α-and β-carotene. Also, 9 aminoacids in the flowers, 8 in the fruits and 7 each in the proteinhydrolysate of flowers and fruits of M. oleifera wereidentified. Alanine, arginine, glutamic acid, glycine, serine,threonine and valine were common in all the parts tested,whereas aspartic acid was present in the flowers as well asthe fruits, and lycine occurred only in the flowers. Theflowers contained both sucrose and d-glucose, whereas thefruits showed the presence of sucrose only (Ram 1994).

The juice from the leaves and stem bark of M. oleiferainhibited Staphylococcus aureus but not Escherichia coli.The 50% ethanolic extract of root bark of M. oleiferashowed antiviral activity against vaccinia virus, but wasinactive against Ranikhet disease virus. M. oleifera rootextract (50% ethanolic) at a dose of 200 mg/kg led to fetal

EL-MOURSI et al. – Effect of stigmasterol and chelated zinc on Moringa oleifera 119

resorption in 60% female pregnant rats. Ethanolic extract(50%) of M. oleifera (whole plant excluding roots) showedanticancer activity against human epidermoid carcinoma ofnasopharynx in tissue culture and P388 lymphocyticleukemia in mice (Jayavardhanan et al. 1994).

The roots are carminative, stomachic, abortifacient,cardiac tonic and also used in paralytic conditions andintermittent fever; also useful as rubefacient in rheumatism,in spasmodic affections of the bowels, hysteria andflatulence as well as in epilepsy. Root bark is used asfermentation to relieve spasm. Bark is considered to be anabortifacient. The fruit is recommended in diseases of liverand spleen, in tetanus and paralysis. Flowers are stimulantand aphrodisiac. Seed oil is applied externally inrheumatism. Leaves are emetic and their juice with blackpepper is used in headache. The poultice of leaves is usedin reducing glandular swellings. The gum is given in dentalcaries with sesamum oil and also for relief of otalgia and itis applied with milk on the temples in headache. Seeds areused in veneral affections and to relieve the pain of goutand acute rheumatism (Eilert et al. 1981; Pal et al.1995).

Stigmasterol is a structural component of the lipid coreof cell membranes and is the precursor of numeroussecondary metabolites, including plant steroid hormones, oras carriers in acyl, sugar and protein transport (Genus 1978).Brassinosteroids (BR) are known as a group of naturallyoccurring polyhydroxysteroids. All brassinosteroids isolatedfrom plants are characterized as 5--cholestane derivativesthat classified as C27, C28, C29 steroids as revealed by(Yokota et al. 1982). BR has the same biological action likegibberellins and auxins. The pollen grains of plant flowerscontained the highest values of BR compared with the otherplant organs (Horgan et al. 1984). Brassinosteroids havebeen found to evoke both cell elongation and cell divisionresulting in elongation, swelling, curvature and splitting ofthe internode (Mandava 1988). Physiological functionsproposed for Brassinosteroids included cell elongation, celldivision, leaf bending, vascular differentiation, proton pump-mediated membrane polarization, sink/source regulationresponses (Sasse 1999). In addition, brassinosteroids causedchanges in enzymatic activities, membrane potential, DNA,RNA, protein synthesis, photosynthetic activity and changesin the balance of endogenous phytohormones (Steven andJeneth 1998). Particular interest in sterols was elicited byenhanced growth characters and yield of chamomile plant(Abdel-Wahed and Gamal El-Din 2004). Recently, thesestudies provided strong evidence that sterols could beessential for normal plant growth and development(Ozdemir et al. 2004).

In recent years, zinc is one of the most important elementsfor the growth and flowering of some plants as reported byChandler (1982). Abou-Leila et al. (1994) found that foliarapplication of Ocimum basilicum L with Zn at 75 mg/lgave the highest values of herbal yield, carbohydrate andoil contents. The effect of Zn on enzyme systemresponsible for biosynthesis of carbohydrates was reportedby Sandmann and Boger (1983). The necessity of Zn formost crops was emphasized by Singh and Ganguar (1973).They mentioned that Zn participates in the production ofIAA which resulted in an increase in growth and sugar

production in sugar beet. Adding Zn fertilizers as foliarapplication was suggested in Egyptian alkaline soils, wherethe availability of Zn and other microelements for plantroots becomes relatively low (El-Sayed 1971).

MATERIAL AND METHODS

Plant materials. Moringa oleifera L. seeds securedfrom the Institute of Horticulture Research, Ministry ofagriculture, Giza, Egypt. Two pot experiments were carriedout in the Screen of the National Research Centre, Dokki,Giza, Egypt, during two successive seasons (2009/2010and 2010/2011), respectively to study the effect of foliarspray with chelated zinc (100, 200 and 300 mg/L) andstigmasterol (50, 100 and 150 mg/L) on growth andchemical constituents of moringa plants. On 15th October2009 and on 19th October 2010, the seeds were sown inpots (35 cm in diameter), each pot contained 12 kg clayloamy soil. Treatments were distributed in completerandomized block design with five replications, five potseach. Fifteen days after sowing, the seedlings were thinnedto the most three uniform plants in each pot. Each potreceived equal and adequate amounts of water andfertilizers. Phosphorous as calcium superphosphate wasmixed with the soil before sowing at the rate of 4.0 g/pot.Three g of nitrogen as ammonium sulphate were added inthree applications (one g each) with intervals of two weeksstarted 30 days after sowing. Also, two g of potassiumsulphate were added as soil application. Other agriculturalprocesses were performed according to normal practice. M.oleifera plants were sprayed with stigmasterol and chelatedzinc solutions 45 days after sowing. The volume of thespraying solution was maintained just to cover completelythe plant foliage till drip. Distilled water was sprayed in thesame previous manner on untreated plants (control plants).The first sample (vegetative stage) was taken 10 days aftertreatment. The second sample (full vegetative stage) wastaken at 15th May 2010 and 19th May 2011, respectively.Measurement of growth parameters; plant height (cm),number of branches per plant, fresh and dry weights ofleaves and stems (g/plant) were determined.

Chemical analysis. Represented samples of the leavesof each treatment were subjected to the following differentchemical analyses. Determination of total sugars wascarried out according to Dubois et al. (1956). Totalnitrogen (modified micro-Kjeldahl) was determined asdescribed by Jackson (1973) and from which protein wascalculated. Potassium, calcium and phosphorous weredetermined according to the procedure described by Brownand Lilliand (1946) and Troug and Meyer (1939),respectively. Iron was determined by atomic absorptionspectrophotometer (Chapman and Pratt 1961).

Data analysis. Data obtained (means of the twogrowing seasons) were subjected to standard analysis ofvariance procedure. The values of LSD were calculated,whenever F values were significant at 5% level as reportedby (Snedecor and Cochran 1980).



Effect on vegetative growthTable 1 clearly revealed that foliar spray of stigmasterol

and chelated zinc significantly increased plant height at alltreatments. The most effective treatments in this respectwas that of 300 mg/L Zn and 150 mg/L stigmasterol whichrecorded the highest values of plant height (35 cm and37.33 cm) compared to 27.25 cm of untreated control.Number of leaves/plant showed the same trend of plantheight which recorded 44.17 and 40.50 at treatments 300mg/L Zn and 150 mg/L stigmasterol, respectivelycompared to (28.75) of untreated plants. The sametreatments resulted in the highest values of fresh and dryweights of leaves which recorded 7.34 and 9.17 g/plantcompared to 3.75 g/plant of untreated plants as freshweights and 1.58 and1.88 g/plant compared 0.93 g/plant ofuntreated plants as dry weights. The least increases wereobtained at treatments 100 mg/L Zn and 50 mg/Lstigmasterol related to slight increases in total sugars% andtotal protein% in the same treatments.

Data presented in Table 2 indicated that foliarapplication of stigmasterol and chelated zinc significantlyincreased plant height, number of leaves, fresh and dryweights of branches/plant, fresh and dry weights ofleaves/plant. The most effective treatments in this concernwas that of 300 mg/L Zn and 150 mg/Lstigmasterol. On the other hand, number ofbranches/plant was not significantlyaffected.

The positive effect of stigmasterol andchelated zinc on plant growth waspreviously reported on wheat plant (Abdel-Wahed et al. 2000), sugar beet plant (Abdel-Wahed and Ali 2001), and Tagetes erectaplant (Balbaa et al. 2008), geranium plant(Ayad et al. 2009), flax plant (El-Lethy et al.2010; Hashem et al. 2011), Matthiolaincana plant (Mahgoub et al. 2011), basilplant (Youssef et al. 2004) and fenugreekplant (Gamal El-Din 2005).

The pronounced increases of vegetativegrowth of moringa plants when treated withstigmasterol could be attributed to its role incell elongation and division (Mandava 1988;Clouse and Sasse 1998). The favorableaction of chelated zinc might be attributed toits role in the synthesis of tryptophan (theprecursor of IAA) which in turn affectedseveral plant phenomena as reported byValke and Wecker (1970).

Effect on chemical constituentsData presented in Table 3 also indicate

that foliar application of chelated zinc andstigmasterol to moringa plants significantlyincreased potassium (K) content in theleaves at treatments 100 mg/L and 150 mg/Lstigmasterol, but treatments 100, 200, 300mg/L chelated zinc and 50 mg/L

stigmasterol were not significant. Treatment 150 mg/Lstigmasterol recorded the highest value of potassiumcontent (51.00 mg/ 100 g dry weight) compared to (41.00mg/100 g dry weight) of untreated control.

Data also indicated that all treatments of chelated zincand stigmasterol resulted in significant increases in calciumcontent and the highest values were recorded in plantstreated with 300 mg/L chelated zinc and 150 mg/Lstigmasterol which recorded 133.35 and 130.38 mg/100 gdry matter compared to 87.10 mg/100 g dry matter ofuntreated control.

Data presented in Table 3 reveal that foliar spray ofchelated zinc and stigmasterol significantly increased ironcontent except in plants treated with 100 mg/L chelatedzinc which was not significantly affected. Application of300 mg/L chelated zinc recorded the highest value of ironcontent (83.67 mg/100 g dry matter) followed by treatmentwith 150 mg/L stigmasterol which recorded 82.00 mg/ 100g dry matter, while untreated control recorded (63.50 mg/gdry matter).

Data also indicated that spraying plants with 200 or 300mg/L chelated zinc and 150 mg/L stigmasterol resulted insignificant increases in phosphorous content of moringaleaves. Meanwhile, treatment of plants with 50 or 100mg/L stigmasterol was non-significant (Table 3).

Treatment of moringa plant with chelated zinc and

Table 1. Physiological effect of chelated zinc and stigmasterol on vegetativegrowth of moringa plants.

Treatments(mg/L)

Plant height(cm)

Number ofleaves

Fresh wt ofleaves

(g/plant)

Dry wt ofleaves

(g/plant)Zn 100 30.67 32.00 5.54 1.44Zn 200 31.33 35.00 6.20 1.48Zn 300 35.00 44.17 7.34 1.58Stigma 50 31.00 31.00 6.12 1.30Stigma100 33.00 38.00 6.15 1.32Stigma150 37.33 40.50 9.17 1.88control 27.25 28.75 3.75 0.93LSD (5%) 3.07 3.18 0.94 0.32

Table 2. Physiological effect of chelated zinc and stigmasterol on growth ofmoringa plants at full vegetative stage.

Treatments(mg/L)

Plantheight(cm)

No. ofleaves

No. ofbranches

Freshweight

ofbranches(g/plant)

Dryweight ofbranches(g/plant)

Freshweight of

leaves(g/plant)

Dryweight of

leaves(g/plant)

Zn 100 106.67 12.33 6. 33 54.35 16.04 58.08 15.74Zn 200 117.67 14.67 7.00 60.10 19.74 65.95 16.94Zn 300 143.33 17.00 7.00 89.61 28.41 74.94 18.11Stigma 50 119.00 14.92 6.00 57.81 17.38 65.79 14.63Stigma100 122.00 16.00 7.00 62.92 22.00 73. 05 16.90Stigma150 122.33 14.00 7.33 64.70 24.26 73. 13 17.72control 99.33 10.00 6.00 32.97 10.06 50.01 17.67LSD (5%) 6.87 1.84 N.S. 9.48 3.03 5.61 3.97


stigmasterol led to significant increases in total sugars% inall treatments, except that treatment 50 mg/L stigmasterolwas non-significant. Treatments 300 mg/L chelated zincand 150 mg/L stigmasterol recorded the highest values oftotal sugars% (3.80 and 3.46%, respectively) comparedwith 2.68% in control plants (Table 3).

Data presented in Table 3 also indicate that foliarspraying moringa plants with chelated zinc or stigmasterolsignificantly increased total protein%, except treatments 50and 100 mg/L stigmasterol which were not significant.Treatments 300 mg/L chelated zinc and 150 mg/Lstigmasterol recorded the highest values of total protein%(3.95 and 3.50%, respectively), compared to 2.45% incontrol plants.

The positive effect of chelatedzinc and stigmasterol on chemicalconstituents was previously reportedon lemon grass plant (Gamal El-Dinet al. 1997), sugar beet plants (Abdel-Wahed and Ali 2001), fenugreekplant (Gamal El-Din 2005), Tageteserecta L. Plant (Balbaa et al. 2008),geranium (Ayad et al. 2009), flaxplant (El-Lethy et al. 2010) andMatthiola incana plants (Mahgoub etal. 2011).

Protein patternTable 4 reveals the changes in the

pattern of protein electrophoresis(SDS-PAGE) extracted from thenewly formed leaves of moringaplants treated with differentconcentrations of chelated zinc (Zn)or stigmasterol (stigma.). Themolecular weights of the proteinsranged between 9.82-108.57 kDa andexhibited a maximum number of 17bands. The scanning profile of suchdetected protein bands revealed thatthe band number 9 having themolecular weight of 31.87 kDaproduced the highest intensity ofprotein which recorded 19.36% inplants treated with chelated Zn atconcentration equal 100 mg/L.

Treatment of moringa plants withchelated Zn at 100 mg/L led to theappearance of 6 protein bandsranging between 11.86-108.57 kDa.Comparing with the features ofprotein banding pattern obtainedfrom the untreated plants, it isevident that such treatment inducedthe appearance of 1 newly proteinband having the molecular weights31.87 kDa.

The electrophoretic pattern of theplants treated with 200 mg/L chelatedZn showed the presence of 7 protein

bands ranging between 11.86-108.57 kDa. It is evident thatthis treatment induced the appearance of 2 new proteinbands having molecular weights of 66.03 and 31.87 kDa.These two new bands have protein intensity 6.05 and7.39%, respectively.

Application of chelated Zn at 300 mg/L showed theexistence of 6 protein bands with the molecular weightsranging between11.86-108.57 kDa as compared to theprotein bands obtained from the control plants. The datashow that this treatment induced the appearance of 1 newlyprotein band having the molecular weight of 31.87 kDa andprotein intensity 7.84%. Meanwhile, one protein banddisappeared having molecular weight of 18.40 kDa.

Table 3. Physiological effect of chelated zinc and stigmasterol on chemical constituentsof moringa plants.

Treat-mentsMg/L

K(mg/100 g

drymatter)

Ca(mg/100 g dry

matter)

Fe(mg/100 g

drymatter)

P(mg/100 g

drymatter)

Totalsugars%

Totalprotein%

Zn 100 43.00 105.68 69.00 98.67 3.47 3.32Zn 200 43.67 120.66 74.33 110.00 3.70 3.48Zn 300 45.00 133.35 83.67 113.33 3.80 3.95Stigma 50 44.00 108.10 72.50 101.00 2. 80 2.80Stigma100 45.33 124.11 76.00 103.00 3.35 2.95Stigma150 51.00 130.38 82.00 111.17 3.46 3.50Control 41.00 87.10 63.50 100.00 2. 68 2.45LSD (5%) 4.24 11.06 5.54 5.66 0.55 0.82

Table 4. Comparative analysis of relative area (%) of each band of the coomassie blue-stained gels of moringa plants treated with different concentrations of chelated Zn andStigmasterol.

Bandnumber

Mol.weight(kDa)

ControlZn at100

mg/L

Zn at200

mg/L

Zn at 300mg/L

Stigmaat 50mg/L

Stigmaat 100mg/L

Stigmaat 150mg/L

1 108.57 7.13 10.12 8.88 7.03 7.42 7.09 8.982 101.883 87..81 3.13 4.63 5.43 3.16 4.10 3.71 4.474 74.235 66.03 - - 6.05* 8.36* 9.36* 7.832* 8.81*6 49.957 42.92 5.13 9.20 6.40 4.74 5.74 7.23 7.448 34.629 31.87 - 19.36* 7.39* 7.84* 7.09* 6.31* 10.32*10 28.5111 22.96 8.10*12 20.8313 18.40 10.43 11.60 11.77 - - - -14 16.6115 14.5016 11.86 6.87 12.01 3.95 10.80 11.80 9.51 11.55

17 9.82 - 7.83*Total no.of bands

- 5 6 7 6 7 6 7

Newbands

- - 1 2 2 3 2 3

Disappearbands

- - - - 1 1 - -

Note: Zn = chelated Zn, Stigma = stigmasterol, *= new bands


The electrophoretic banding pattern of proteins resultedfrom the application of stigmasterol at 50 mg/L on moringaplants showed the appearance of 7 protein bands withmolecular weights ranging from11.86-108.57 kDa. Threenew bands were shown to be induced as a result of thistreatment having molecular weights of 66.03, 31.87 and22.96 kDa. The three new bands have protein intensity9.36, 7.09 and 8.10, respectively. Meanwhile, one proteinband disappeared having molecular weight of 18.40 kDa.

Spraying the plants with 100 mg/L stigmasterol resultedin the induction of two new protein band having molecularweights of 66.03 and 31.87 kDa. This treatment alsoresulted in the disappearance of one protein bands ofmolecular weight of 18.40 kDa.

Application of stigmasterol at 150 mg/L on moringaplants led to the appearance of 17 protein bands rangingbetween 9.82-108.57 kDa and induced the appearance of 3newly protein bands having the molecular weights of66.03, 31.87 and 9,82 kDa, respectively. It is necessary tomention here that, this treatment led to appearance of 1new band having molecular weight 9.82 kDa, which didnot appear in the control and all other treatments withprotein intensity 7.83%. Meanwhile, one protein banddisappeared having molecular weights of 18.40 kDa.

The outcome of the obtained results clearly indicate thatspraying moringa plants with different concentrations ofchelated Zn or stigmasterol led to the appearance of newprotein bands which varied according to the appliedconcentration. The existence of such newly formed proteinbands in treated moringa plants might be explained basingon the potentiality of chelated Zn and stigmasterol totrigger the expression of specific genes along DNAmolecule in the target cells, a process which appears toplay a key role in regulating a cascade of biochemicalreactions which might determine the ultimate appearanceof growth patterns and yield of the produced plants. Thismight be accompanied by a persistent effect carrying overto the progeny via alteration of DNA-binding proteinreceptors mechanism which might amplify the signal-transduction pathway, this suggestion is reinforced by thefindings of Jacobsen and Beach (1985) and Abdel-Hamid(2002).

Bekheta (2004) showed that application of paclo-putrazol accompanied with gibberellic acid (GA3) on wheatplants changed the electrophoretic profile of proteinpatterns. In addition, Bekheta and Talaat (2009) showedchanges in the pattern of protein electrophoresis (SDS-PAGE) extracted from the newly formed leaves of mungbean plants treated with different concentrations ofsalicylic acid, glutahione or pacloputraszol. The molecularweights of the proteins ranged between 8.094-2455.534kDa and exhibited a maximum number of 21 bands. Thescanning profile of such detected protein bands revealedthat the band number 20 having the molecular weight of8.291 KDa produced the highest intensity of protein whichrecorded 42.13% in plants treated with 100 mg/Lglutathione.

In conclusion, foliar treatments of moringa plants withdifferent concentrations of chelated Zn or stigmasterol had

beneficial influences for improving plant growth, leavesquality and quantity.

CONCLUSION

From the obtained data, it could be concluded thatstigmasterol or chelated zinc might play an important rolein plant phytochemical mechanism through its effect on theelectrophoretic pattern of protein electrophoresis and/or onthe mineral ions content, but further studies are needed tolearn more about these mechanisms.

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Review:Biological fertilization and its effect on medicinal and aromatic plants

KHALID ALI KHALIDDepartment of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., 12311 Dokki, Cairo, Egypt. Tel. +202-3366-9948, +202-

33669955, Fax: +202-3337-0931, e-mail: [email protected]

Manuscript received: 18 October 2012. Revision accepted: 10 November 2012.

Abstract. Khalid KA. 2012. Review: Biological fertilization and its effect on medicinal and aromatic plants. Nusantara Bioscience 4:124-133. The need of increase food production in the most of developing countries becomes an ultimate goal to meet the dramaticexpansion of their population. However, this is also associated many cases with a reduction of the areas of arable land which leaves noopinion for farmers but to increase the yield per unit area through the use of improved the crop varieties, irrigation and fertilization. Themajor problem facing the farmer is that he cannot afford the cost of these goods, particularly that of chemical fertilizers. Moreover, incountries where fertilizer production relies on imported raw materials, the costs are even higher for farmer and for the country. Besidesthis, chemical fertilizers production and utilization are considered as air, soil and water polluting operations. The utilization of bio-fertilizers is considered today by many scientists as a promising alternative, particularly for developing countries. Bio-fertilization isgenerally based on altering the rhizosphere flora, by seed or soil inoculation with certain organisms, capable of inducing beneficialeffects on a compatible host. Bio-fertilizers mainly comprise nitrogen fixes (Rhizobium, Azotobacter, Azospirillum, Azolla or blue greenalgae), phosphate dissolvers or vesicular-arbuscular mycorrhizas and silicate bacteria. These organisms may affect their host plant byone or more mechanisms such as nitrogen fixation, production of growth promoting substances or organic acids, enhancing nutrientuptake or protection against plant pathogens. Growth characters, yield, essential oil and its constituents, fixed oil, carbohydrates, solublesugars and nutrients contents of medicinal and aromatic plants were significantly affected by adding the biological fertilizers comparedwith recommended chemical fertilizers.

Key words: biological fertilizers, medicinal, aromatic, plants

Abstrak. Khalid KA. 2012. Review: Pemupukan hayati dan pengaruhnya terhadap tanaman obat dan aromatik. Nusantara Bioscience4: 124-133. Peningkatan produksi pangan menjadi tujuan utama sebagian besar negara-negara berkembang karena pertambahan jumlahpenduduk yang dramatis. Namun, hal ini terhambat oleh berkurangnya lahan garapan, yang tidak memberi pilihan kepada petani, kecualimeningkatkan hasil panen per satuan luas melalui penggunaan varietas unggul, irigasi dan pemupukan. Namun banyak petani yang tidakmampu membayar biaya-biaya tersebut, terutama pupuk kimia. Di negara-negara yang mengandalkan bahan baku impor untuk produksipupuk, biaya yang ditanggung petani dan negara bahkan lebih tinggi lagi. Di samping itu, produksi pupuk kimia dan pemanfaatannyadianggap sebagai penyebab pencemaran udara, tanah dan air. Pemanfaatan pupuk hayati dianggap oleh banyak ilmuwan sebagaialternatif yang menjanjikan, khususnya pada negara-negara berkembang. Pupuk hayati umumnya didasarkan pada perubahan florarhizosfer, dengan biji atau inokulasi tanah yang mengandung organisme tertentu, yang mampu merangsang efek menguntungkan padainang sasaran. Pupuk hayati terutama terdiri dari organisme yang memfiksasi nitrogen (Rhizobium, Azotobacter, Azospirellum, Azollaatau ganggang biru hijau), pelarut fosfat atau mikoriza vesikular-arbuskular dan bakteri silikat. Organisme-organisme tersebut dapatmempengaruhi tanaman inang dengan satu atau beberapa mekanisme seperti fiksasi nitrogen, produksi zat pengatur tumbuh atau asam-asam organik, meningkatkan penyerapan nutrisi atau melindungi terhadap penyakit tanaman. Karakter pertumbuhan, hasil panen,minyak atsiri dan kandungannya, minyak, karbohidrat, gula larut dan kandungan nutrisi tanaman obat dan aromatik secara signifikanlebih dipengaruhi oleh menambahkan pupuk hayati dari pada pupuk kimia.

Kata kunci: pupuk hayati, obat, aromatik, tanaman

INTRODUCTION

A biological fertilizer (also bio-fertilizer) is a substancewhich contains living microorganisms, when applied toseed, plant surfaces, or soil colonizes the rhizosphere or theinterior of the plant and promotes growth by increasing thesupply or availability of primary nutrients to the host plant(Vessey 2003). Bio-fertilizers add nutrients through thenatural processes of nitrogen fixation, solubilizingphosphorus, and stimulating plant growth through the

synthesis of growth-promoting substances. Bio-fertilizerscan be expected to reduce the use of chemical fertilizersand pesticides. The microorganisms in bio-fertilizersrestore the soil's natural nutrient cycle and build soilorganic matter. Through the use of bio-fertilizers, healthyplants can be grown, while enhancing the sustainability andthe health of the soil. Since they play several roles, apreferred scientific term for such beneficial bacteria isplant-growth promoting rhizobacteria (PGPR). Therefore,they are extremely advantageous in enriching soil fertility

KHALID – Biological fertilization of medicinal and aromatic plants 125

and fulfilling plant nutrient requirements by supplying theorganic nutrients through microorganism and theirbyproducts. Hence, bio-fertilizers do not contain anychemicals which are harmful to the living soil. Bio-fertilizers are eco-friendly organic agro-input and morecost-effective than chemical fertilizers. Bio-fertilizers suchas Rhizobium, Azotobacter, Azospirillum and blue greenalgae (BGA) have been in use a long time. Rhizobiuminoculants’ is used for leguminous crops. Azotobacter canbe used with crops like wheat, maize, mustard, cotton,potato and other vegetable crops. Azospirillum inoculationsare recommended mainly for sorghum, millets, maize,sugarcane and wheat. Blue green algae belongings generalcyanobacteria genus, Nostoc, Anabaena, Tolypothrix orAulosira, fix atmospheric nitrogen and are used asinoculations for paddy crop grown both under upland andlow-land conditions. Anabaena in association with waterfern Azolla contributes nitrogen up to 60 kg/ha/season andalso enriches soils with organic matter (Vessey 2003).Other types of bacteria, so-called phosphate-solubilizingbacteria, such as Pantoea agglomerans strain P5 orPseudomonas putida strain P13 are able to solubilize theinsoluble phosphate from organic and inorganic phosphatesources (Subba Rao 1984). In fact, due to immobilizationof phosphate by mineral ions such as Fe, Al and Ca ororganic acids, the rate of available phosphate (Pi) in soil iswell below plant needs. In addition, chemical Pi fertilizersare also immobilized in the soil, immediately, so that lessthan 20 percent of added fertilizer is absorbed by plants.Therefore, reduction in Pi resources, on one hand, andenvironmental pollutions resulting from both productionand applications of chemical Pi fertilizer, on the otherhand, have already demanded the use of new generation ofphosphate fertilizers globally known as phosphate-solubilizing bacteria or phosphate bio-fertilizers. Atpresent, however, the yield of many crops in the world hasreached a plateau. Moreover, the negative effects of heavyapplications of chemical inputs are becoming apparent, interms of both production and the environment, especially inthe case of medicinal and aromatic plants. Physiologicaldisturbance of plant metabolism is common, due to theaccumulation of excess plant nutrients in the soil. Thespread of soil-borne diseases is a threat to medicinal andaromatic plants production, especially where monocultureis prevailing. Pollution of underground and surface waterby nitrates is sometimes reported from medicinal andaromatic plants producing areas. Quality deterioration, interms of a decrease in the content of vitamins, sugars activeprincipals, is becoming a subject of concern. All thesefactors are giving farmers an interest in the function andutilization of soil microorganisms, as a way of repairing thedamage from the overuse of chemical inputs. Many farmersin world are showing a strong interest in the utilization ofmicroorganisms to help stimulate plant nutrient uptake;provide biological control of soil-borne diseases; hasten thedecomposition of straw and other organic wastes; improvesoil structure; and promote the production ofphysiologically active substances in the rhizosphere or inorganic matter. The main incentive for farmers to usemicroorganisms seems to be that they hope to increase the

yield or quality of their crops at a relatively low cost,without a large investment of money and labor. Althoughmany microbial materials are sold commercially, most ofthem are not microbiologically defined, i.e. themicroorganisms contained in the products are notidentified, and the microbial composition is not fixed.Many of these commercial products are advertised as ifthey could solve any problem a farmer is likely toencounter. Because most extension advisors lack anyknowledge of microbial products, confusion and troublefrequently occur.

The main objectives of this manuscript are: (i) Types ofbiological fertilization, i.e. nitrogen fixation, phosphatesolubilizing microorganisms, sulphur oxidizing bacteria,silicate bacteria, plant growth promoting rhizobacteria(PGPR), mycorrhizal fungi, decomposition, and (ii) Effectof biological fertilization on medicinal and aromatic plants.This manuscript concentrates on biological fertilization itseffects on medicinal and aromatic plants because of thereare no review article was published before concentrated onthese items.

TYPES OF BIOLOGICAL FERTILIZATION

Nitrogen fixationNitrogen can be found in many forms in our

environment. Nitrogen is also very important for plants tolive. The earth's atmosphere is made up of 78 percentnitrogen in the form of a colorless, odorless, nontoxic gas.The same nitrogen gas found in the atmosphere can befound in spaces between soil particles. However, plants areunable to use this form of nitrogen. Certainmicroorganisms found in the soil are able to convertatmospheric nitrogen into forms plants can use. This iscalled biological nitrogen fixation (Subba Rao 1984). Oneof the most interesting forms of biological nitrogen fixationis that which takes place by microorganisms living in verysmall nodules on the roots of certain plants such aslegumes. This is called symbiotic nitrogen fixation. Asymbiotic relationship is an association or relationshipwhere both organisms mutually benefit. In this case,microorganisms obtain food and energy from the root ofthe plant while producing nitrogen the plant can use forgrowth and development. The form of nitrogen produced isthe same form of nitrogen that is found in several types ofcommercial nitrogen fertilizers (Subba Rao 1984). Themicroorganism's ability to fix atmospheric nitrogen is oftendiscussed in terms of the plant's ability to fix nitrogen. Theamount of fixation that takes place is strongly influencedby soil conditions. Factors such as moisture, temperature,oxygen supply and fertility in the soil can influencefixation. Diseases and insects can also affect the degree ofnitrogen fixation (Subba Rao 1984). One of the mostcommon groups of plants that fix nitrogen is legumes. Ofthe total nitrogen required by legumes, generally about halfis nitrogen fixed from the atmosphere, with the remainderbeing taken up from residual nitrate in the soil. This meansthat where legumes are grown, outside applications ofmanure or fertilizer nitrogen are not needed. Different


legumes also vary in the amount of total nitrogen they canfix (Subba Rao 1984).

Phosphate solubilizing microorganismsPhosphorous is added to cultivated soil in different

forms as mineral phosphate fertilizers or organic manure,the soluble P in these fertilizers is quickly turns intounavailable form for plant nutrition, this problem is wellknown in Egyptian soils specially those rich in CalciumCarbonate (El-Gamal 1996), phosphorous is commonlydeficient in most natural soils, since it is fixed as calciumphosphates in alkaline soils (Cunningham and Kuiack1992; Goldstein 1995). Faisal and El-Dawwy 1999indicated that inadequate phosphorous is a widespreadproblem in crop production in Egypt and elsewhere. InEgypt’s alkaline soils, low solubility calcium triphosphateare formed following the application of P fertilizer, solublephosphate ions also are adsorbed on solid calciumcarbonate surfaces. Fortunately, soil microorganismsknown as phosphate solubilizing microorganisms play afundamental role in converting P fixed form to be solubleready available for plant nutrition. The microbialbreakdown of soil organic matter is associated with anincrease organic, inorganic acids and CO2 production withpossibly increases the solubility of soil phosphate (Quastel1965; Taha et al. 1969; Mishustin et al. 1972; Alexander1977; Pamela and Hayasaka 1982; Subba Rao 1984 andCurl and Truelove 1985). However Cunningham andKuiack (1992) and Goldstein (1995) reported that,insoluble calcium phosphate can be dissolved and madeavailable to plants by rhizosphere microorganisms via amechanism that thought to involve the release of organicacids. Most studies of phosphate solubilizingmicroorganisms involve inoculating the soils (Mishustin etal. 1972). Inoculation with phosphate-dissolvers is claimedto increase the yield of many agriculture crops (Taha et al.1969; Ewada 1976; Ocampo et al. 1978; Guar et al. 1980;Abdel-Nasser et al. 1982; Subba Rao 1984). Several reportshave examined the ability of different bacterial species tosolubilize insoluble inorganic phosphate compounds, suchas tricalcium phosphate, dicalcium phosphate,hydroxyapatite, and rock phosphate (Goldstein 1986).Among the bacterial genera with this capacity arePseudomonas, Bacillus, Rhizobium, Burkholderia,Achromobacter, Agrobacterium, Micrococcus, Aerobacter,Flavobacterium and Erwinia. There are considerablepopulations of phosphate-solubilizing bacteria in soil and inplant rhizospheres (Sperberg 1958; Katznelson et al. 1962;Raghu and MacRae 1966; Alexander 1977). These includeboth aerobic and anaerobic strains, with a prevalence ofaerobic strains in submerged soils (Raghu and MacRae1966). A considerably higher concentration of phosphatesolubilizing bacteria is commonly found in the rhizospherein comparison with nonrhizosphere soil (Katznelson et al.1952; Raghu and MacRae 1966).

Sulphur oxidizing bacteriaSulphur is one of the essential plant nutrients and it

contributes to yield and quality of crops. Sulphur occurs ina wide variety of organic and inorganic combinations. The

transfer of sulphur between the inorganic and organic poolis entirely caused by the activity of the soil biota,particularly the soil microbial biomass, which has greatestpotential for both mineralization and also for subsequenttransformation of the oxidation state of sulphur.Thiobacillus play an important role in sulphur oxidation insoil. Sulphur oxidation is the most important step ofsulphur cycle, which improves soil fertility. It results in theformation of sulphate, which can be used by the plants,while the acidity produced by oxidation helps to solubilizeplant nutrients and improves alkali soils (Wainwright1984). The sulphur oxidizing microorganisms are primarilythe gram negative bacteria currently classified as species ofThiobacillus, Thiomicrospira and Thiosphaera, butheterotrophs, such as some species of Paracoccus,Xanthobacter, Alcaligenes and Pseudomonas can alsoexhibit chemolithotrophic growth on inorganic sulphurcompounds (Kuenen and Beudeker 1982). Two clearmetabolic types exist in this group: The obligatechemolithotrophs, which can only grow when suppliedwith oxidizable sulphur compounds (and CO2 as the sourceof metabolic carbon) and heterotrophs that can also use thechemolithoautotrophic mode of growth. The obligatechemolithotrophs include Thiobacillus thioparus, T.neapolitanus, T. denitrificans (facultative denitrifier),Thiobacillus thiooxidans (extreme acidophile), Thiobacillusferrooxidans (acidophilic ferrous iron-oxidizer), Thiobacillushalophilus (halophile) and some species of Thiomicrospira.The heterotrophs include Thiobacillus novellus, T. acidophilus(acidophile), Thiobacillus aquaesulis (moderate thermophile),Thiobacillus intermedius, Paracoccus denitrificans, P.versutus, Xanthobacter tagetidis, Thiosphaera pantotrophand Thiomicrospira thyasira.

Several Thiobacillus species are able to utilize mixturesof inorganic and organic compounds simultaneously, oftenreferred to as mixotrophic growth (Kuenen and Beudeker1982). Depending on the ratio of inorganic and organicsubstrates, CO2 may serve as an additional carbon source.Of the 13 species of the genus Thiobacillus recognized,occurring in diverse habitats, only five species areimportant in sulphur oxidation in soil (Starkey 1966). Fourof these Thiobacillus thiooxidans, T. ferrooxidans, T.thioparus and T. denitrificans are obligate chemo-autotrophs while T. novellus is considered a facultativechemoautotroph (Taylor and Hoare 1969). Also fungi arecapable of oxidizing elemental sulphur and thiosulphate,which include, Alternaria tenuis, Aureobasidium pullulans,Epicoccum nigrum, a range of Penicillium species(Wainwright 1984), Scolecobasidium constrictum,Myrothecium cinctum and Aspergillus (Shinde et al. 1996).

Though sulphur traces may be oxidized in soil byvarious species of microorganisms and fungi, Waksman(1932) pointed out Thiobacillus as the most characteristicgroup of microorganism performing the oxidative part ofsulphur transformation in soil. Studies on the distributionof Thiobacillus thiooxidans and T. thioparus showed thatthese bacteria are found in an active state mainly in soilsfertilized with sulphur. The distribution of T. thioparus insoils was studied by Starkey (1934) who demonstrated thealmost ubiquitous presence of bacteria in alkaline and


neutral soils and their absence in strongly acid soilsfertilized with sulphur. The widespread occurrence ofThiobacillus in soils fertilized with sulphur or in soils inwhich accumulation of sulphur compounds occurs undernatural conditions (marshes and peats) indicates that thesebacteria play an important role in the oxidation of sulphurand its compounds in soils. Inoculation of Thiobacillusgenerally increases the rate of sulphur oxidation (Kapoor,and Mishra 1989). Kapoor and Mishra (1989) observed thatsulphur was rapidly oxidized in a field soil of pH 8.0 andthe rate of oxidation could be enhanced by inoculation withT. thiooxidans.

Silicate bacteriaSilicate bacteria or biological potassium fertilizer can

activate the fixed potassium for plant nutrition, as well asprevention and control of plant diseases; also it caneffectively prevent crops from early aging and have astrong resistance to drought and cold (Subba Rao 1984).Zahra et al. (1984) reported that, silicate-dissolving bacteriaplayed a pronounced role in the biological weathering ofsoil minerals and it can promote K and Si releasing fromfeldspar. Sheng et al. (2003) showed that, silicate-dissolving bacteria could activate soil P, K, andmicronutrients reserves and promote plant growth.Styriakova et al. (2003) reported that, the activity of silicatedissolving played a pronounced role in release of Si, Fe andK from feldspar and Fe oxyhydroxides.

Plant growth promoting rhizobacteria (PGPR)Plant growth promoting rhizobacteria (PGPR) comprise

a diverse group of rhizosphere-colonizing bacteria anddiazotrophic microorganisms which, when grown in asso-ciation with a plant, stimulate growth of the host. PGPRcan affect plant growth and development indirectly ordirectly (Glick 1995; Vessey 2003). In indirect promotion,the bacteria decrease or eliminate certain deleterious effectsof a pathogenic organism through various mechanisms,including induction of host resistance to the pathogen (VanLoon 2007). direct promotion, the bacteria may provide thehost plant with synthesized compounds; facilitate uptake ofnutrients; fix atmospheric nitrogen; solubilize minerals suchas phosphorus; produce siderophores, which solubilize andsequester iron; synthesize phytohormones, including auxins,cytokinins, and gibberellins, which enhance various stagesof plant growth; or synthesize enzymes that modulate plantgrowth and development (Lucy et al. 2004; Gray and Smith2005). Bradyrhizobium and Sinorhizobium, of the bacterialfamily Rhizobiaceae, are known for their ability to fixatmospheric nitrogen while living symbiotically on andnodulating the roots of leguminous plants. However, membersof this family also display non-specific associative interactionswith roots of other plants, without forming nodules (VanLoon 2007). These rhizobial strains are presumed to produceplant growth regulators, and are classified as PGPR(Vessey 2003).

Mycorrhizal fungi

Mycorrhizal typesOf the many types of mycorrhizal association (Harley

and Smith 1983), two are of major economic and ecologicalimportance: ectomycorrhizal associations, and the endo-mycorrhizal association of the vesicular-arbuscular (VA)type. In ectomycorrhizal associations, the fungi invade thecortical region of the host root without penetrating corticalcells. The main diagnostic features of this type ofmycorrhiza are (i) the formation within the root of a hyphalnetwork known as the Hartig net around cortical cells and(ii) a thick layer of hyphal mat on the root surface knownas sheath or mantle, which covers feeder roots. Infection ofhost plants by ectomycorrhizal fungi often leads to changesin feeder roots that are visible to the naked eye. Feederroots colonized by the fungi are thicker and more branchedthan uncolonized roots; ectomycorrhizal feeder roots alsotend to be colored differently.

In endomycorrhizal associations of the VA type, thefungi penetrate the cortical cells and form clusters of finelydivided hyphae known as arbuscules in the cortex. Theyalso form vesicles, which are membrane-bound organellesof varying shapes, inside or outside the cortical cells.Arbuscules are believed to be the sites where materials areexchanged between the host plant and the fungi. Vesiclesgenerally serve as storage structures, and when they are oldthey can serve as reproductive structures. Vesicles andarbuscules, together with large spores, constitute thediagnostic features of the VA mycorrhizas. Roots have tobe cleared and stained in specific ways and examined undera microscope to see that they are colonized by VA mycor-rhizal fungi. Because vesicles are not always found in thesetypes of mycorrhizal associations, some researchers nowprefer the designation arbuscular mycorrhiza (AM) overthe term vesicular-arbuscular (VA) mycorrhiza. Both AMfungi and ectomycorrhizal fungi extend hyphae from theroot into the soil, and these external (or extraradical)hyphae are responsible for translocating nutrients from thesoil to the root.

Most ectomycorrhizal fungi belong to several generawithin the class Basidiomycetes, while some belong to thezygosporic of Zygomycetes and ascomycetes. On the otherhand, AM fungi belong to six genera within theazygosporous of Zygomycetes.

Host specificityAM associations occur in a wide spectrum of tropical

and temperate tree species. They are known not to occuronly in a few plants, namely members of the familiesAmaranthaceae, Pinaceae, Betulaceae, Cruciferae,Chenopodiaceae, Cyperaceae, Juncaceae, Proteaceae, andPolygonaceae. The ectomycorrhizas, on the other hand,occur primarily in temperate forest species, although theyhave been reported to colonize a limited number of tropicaltree species.

Functions of mycorrhizal fungiResults of experiments suggest that AM fungi absorb N,

P, K, Ca, S, Cu, and Zn from the soil and translocate them


to associated plants (Tinker and Gilden 1983). However,the most prominent and consistent nutritional effect of AMfungi is in the improved uptake of immobile nutrients,particularly P, Cu, and Zn (Pacovsky 1986; Manjunath andHabte 1988). The fungi enhance immobile nutrient uptakeby increasing the absorptive surfaces of the root. Thesupply of immobile nutrients to roots is largely determinedby the rate of diffusion. In soils not adequately suppliedwith nutrients, uptake of nutrients by plants far exceeds therate at which the nutrients diffuse into the root zone,resulting in a zone around the roots depleted of thenutrients. Mycorrhizal fungi help overcome this problemby extending their external hyphae to areas of soil beyondthe depletion zone, thereby exploring a greater volume ofthe soil than is accessible to the unaided root. Enhancednutrient uptake by AM fungi is often associated withdramatic increase in dry matter yield, typically amountingto several-fold increases for plant species having highdependency on mycorrhiza.

AM fungi may have biochemical capabilities forincreasing the supply of available P and other immobilenutrients. These capabilities may involve increases in rootphosphatase activity, excretion of chelating agents, andrhizosphere acidification. However, these mechanisms donot appear to explain the very pronounced effect the fungihave on plant growth (Habte and Manjunth 1991).

AM fungi are often implicated in functions which mayor may not be related to enhance nutrient uptake. Forexample, they have been associated with enhancedchlorophyll levels in leaves and improved plant toleranceof diseases, parasites; water stress, salinity, and heavymetal toxicity (Bethlenfalvay and Gabor 1992). Moreover,there is increasing evidence that hyphal networks of AMfungi contribute significantly to the development of soilaggregates, and hence to soil conservation (Miller andJastrow 1992).

Charcoal applicationThe amount of nutrients (N, P, and K) absorbed by the

shoots showed a trend similar to that of the shoot freshweight. The amount of N fixed by the nodules andtransported to the shoots was calculated by subtracting theN content of the shoots of the plants not inoculated withrhizobia from the N content of the inoculated plants. Theaddition of charcoal increased this amount of N 2.8-4.0times. Added charcoal also increased the nodule weight by2.3 times. Significant correlation was observed between theincrements of P and N, suggesting that the stimulation ofnitrogen fixation by charcoal addition may be due to thestimulation of P uptake. Charcoal may stimulate the growthof AMF by the following mechanism. Charcoal particleshave a large number of continuous pores with a diameter ofmore than 100µm. They do not contain any organicnutrients, because of the carbonization process. The largepores in the charcoal may offer a new microhabitat to theAMF, which can obtain organic nutrients through myceliaextended from roots. This may enable the AMF to extendtheir mycelia far out from the roots, thus collecting a largeramount of available phosphate (Nishio and Okano 1991).

DecompositionOrganic matter decomposition serves two functions for

the microorganisms, providing energy for growth andsupplying carbon for the formation of new cells. Soilorganic matter (SOM) is composed of the “living”(microorganisms), the “dead” (fresh residues), and the“very dead” (humus) fractions. The “very dead” or humusis the long-term SOM fraction that is thousands of yearsold and is resistant to decomposition. Soil organic matterhas two components called the active (35%) and thepassive (65%) SOM. Active SOM is composed of the“living” and “dead” fresh plant or animal material which isfood for microbes and is composed of easily digestedsugars and proteins. The passive SOM is resistant todecomposition by microbes and is higher in lignin.Microbes need regular supplies of active SOM in the soil tosurvive in the soil. Long-term no-tilled soils havesignificantly greater levels of microbes, more activecarbon, more SOM, and more stored carbon thanconventional tilled soils. A majority of the microbes in thesoil exist under starvation conditions and thus they tend tobe in a dormant state, especially in tilled soils. Dead plantresidues and plant nutrients become food for the microbesin the soil. Soil organic matter (SOM) is basically all theorganic substances (anything with carbon) in the soil, bothliving and dead. SOM includes plants, blue green algae,microorganisms (bacteria, fungi, protozoa, nematodes,beetles, springtails, etc.) and the fresh and decomposingorganic matter from plants, animals, and microorganisms(Hoorman and Islam 2010).

Climate, Temperature, and pH effects on SOMSOM is affected by climate and temperature. Microbial

populations double with every 10 degree Fahrenheit changein temperature. If we compare the tropics to colder arcticregions, we find most of the carbon is tied up in trees andvegetation above ground. In the tropics, the topsoil hasvery little SOM because high temperatures and moisturequickly decompose SOM. Moving north or south from theequator, SOM increases in the soil. The tundra near theArctic Circle has a large amount of SOM because of coldtemperatures. Freezing temperatures change the soil so thatmore SOM is decomposed then in soils not subject tofreezing.

Moisture, pH, soil depth, and particle size affect SOMdecomposition. Hot, humid regions store less organiccarbon in the soil than dry, cold regions due to increasedmicrobial decomposition. The rate of SOM decompositionincreases when the soil is exposed to cycles of drying andwetting compared to soils that are continuously wet or dry.Other factors being equal, soils that are neutral to slightlyalkaline in pH decompose SOM quicker than acid soils;therefore, liming the soil enhances SOM decomposition andcarbon dioxide evolution. Decomposition is also greatestnear the soil surface where the highest concentration ofplant residues occur. At greater depths there is less SOMdecomposition, which parallels a drop in organic carbonlevels due to less plant residues. Small particle sizes aremore readily degraded by soil microbes than large particles


because the overall surface area is larger with smallparticles so that the microbes can attack the residue.

A difference in soil formation also occurs traveling eastto west across the United States. In the east, hardwoodforests dominated and tree tap roots were high in lignin,and deciduous trees left large amounts of leaf litter on thesoil surface. Hardwood tree roots do not turn over quicklyso organic matter levels in the subsoil are fairly low. Inforest soils, most of the SOM is distributed in the top fewinches. As you move west, tall grassland prairiesdominated the landscape and topsoil formed from deepfibrous grass root systems. Fifty percent of a grass root diesand is replaced every year and grass roots are high insugars and protein (higher active organic matter) and lowerin lignin. So soils that formed under tall grass prairies arehigh in SOM throughout the soil profile. These prime soilsare highly productive because they have higher percentageof SOM (especially active carbon), hold more nutrients,contain more microbes, and have better soil structure due tolarger fungal populations.

Carbon to Nitrogen(C:N) ratioLow nitrogen content or a wide C:N ratio is associated

with slow SOM decay. Immature or young plants havehigher nitrogen content, lower C:N ratios and faster SOMdecay. For good composting, a C:N ratio less than 20allows the organic materials to decompose quickly (4 to 8weeks) while a C:N ratio greater than 20 requiresadditional N and slows down decomposition.

The C:N ratio of most soils is around 10:1 indicatingthat N is available to the plant. The C:N ratio of most plantresidues tends to decrease with time as the SOM decays.This results from the gaseous loss of carbon dioxide.Therefore, the percentage of nitrogen in the residual SOMrises as decomposition progresses. The 10:1 C:N ratio ofmost soils reflects an equilibrium value associated withmost soil microbes (bacteria 3:1 to 10:1, fungus 10:1 C:Nratio).

Bacteria are the first microbes to digest new organicplant and animal residues in the soil. Bacteria typically canreproduce in 30 minutes and have high N content in theircells (3 to 10 carbon atoms to 1 nitrogen atom or 10 to 30%nitrogen). Under the right conditions of heat, moisture, anda food source, they can reproduce very quickly. Bacteriaare generally less efficient at converting organic carbon tonew cells. Aerobic bacteria assimilate about 5 to 10 percentof the carbon while anaerobic bacteria only assimilate 2 to5 percent, leaving behind many waste carbon compoundsand inefficiently using energy stored in the SOM.

Fungus generally release less carbon dioxide into theatmosphere and are more efficient at converting carbon toform new cells. The fungus generally captures more energyfrom the SOM as they decompose it, assimilating 40 to 55percent of the carbon. Most fungi consume organic matterhigher in cellulose and lignin, which is slower and tougherto decompose. The lignin content of most plant residuesmay be of greater importance in predicting decompositionvelocity than the C:N ratio.

Mycorrhizal fungi live in the soil on the surface of orwithin plant roots. The fungi have a large surface area and

help in the transport of mineral nutrients and water to theplants. The fungus life cycle is more complex and longerthan bacteria. Fungi are not as hardy as bacteria, requiring amore constant source of food. Fungi population levels tendto decline with conventional tillage. Fungi have a highercarbon to nitrogen ratio (10:1 carbon to nitrogen or 10%nitrogen) but are more efficient at converting carbon to soilorganic matter. With high C:N organic residues, bacteriaand fungus take nitrogen out of the soil.

Protozoa and nematodes consume other microbes.Protozoa can reproduce in 6-8 hours while nematodes takefrom 3 days to 3 years with an average of 30 days toreproduce. After the protozoa and nematodes consume thebacteria or other microbes (which are high in nitrogen),they release nitrogen in the form of ammonia. Ammonia(NH4+) and soil nitrates (NO3-) are easily converted backand forth in the soil. Plants absorb ammonia and soilnitrates for food with the help of the fungi mycorrhizalnetwork.

Microorganism populations change rapidly in the soil asSOM products are added, consumed, and recycled. Theamount, the type, and availability of the organic matter willdetermine the microbial population and how it evolves.Each individual organism (bacteria, fungus, and protozoa)has certain enzymes and complex chemical reactions thathelp that organism assimilate carbon. As waste productsare generated and the original organic residues aredecomposed, new microorganisms may take over, feedingon the waste products, the new flourishing microbialcommunity (generally bacteria), or the more resistantSOM. The early decomposers generally attack the easilydigested sugars and proteins followed by microorganismsthat attack the more resistant residues.

It can be summarized that (i) Microorganisms aboundin the soil and are critical to decomposing organic residuesand recycling soil nutrients. Bacteria are the smallest andmost hardy microbe in the soil and can survive under harshconditions like tillage. Bacteria are only 20-30% efficientat recycling carbon, have high nitrogen content (3 to 10carbon atoms to nitrogen atom or 10 to 30% nitrogen),lower carbon content, and a short life span. Carbon useefficiency is 40-55% for mycorrhizal fungi so they storeand recycle more carbon (10:1 carbon to nitrogen ratio) andless nitrogen (10%) in their cells than bacteria. Fungi aremore specialized but need a constant food source and growbetter under no-till conditions. (ii) Soil organic matter(SOM) is composed of the “living” (microorganisms), the“dead” (fresh residues), and the “very dead” (humus)fractions. Active SOM is composed of the fresh plant oranimal material which is food for microbes and iscomposed of easily digested sugars and proteins. Thepassive SOM is resistant to decomposition by microbes(higher in lignin). Active SOM improves soil structure andholds plant available nutrients. Every 1% SOM contains1,000 pounds of nitrogen, 100 pounds of phosphorus, 100pounds of potassium, and 100 pounds of sulfur along withother essential plant nutrients. Tillage destroys SOM byoxidizing the SOM, allowing bacteria and other microbesto quickly decompose organic residues. Highertemperatures and moisture increase the destruction of SOM


by increasing microbial populations in the soil. Organicresidues with a low carbon to nitrogen (C:N) ratio (lessthan 20) are easily decomposed and nutrients are quicklyreleased (4 to 8 weeks), while organic residue with a highC:N ratio (greater than 20) decompose slowly and themicrobes will tie up soil nitrogen to decompose theresidues. Protozoa and nematodes consume other microbesin the soil and release the nitrogen as ammonia, whichbecomes available to other microorganisms or is absorbedby plant roots.

EFFECT OF BIOOGICAL FERTILIZATION ONMEDICINAL AND AROMATIC PLANTS

Growth characters of rosemary, K, N content essentialoil constituents (alpha-pinene, β-pinene, limonene,1,8-cineole, linalool, campor, β-terpineol, borneol, terpinen 4-ol,carvone, thymol, carvacrol, linalylacetate, geranylacetate,β-caryophyllene, caryophyllene oxide) were significantlyincreased under biofertilizer (Azotobacter vinelandii)treatments (Leithy and El-Meseiry 2006). The effects ofbiofertilization on growth, fruit yield, and oil compositionof fennel plants were investigated by Mahfouz and Sharaf-Eldin (2007). Application of biofertilizer, which was amixture of Azotobacter chroococcum, Azospirillum lipoferum,and Bacillus megaterium applied with chemical fertilizers(only 50% of the recommended dosage of NPK) increasedvegetative growth (plant height, number of branches, andherb fresh and dry weight per plant) compared to chemicalfertilizer treatments only. The tallest plants, the highestnumber of branches per plant, and the highest fresh and dryweights of plants were obtained from the treatment ofbiofertilizer plus a half dose of chemical fertilizer (357 kgammonium sulphate + 238 kg calcium super phosphate +60 kg potassium sulphate ha-1). The lowest fresh and dryweights of plants occurred with the 50% NPK. Also,addition of biofertilizer with the chemical fertilizerincreased these characters more than the half dose ofchemical fertilizer alone. Total carbohydrates in the dryplant material were influenced by the biofertilizer. Thehighest values of total carbohydrates were found in thetreatment with biofertilizer plus a half dose of nitrogen andphosphorus. Nitrogen, phosphorus, and potassium levels inthe plant tissue increased when soil was inoculated bynitrogen-fixing bacteria, phosphate dissolving bacteria, anda mixture of all strains, respectively. The least amount ofN, P and Kin the plant tissue occurred with the half dose ofchemical fertilizer. Essential oil content in the fennel fruitswas increased due to inoculation compared to the half doseof chemical fertilizer. The highest oil yield per plant wasobserved with the treatment of biofertilizer plus a half doseof nitrogen and phosphorus. The lowest amount of essentialoil yield was obtained with the half dose of chemicalfertilizer. Oxygenated compounds were increased as a resultof using biofertilizer. The highest anethol (trans-1-methoxy-4-(prop-1-enyl) benzene; C10H12O) in fennel essential oiloccurred with the half dose of N, P, and K and inoculationwith Bacillus megaterium. The effect of compost and bio-fertilizers on the growth, yield and essential oil constituents

of marjoram (Majorana hortensis L.) was investigated byGharib et al. (2008). Forty five days old seedling weretransplanted in soil treated with 15 and 30% aqueous extractsof compost and/or biofertilizers (mixture of Azospirillumbrasiliensis, Azotobacter chroococcum, Bacillus polymyxaand B. circulans) in addition to the recommended nitrogen,phosphorus and potassium (NPK) doses as control. Use ofcombined treatment of bio-fertilizers gave better results forall studied traits than those obtained from either nitrogenfixers (Azospirillum brasiliensis, Azotobacterchroococcum, B. polymyxa) or B. circulans alone). Theessential oil percentage and yield per plant for threecuttings was almost two fold higher on fresh weight basisas a result of aqueous extracts of compost at low level +bio-fertilizers compared with control, indicating thatcombinations of low input system of integrated nutrientmanagement could be beneficial to obtain relatively goodyields of essential oil. Essential oil composition usingGC/MS revealed that marjoram belongs to the cis-sabinenehydrate/terpinene-4-ol chemotype. The chemicalcomposition of marjoram essential oil did not change due tothe fertilization type or level; rather the relative percentages ofcertain constituents were affected. The highest level of cis-sabinene hydrate (18.47%) and terpinene-4-ol (24.24%)was obtained with aqueous extracts of compost at 30% + B.circulans and aqueous extracts of compost at 30% + (A.brasiliense + A. chroococcum + B. polymyxa), respectively.

Most studies of phosphate solubilizing microorganismsinvolve inoculating the soils (Mishustin et al. 1972).Inoculation with phosphate-dissolvers is claimed to increasethe yield of many agriculture crops (Taha et al. 1969; Ewada1976; Ocampo et al. 1978; Guar et al. 1980; Abdel-Nasseret al. 1982; Subba Rao 1984). Gomma (1989) demonstratedthat, some effects of phosphate solubilizing microorganism’sinoculation have been observed in terms of increasing theamounts of available P and plant growth of cropproduction. Hauka et al. (1990) stated that, phosphatesolubilizing microorganisms significantly increased dryyields and protein content of Barley and Tomato plant. El-Gamal (1996) revealed that, P level or inoculation withphosphorene (phosphate solubilizing microorganisms)significantly increased soil available P, tuber N and Pcontents and their uptake, foliage dry weight, foliage Pcontent and P uptake, total P uptake and dry matter yield ofPotato plants. Applying phosphorene improved growth andP uptake by the Olive seedlings in comparison to thephosphate fertilizer alone (Faisal and El-Dawwy 1999).Applying phosphate solubilizing microorganisms withcalcium superphosphate to mustard (Sinapis alba L.) plantsimproved growth, seed yield, lipids content, N, P and theiruptake, and protein content but total carbohydrates, solublesugars and insoluble sugars were decreased, also saturatedand unsaturated fatty acids content were changed comparedwith applying calcium superphosphate fertilizer alone. Itrecommended that using phosphate solubilizing micro-organisms because it increases the production (quantity andquality) and medicinal properties. Also it is very cheap andexpressed cash money improving the income of farmer, inaddition, uses biofertilizer (phosphate solubilizingmicroorganisms) is safe for human health (Khalid 2004).


Awad and Khalil (2003) reported that, the biofertilizer(Thiobacillus thiooxidans) and sulphur significantlyincreased the growth of squash and raised their nutrientcontent than Sulphur fertilizer alone. Treated celery (Apiumgraveolens L cv. dulce) plants with different levels ofsulphur and sulphur-oxidizing bacteria resulted in asignificant increase in growth and yield characters, i.e.plant height, branch number, leaf number, umbel number,fresh weight, dry weight and fruit yield/plant in comparisonwith control plants. Khalid (2005) states that chemicalcomposition analysis of treated plants showed an increasein the essential and fixed oil content, total carbohydrates,crude protein and nutrients content (NPKS) and its uptake.Also treated plants showed an increase in the maincomponents of the essential oil (limonene and β-selinene)extracted from the fruits, comparison to untreated plants.

Evaluate the effect of natural products as a source ofsome important elements such as rock phosphate as asource of phosphorous and feldspar mica as a source ofpotassium with biological potassium phosphorousfertilizers or biological potassium fertilizers (Silicatebacterium) at different levels (0.0, 25, 50 and 100 g/L) onRuta graveolens L. plant instead of the chemical fertilizeswere investigated by Khalid et al. (2007). Addingbiological fertilizer with feldspar or rock phosphateimproved vegetative growth characters such as plant height(cm), branches number/plant, fresh and dry weights ofdifferent plant parts i.e. leaves, stems and roots (g/plant), inaddition to some chemical constituents as essential oil, totalflavonoides, P, K, Fe, Zn and Cu content. On the otherhand, the main constituents of essential oil and N contentwere decreased compared with adding recommendedchemical fertilizers.

According to Banchio et al. (2008) the effects of rootcolonization by plant growth promoting rhizobacteria(PGPR) on biomass, and qualitative and quantitativecomposition of essential oils were determined in thearomatic crop Origanum majorana L. (sweet marjoram).PGPR strains evaluated were Pseudomonas fluorescens,Bacillus subtilis, Sinorhizobium meliloti, and Brady-rhizobium sp. Only P. fluorescens and Bradyrhizobium sp.showed significant increases in shoot length, shoot weight,number of leaf, number of node, and root dry weight, incomparison to control plants or plants treated with otherPGPR. Essential oil yield was also significantly increasedrelative to non-inoculated plants, without alteration of oilcomposition. P. fluorescens has clear commercial potentialfor economic cultivation of O. majorana.

In studies on Coriandrum sativum, Anethum graveolensand Foeniculum vulgare, it was shown that AMF rootcolonization enhances the essential oil quality by alteringessential oil components (Kapoor et al. 2002, 2004). Fourorganic amendments: leaf compost (LC), vegetable compost(VC), poultry manure (PM) and sewage sludge (SSL)applied at four doses (40, 80, 100 and 120 t ha-1) wereevaluated for their effect on the herbage yield, essential oilcontent and inoculum potential (IP) of native arbuscularmycorrhizal fungi (AMF) on three varieties of Javacitronella, Cymbopogon winterianus Jowitt (Manjusha,Mandakini, and Bio-13). PM applied at 100 t ha-1 followed

by SSL increased the herbage, essential oil content and drymatter yield significantly. Bio-13 performed better andproduced the highest herbage, essential oil and dry matteryield. The type and dose of the various organicamendments also significantly influenced the indigenousAMF infectious propagules in soil. Highest number ofAMF propagules were recorded in the LC amended plots inall the three varieties. Amongst the varieties, highest nativemycorrhizal inoculum was recorded in the Bio-13. Leastnumber of AM infectious propagules was recorded in theMandakini plants grown in 40 t ha-1 SSL (Tanu 2004).Khaosaad et al. (2006) observed that essential oil levels inOriganum species are increased in the presence ofarbuscular mycorrhizal fungi. A field experiment wasconducted to study and compare the effectiveness of twoarbuscular mycorrhizal fungi (AMF), Glomusmacrocarpum (GM) and Glomus fasciculatum (GF) onthree accessions of Artemisia annua. The AM inoculationsignificantly increased the production of herbage, dryweight of shoot, nutrient status (P, Zn and Fe) of shoot,concentration of essential oil and artemisinin in leaves ascompared to non-inoculated plants. The extent of growth,nutrient concentration and production of secondary plantmetabolites varied with the fungus-plant accessioncombination. The mycorrhizal dependency of the threeaccessions was related to the shoot: root ratio. Comparingthe two fungal inoculants in regard to increase in essentialoil concentration in shoot, the effectiveness of GF wasmore than that of GM. While in two accessions, GM wasmore effective in enhancing artemisinin concentration thanGF. Increase in concentration of essential oil was found tobe positively correlated to P-status of the plant conversely(Chaudhary et al. 2008).

Five strains of bacteria (Azotobacter chroococcum,Azospirillum lipoferum, Bacillus polymyxa, Bacillusmegaterium and Pseudomonas fluorescens) were mixed inequal parts and used as biofertilizer in this experiment. Thebiofertilizer treatment was applied alone or in combinationwith 1/3, 2/3 or full recommended dose of mineral nitrogenfertilizer. The results indicated that applying biofertilizertreatment alone or in combination with chemical Nfertilizer increased the growth, yield and chemicalconstituents of dill plant compared to the untreated control.The highest values of vegetative growth, oil yield,chlorophyll content and NPK percentages were recorded bythe treatment of bio-fertilizer plus two third ofrecommended dose of nitrogen fertilizer. The lowest valuesin this respect were obtained by control plants during twoseasons. The GC analysis of volatile oil indicated that themain components were carvone, limonene and apiol. Thesecomponents were affected by biofertilization and chemicalN treatments. Partial substitution of mineral nitrogenfertilizer by bio-fertilizer was recommended to increase theyield as well as the quality of dill plant (Hellal et al. 2011).Bio-fertilizer treatments increased the growth charactersand essential oil composition of coriander compared withthe chemical fertilizers treatments (Hassan et al. 2012).Bio-fertilizer treatments (mycorrhizal and phosphatebacteria) increased the seed yield and essential oil of fennelplants compared with vermicompost treatments (Darzi


2012). Application of phosphate bio-fertilizer and phosphoruswere significant on the vegetative growth characters ofTagetes erecta L. plants (Hashemabad et al. 2012).

Adding dry yeast at the rate of 6 g/L. was the mosteffective on growth parameters and oil-percent of Boragoofficinalis plant (Ezz El-Din and Hendawy 2010). AnIranian investigation revealed that inoculation of Ocimumbasilicum roots with plant growth-promoting rhizobacteria(PGPR) improved growth and accumulation of essentialoils. Treatments were Pseudomonas putida strain 41,Azotobacter chroococcum strain-5 and Azospirillum lipoferumstrain. In comparison to the control treatment, all factorswere increased by PGPR treatments. The maximum Rootfresh weight (3.96 g/plant), N content (4.72%) and essentialoil yield (0.82%) were observed in the Pseudomonas +Azotobacter + Azospirillum treatment. All factors werehigher in the Pseudomonas + Azotobacter + Azospirillumand Azotobacter + Azospirillum treatments (Ordookhani etal. 2011).

CONCLUSION

What is the key behind specificity of certain Nitrogenfixing microorganisms to selected plants? Why not otherNon-Nitrogen fixing microorganisms acquire the propertyof nitrogen fixation? Can we evolve nitrogen fixing plants?The search for new microorganisms capable of fixingnitrogen. Exploiting other plant microorganisms associations.Proper utilization of fertilizer nitrogen by means of slowrelease nitrogen fertilizer. Domestication and cultivation ofpromising nodulated legume species. Recycling of wastesfor elements; microorganisms abound in the soil and arecritical to decomposing organic residues and recycling soilnutrients. The above questions and statements are anoutlook of future research.

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Short Communication:Effects of temperature on growth, pigment composition and protein

content of an Antarctic Cyanobacterium Nostoc commune

RANJANA TRIPATHI, UMESH P. DHULDHAJ, SURENDRA SINGH♥

Centre of Advanced Study in Botany, Faculty of Science, Banaras Hindu University, Varanasi 221005, Utar Pradesh, India. Tel: +91-542-6701101; Fax:+91-542-2368174. e-mail: [email protected]

Manuscript received: 14 November 2012. Revision accepted: 30 November 2012.

Abstract. Tripathi R, Dhuldhaj UP, Singh S. 2012. Short Communication: Effects of temperature on growth, pigment composition andprotein content of an Antarctic Cyanobacterium Nostoc commune. Nusantara Bioscience 4: 134-137. Effect of temperature variation onbiomass accumulation, pigment composition and protein content were studied for the cyanobacterium Nostoc commune, isolated fromAntarctica. Results confirmed the psychrotrophic behavior (optimum growth temperature 250C) of the cyanobacterium. Lowtemperature increased the duration of lag phase and exponential growth phase. Maximum increase in biomass was recorded on 24th dayat 250C and on 12th day at 50C. The downshift from 25 to 50C had almost negligible effect on chl a content. Maximal protein contentwas recorded for cultures growing at 50C on 12th day. The carotenoids/chl a ratio was maximum (2.48) at 50C on 9th day. It remainedalmost constant for cultures growing at 5 and 350C. There was an induction in protein synthesis following downshift in temperaturefrom 25 to 5◦C.

Key words: Cyanobacterium, low-temperature, growth rate, phycobiliproteins, pigments

Abstrak. Tripathi R, Dhuldhaj UP, Singh S. 2012. Komunikasi singkat: Pengaruh suhu terhadap pertumbuhan, komposisi pigmen dankandungan protein cyanobacterium dari Antartika Nostoc commune. Bioscience Nusantara 4: 134-137. Pengaruh variasi suhu terhadapakumulasi biomassa, komposisi pigmen dan kandungan protein dipelajari pada cyanobacterium Nostoc komune, yang diisolasi dariAntartika. Hasil penelitian menegaskan perilaku psikrotrofik (suhu pertumbuhan optimum 25◦C) dari cyanobacterium tersebut. Suhurendah meningkatkan durasi fase lambat dan fase pertumbuhan eksponensial. Peningkatan biomassa maksimal tercatat hari ke-24 pada25◦C dan hari ke-12 pada 5◦C. Penurunan suhu dari 25 ke 5◦C hampir tidak berpengaruh pada kandungan chl a. Kandungan proteintertinggi tercatat pada kultur yang tumbuh pada 5◦C, hari ke-12. Rasio karotenoid/chl a tertinggi (2,48) terjadi pada 5◦C, hari ke-9. Halini hampir selalu konstan untuk kultur yang tumbuh pada 5-35◦C. Terdapat induksi dalam sintesis protein mengikuti penurunan suhudari 25 ke 5◦C.

Kata kunci: cyanobacterium, suhu rendah, tingkat pertumbuhan, phycobiliproteins, pigmen

Cold-stress is often lethal to living organisms. Forgrowth to occur in low temperature environments, cellularcomponents such as membranes, proteins and nucleic acidshave to adapt to the cold (Cavicchioli et al. 2002).Microbial diversity of the Antarctica is composed of eitherpsychrophilic (optimum growth temperature <150C) orpsychrotrophic (optimum growth temperature >150C)(Morita 1975; Veerapaneni 2009). Psychrotrophs constitutethe bulk of continental Antarctica microflora. Adaptiveresponses of the Antarctic microbes growing in thepermanently cold environments, especially at 0-40C arelittle studied (Chattopadhyay 2006). Majority of Antarcticcyanobacteria grow in a wide range of temperature(Nadeau and Castenholz 2000; Nadeau et al. 2001). Thetemperature-growth response suggests their closerelationship with moderate regions of the Antarctic(Seaberg 1981).

Nostoc commune (Nostocales) was collected fromSchirmacher Oasis, Antarctica by Dr. Suresh Chandra

Singh, a member of the Seventeenth Indian Expedition toAntarctic (Pandey and Upreti 2000). The cyanobacteriumwas isolated, purified using standard microbiologicaltechniques, and is maintained in nitrogen free BG-11-medium in a culture room set at 25±10C, illuminated withday-light fluorescent tubes having the photon fluency rateof 35E m-2 s-1 at the surface of vessels. Here, we studiedthe biomass accumulation (in terms of fresh weight and dryweight ml-1 volume of liquid culture harvested at 3 dayintervals) and pigment composition of N. commune, anAntarctic cyanobacterial to different temperatures (i.e., 5, 15,25, 350C).

Growth of N. commune was estimated in terms ofchlorophyll a (chl a) content and expressed in terms ofspecific growth rate computed as per the method of Myersand Kratz (1955). Growth rate (µ) of N. commune at eachtemperature was estimated as changes in biomass over timefrom the log-linear portion of the curve using non linearcurve fit method with Boltzmann constant. Chl a was

TRIPATHI et al. – Physiology of an Antarctic cyanobacterium Nostoc commune 135

quantified using the formula of Talling and Driver (1963),with 12.7 as extinction coefficient for chl a at 663 nm.Carotenoids were estimated according to Myers and Kratz(1955). Phycobilliproteins (PBPs) (both qualitatively andquantitatively) were analysed by recording the absorptionspectra and absorbance at various wavelengths in a UV-VIS spectrophotometer (Varian, Cary100-Bio, USA) with a1 cm light path. Fluorescence excitation and emissionspectra were recorded in a fluorescence spectrophotometer(Hitachi F-2500, Japan). The amounts of different PBPsnamely, phycocyanin (PC, Amax 565 nm), allophycocynain(APC, Amax 620 nm) and phycoerythrin (PE, Amax 650 nm)were determined according to the equations given byTandeau de Marsac and Houmard (1993). Protein wasestimated following the method of Lowry et al. (1951)using lysozyme as the standard.

The specific growth rate (K) and log10 specific growthrate (log10 K) of N. commune growing at differenttemperatures (5 to 350C) are shown in Table 1. Theoptimum temperature (Topt) for growth of N. communeranged in between 15 and 25oC with less difference in µmax

values. The specific growth rate of N. commune wasmaximal at 250C. Maximum growth rate was 1.9590 day-1

at 150C. The Q10 value ranged from 2-3. Results suggestpsychrotrophic behavior of N. commune.

Table 1. Effect of temperature on growth characteristics of N.commune.

Temperature (0C) µ Log K10

5 0.48 0.597215 1.0072 1.959025 1.48 1.290035 0.48 0.4800

Curve fitting results suggest that N. commune grewexponentially from 3 to 9 day at 250C, and 9 to 15 day at50C (Figure 1). After a downshift in temperature from 25 to50C, exponential phase of the cyanobacterium moved from3 to 9 day. Duration of lag phase also increased followingthe downshift. The stationary growth phase started from15th day for cultures growing at 50C, and continued withincrease in incubation period. This indicates theadaptability of N. commune to low temperature (50C), andits preference for 250C. Maximum increase in biomass wasrecorded at 24th day at 250C and on 12th day at 50C (Figure 2).

A BFigure 1. Best fit curve of Nostoc commune. A. at 250C, B. at 50C.

Figure 2. Biomass accumulation by N. commune at differenttemperatures (5-350C).

Maximum amount of chl a was recorded on 27th day forcultures growing at 250C. The downshift from 25 to 50Chad almost negligible effect on chl a content. The chl acontent increased from 0.49 to 0.703 µg mL-1 at 350Cduring first 6 days of incubation and remained constantthereafter (Figure 3). Maximal protein content wasrecorded for cultures growing at 50C on 12th day. Proteinsynthesis was exponential during 9 to 15 days (Figure 4).

Incubation period (days)

amou

nt o

f chl

a (mi

crog

ram

g-1 ml-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

50C150C250C350C

3 6 9 12 15 18 21 24 270

Figure 3. Effect of temperature (5-350C) on chl a content of N.commune.


Prot

ein co

nten

t (m

g g-1

)

0

2

4

6

8

10

12

14

50C150C250C350C50C

3 6 9 12 21 24 270 15

Figure 4. Effect of temperature (5-350C) on protein content of N.commune.


Biom

ass

(dry

weig

ht g

ml-1 )

0.0

0.2

0.4

0.6

0.8

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50C150C250C350C

3 6 9 120 15 21 2418

Dry

wei

ght (

g) a

t 25C

Dry

wei

ght (

g) a

t5C


PC/chl a ratio was highest on 21st day at 350C. At 50C,the ratio of PC/chl a increased upto 6th day, decreasedthereafter. A minimum increase in PC/chl a ratio (0.0148and 0.0126) was recorded on day 15th (also 18 day) at 50C.The maximum increase in PC/chl a ratio (0.1876) wasrecorded on 21 day at 250C (Figure 5A). PE/chl a ratio wasmaximum on 21st day for cultures growing at 350C.However, cultures growing at 5 and 150C exhibitedminimum PE/chl a ratio on 15th and 18th day, respectively.The ratio increased upto 12th day at 50C and declinedthereafter (Figure 5B). APC/chl a ratio was found to behighest on 24th day at 150C. Its value for cultures growingat 50C increased from 12th day (Figure 5C).

A

B

C

Figure 5. Effect of temperature on N. commune: A. PC/chl aratio, B. PE/chl a ratio, C. APC/chl a ratio.

The carotenoids/chl a ratio was maximum (2.48) on 9th

day at 50C. It remained almost constant for culturesgrowing at 5 and 350C (Figure 6). Protein/chl a ratioincreased up to 24th day at 50C. There was an induction inprotein synthesis following downshift in temperature from25 to 50C (Figure 7).


Carte

noid

s / ch

lara

tio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

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150C250C350C

0 3 6 9 12 15 18 21 24 27

Figure 6. Effect of temperatures on carotenoids/chl a ratio of N.commune.


prot

ein

/ chl

a ra

tio

0.01

0.1

150C150C250C350C

0 3 6 9 12 15 18 21 24

Figure 7. Effect of different temperature on protein/chl a ratio ofN. commune.

The growth and survival of cyanobacteria inhabitingAntarctic environments are less understood. N. communegrew within a range of 10-300C, suggesting eurythermal(broad tolerance range) nature of the cyanobacterium.Reduced growth at low temperature (e.g. ≤ 50C) andresponsiveness to the temperature changes suggest that inAntarctica cyanobacteria could accumulate biomass duringthe brief periods of summer.

The increase in carotenoids/chl a ratio at lowtemperature could help the cyanobacterium from protectionagainst photooxidation, resulting at low temperature(Young 1991; Chalifour and Juneau 2011). The consistentdecrease in phycobilliproteins/chl a ratio with increasingtemperature suggests that all pigment ratios (includingcarotenoids/chl a) are controlled primarily by cellular chl acontent (biosynthesis).

The algal communities in the polar environments areexposed to continuous high irradiance during the summer.This, in combination with the low temperature, it increasesthe chances of photoinhibition (Bascuñán-Godoy et al.2012). It was reported that carbon fixation limits growthand photosynthesis at low temperature, and algae tend todirect resources away from the synthesis of light-harvestingcomponents at low temperature (Alves et al. 2002)

TRIPATHI et al. – Physiology of an Antarctic cyanobacterium Nostoc commune 137

ACKNOWLEDGEMENTS

We thank the Head, Department of Botany, BanarasHindu University, Varanasi, UP, India for providingnecessary facilities. This manuscript is dedicated to lateProf. S. N. Tripathi.

REFERENCES

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Bascuñán-Godoy L, Sanhueza C, Cuba M, Zuñiga GE, Corcuera LJ,Bravo LA. 2012. Cold-acclimation limits low temperature inducedphotoinhibition by promoting a higher photochemical quantum yieldand a more effective PSII restoration in darkness in the Antarcticrather than the Andean ecotype of Colobanthus quitensis Kunt Bartl(Cariophyllaceae). BMC Plant Biol 12:114.

Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. 2002. Lowtemperature extremophiles and their applications. Curr Op Biotechnol13: 253-261.

Chalifour A, Juneau P. 2011. Temperature-dependent sensitivity ofgrowth and photosynthesis of Scenedesmus obliquus, Naviculapelliculosa and two strains of Microcystis aeruginosa to the herbicideatrazine. Aqua Toxicol 103: 9-17.

Chattopadhyay MK. 2006. Mechanism of bacterial adaptation to lowtemperature. J Biosci 31 (1): 157-165.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Proteinmeasurement with the folin-phenol reagent. J Biol Chem 193: 265-275.

Morita RY. 1975. Psychrophilic bacteria. Bacteriol Rev 39: 144-167.Myers J, Kratz WA. 1955. Relationship between pigment content and

photosynthetic characteristics in blue-green algae. J Gen Physiol 39:11-21.

Nadeau TL, Castenholz RW. 2000. Characterization of psychrophilicOscillatorians (Cyanobacteria) from Antarctic meltwater ponds. JPhycol 36: 914-923.

Nadeau TL, Mibrandt EC, Castenholz RW. 2001. Evolutionaryrelationships of cultivated Antarctic Oscillatiorians (cyanobacteria). JPhycol 37: 353-360.

Pandey V, Upreti DK. 2000. Seventeenth Indian Expedition to Antarctica,Scientific Report. DOD, New Delhi.

Seaberg KG, Parked BC, Wharton RA, Simmons GM. 1981.Temperature-growth responses of algal isolates from Antarctic oases.J Phycol 17: 353-360.

Talling, JF, Driver D. 1963. Some problems in the estimation ofchlorophyll-A in phytoplankton. In Proceedings of the Conference onPrimary Productivity Measurement, Marine and Freshwater, held atHonolulu in 1961. U.S. Atomic Energy Commission, Division ofTechnical Information TID-7633, Biology and Medicine, p. 142-146.

Tandeau de Marsac N, Houmard J. 1993. Adaptation of cyanobacteria toenvironmental stimuli: new steps towards molecular mechanisms.FEMS Microbiol Rev 104: 119-190.

Veerapaneni R. 2009. Analysis and characterization of microbes fromancient glacial ice. A Dissertation Submitted to the Graduate Collegeof Bowling Green State University in partial fulfillment of therequirements for the degree of Doctor of philosophy.

Young AJ. 1991. The photoprotective role of carotenoids in higher plants.Physiol. Plant 83: 702-708.

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A-1

Authors Index

Ade R 105Al-Hammady MAM 62Ammar MSA 62Arkhipova AV 97Aryana IGPM 113Balbaa LK 11Bekheta MA 118Bonde S 45Budianto A 113Dar M 86Deshmukh S 105,109Dhuldhaj UP 109Dhuldhaj UP 134El-Moursi A 11, 118Farid F 76Gade A 45, 86, 109Gaikwad S 45Gamal El-Din K 118Ghodile NG 81Hajizadeh G 27, 76Hedayati A 6Hushare VJ 81Irnidayanti Y 1Isyadinyati NF 57Kavosi MR 27, 76Khalid KA 36, 124Kitova AE 97

Kon K 50Kratasyuk VA 97Laily AN 16Malpani MO 81Obuid-Allah AH 62Rai M 45, 50, 86, 97, 109Rajput PR 81Rathod D 86Reshetilov AN 97Sable N 45Santoso BB 113Sedayu A 57Shadi A 6Shekhawat P 101Sigit DV 57Singh S 134Solanki P 101Sugiyarto 16Sulistyarsi A 32Supriyadi 32Suranto 16, 32Talaat IM 11, 118Tarkhani R 6Tripathi R 134Varma A 86Yaherwandi 22Yashpal M 109

A-2

Subject Index

3-alkoylchromanone 813-alkoylchromone 813-aroylflavones 81acetic acid 33, 41, 65, 83, 97, 98, 99,

100, 101, 105Acropora humilis 62, 63, 64, 65, 66, 67, 68, 69,

70, 71, 72, 73Ag-NPs 45, 46, 47, 48antioxidant capacity 16, 17, 18, 19, 21antioxidant polyphenols 11, 14, 15Arceuthobium oxycedri 76, 77, 78, 79,aromatic 11, 36, 40, 81, 92, 102, 103,

124, 125, 130, 131biogeography 57, 59Biological fertilizers 124, 131biosensor 97, 98, 99, 100Brassicaceae 22, 23, 24, 25Carica pubescens 16, 17, 18, 19, 20, 21,chelated zinc 118, 119, 120, 121, 122chlorosubstituted 3-aroylflavanones

81

combinations 50, 51, 52, 54, 55, 126, 130community structure 22corals 62, 63, 64, 65, 71, 72, 73curcuminoids. 11, 12, 13, 14, 15Cyanobacterium 36, 134, 135cyclodehydration 101, 103deltamethrin 6, 7, 8, 9diazinon 6, 7, 8, 9disinfestation 36, 37, 42dispersal 22, 57, 58, 60dwarf mistletoe 76, 77, 78, 79,eco-friendly 48, 62, 73, 86, 87, 90, 101,

102, 104, 109, 111, 125egg masses 27, 29, 30epiphyte 76Escherichia coli 50, 51, 52, 53, 54, 55, 118essential oils 50, 51, 52, 53, 54, 131, 132ethanol 12, 17, 32, 33, 65, 81, 82, 82,

83, 97, 98, 99, 100, 102, 103,118, 119

fertility 2, 62, 63, 64, 65, 69, 70, 79,105, 124, 125, 126

fish 6, 7, 8, 9, 62, 69, 72, 73,follicle cells 1, 4FTIR 45, 46, 47, 48fungal diversity 86

germination rate 113, 114, 115, 117Gluconobacter 97, 98, 100,growth rate 134, 135guppy 6, 7, 8, 9herbicide 76, 77, 78, 79Hydrilla 45, 46, 47, 48in vitro 50, 89, 91, 92, 101, 102, 103,

104, 105, 106, 107integrated management 27, 29, 30isoxazoles 81, 82, 83, 84Jatropha curcas 113, 114, 115, 116, 117Knoevenagelcondensation

101

landscape 22, 23, 24, 25, 129LC50 6, 7, 8, 9Leydig cells 1, 2, 3, 4low-temperature 16, 18, 134, 135, 136Lymantria dispar 27, 28, 29,mangrove 57, 58, 59, 60, 91mating disruption 27, 29, 30mechanical method 27, 29medicinal 11, 36, 40, 86, 87, 88, 89, 90,

91, 92, 124, 125, 130medicinal plants 86, 87, 88, 90, 91, 92, 105micropropagation 105, 107microwave 101, 102, 103, 104Moringa oleifera 118, 119, 121morphological characters 16, 17, 18, 19, 21mulches 36, 37, 38, 39multiplication 103, 105, 106, 107, 108, 113mycoendophyte 86, 87, 89, 90, 91, 92, 94nanoparticles 45, 46, 48, 109, 110, 111Nephotettix virescens 32, 33, 35non-crop vegetation 22, 23parasitoid Hymenoptera 22, 23, 24, 25pheromone traps 27, 28, 29, 30phycobiliproteins 134phytofabrication 45, 46phytosynthesis 45, 109pigments 38, 134, 135, 136plants 11, 12, 13, 14, 16, 17, 18, 20,

22, 23, 25, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 45, 46,48, 50, 51, 52, 55, 57, 60, 76,77, 78, 79, 81, 82, 84, 85, 86,87, 88, 89, 90, 91, 92, 94,105, 106, 107, 108, 109, 110,

A-3

111, 113, 114, 118, 119, 120,121, 122, 124, 125, 26, 27,28, 29, 30, 31, 132

plastic 23, 36, 39, 40, 65, 106, 114pollution 6, 22, 36, 39, 62, 63, 64, 69,

72, 73, 79, 125primordial follicle 1, 4propagules 57, 60, 131protein banding pattern 16, 17, 20, 34, 35, 121,protein banding. 16, 17, 20, 32, 34, 35, 121Red Sea 62, 63, 64, 65, 72, 73rice 23, 24, 25, 32, 33, 34, 35, 87room condition 107, 113, 114, 115, 116, 117secondary metabolites 1, 86, 87, 89, 90, 91, 119seed oil content 113, 114, 115, 116, 117seed quality 113, 116SEM 45, 46, 47, 48semi-parasitic plant 76, 77, 78, 79

soil borne diseases 36, 37, 125solarization 36, 37, 38, 39, 40, 41, 42Staphylococcus aureus 50, 51, 52, 53, 54, 55, 118Stevia rebaudiana 105, 106, 107stigmasterol 118, 119, 120, 121, 122Stylophora pistillata 62, 63, 64, 65, 66, 67, 68, 69,

70, 71, 72, 73sweet marjoram 11, 12, 13, 14, 131sweetener 105Tagetes erecta 109, 110, 111, 120, 121, 131TEM 109, 110Thymus vulgaris 50, 51, 53, 54, 55toxicity 6, 7, 9, 54, 87.91, 92, 94, 101,

128tungro 32, 33, 34, 35uterus 1, 2, 3, 4zearalenone 1, 2, 3, 4

A-4

List of Peer Reviewer

Abdel Fattah N. Abd Rabou Department of Biology, Faculty of Science, Islamic University of Gaza, Gaza Strip,Palestine

Agus Dana Permana School of Life Sciene and Technology, Institut Teknologi Bandung, Bandug 40132, WestJava, Indonesia

Ahmad Dwi Setyawan Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas MaretUniversity. Surakarta 57126, Central Java, Indonesia

Alka Karwa Jajoo Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati444602, Maharashtra, India

Amol D.Bhoyar Department of Chemistry, P.R. Patil College of Engineering and Technology(P.R.P.C.E.&T.), Kathora Road, Amravati 444607, Maharasthra, India.

Aniket K. Gade Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati444602, Maharashtra, India

Bogdan Costea Faculty of Horticulture and Forestry, University of Agricultural Science and VeterinaryMedicine of the Banat Timişoara, Timisoara, Romania

Eddy Nurtjahja Department of Biology, Faculty of Agriculture, Fisheries and Biology, State Universityof Bangka Belitung, Sungailiat 33211, Bangka Belitung, Indonesia

Francisca Fernández Piñas Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid,Cantoblanco 28049, Madrid, Espana

Kateryna Kon Department of Microbiology, Virology, and Immunology, Kharkiv National MedicalUniversity, 61022 Pr. Lenina, 4, Kharkiv, Ukraine

Mahendra Rai Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati444602, Maharashtra, India

Malcolm R. Clark National Institute of Water and Atmospheric Research (NIWA), Wellington 6021, NewZealand.

Novri Nelly Department of Plant Pest and Disease, Faculty of Agriculture, Andalas University,Padang 25161, West Sumatra, Indonesia

Nurbalis Department of Plant Pest and Disease, Faculty of Agriculture, Andalas University,Padang 25161, West Sumatra, Indonesia

Syamsul A. Siradz Department of Soil Science, Faculty of Agriculture, Gadjah Mada University, Sleman55281, Yogyakarta, Indonesia

Sugiyarto Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas MaretUniversity. Surakarta 57126, Central Java, Indonesia

Suranto Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas MaretUniversity. Surakarta 57126, Central Java, Indonesia

Wiryono Department of Forestry, Faculty of Agriculture, University of Bengkulu. Bengkulu38371A, Bengkulu, Indonesia.

A-5

Table of Contents

Vol. 4, No. 1, Pp. 1-44, March 2012

The effect of zearalenone mycotoxins administration at late gestation days on the development and reproductiveorgans of miceYULIA IRNIDAYANTI

1-5

Toxicity response of Poecilia reticulata Peters 1859 (Cyprinodontiformes: Poeciliidae) to some agriculturalpesticidesALIAKBAR HEDAYATI, REZA TARKHANI, AHMAD SHADI

6-10

Physiological effect of some antioxidant polyphenols on sweet marjoram (Majorana hortensis) plantsABDALLA EL-MOURSI, IMAN MAHMOUD TALAAT, LAILA KAMAL BALBAA

11-15

Characterization of Carica pubescens in Dieng Plateau, Central Java based on morphological characters, antioxidantcapacity, and protein banding patternAINUN NIKMATI LAILY, SURANTO, SUGIYARTO

16-21

Community structure of parasitoids Hymenoptera associated with Brassicaceae and non-crop vegetationYAHERWANDI

22-26

Evaluation of the effectiveness of integrated management and mating disruption in controlling gypsy moth Lymantriadispar (Lepidoptera: Lymantriidae) populationsGOODARZ HAJIZADEH, MOHAMMAD REZA KAVOSI

27-31

The total protein band profile of the green leafhoppers (Nephotettix virescens) and the leaves of rice (Oryza sativa)infected by tungro virusANI SULISTYARSI, SURANTO, SUPRIYADI

32-35

Review: Soil solarization and its effects on medicinal and aromatic plantsKHALID ALI KHALID

36-44

Vol. 4, No. 2, Pp. 45-96, July 2012

Phytofabrication of silver nanoparticles by using aquatic plant Hydrilla verticilataNEILESH SABLE, SWAPNIL GAIKWAD, SHITAL BONDE, ANIKET GADE, MAHENDRA RAI

45-49

Antibacterial activity of Thymus vulgaris essential oil alone and in combination with other essential oilsKATERYNA KON, MAHENDRA RAI

50-56

Adult mangrove stand does not reflect the dispersal potential of mangrove propagules: Case study of small islets inLampung, SumatraAGUNG SEDAYU, NOVITA FARAH ISYADINYATI, DIANA VIVANTI SIGIT

57-61

Patterns of fertility in the two Red Sea Corals Stylophora pistillata and Acropora humilisMOHAMMED S.A. AMMAR, AHMED H. OBUID-ALLAH, MONTASER A.M. AL-HAMMADY

62-75

Effects of foliar application herbicides to control semi-parasitic plant Arceuthobium oxycedriMOHAMMAD REZA KAVOSI, FERIDON FARIDI, GOODARZ HAJIZADEH

76-80

Synthesis, characterization and physiological activity of some novel isoxazolesVINAYSINGH J. HUSHARE, PRITHVIRAJSINGH R. RAJPUT, MANOJKUMAR O. MALPANI,NITIN G. GHODILE

81-85

Review: Mycoendophytes in medicinal plants: Diversity and bioactivitiesMAHENDRA RAI, ANIKET GADE, DNYANESHWAR RATHOD, MUDASIR DAR, AJIT VARMA

86-96

A-6

Vol. 4, No. 3, Pp. 97-137, November 2012

Determination of ethanol in acetic acid-containing samples by a biosensor based on immobilized GluconobactercellsANATOLY N. RESHETILOV, ANNA E. KITOVA, ALENA V. ARKHIPOVA, VALENTINA A.KRATASYUK, MAHENDRA K. RAI

97-100

Eco-friendly synthesis and potent antifungal activity of 2-substituted coumaran-3-onesPRABHA SOLANKI, PRACHI SHEKHAWAT

101-104

In vitro rapid multiplication of Stevia rebaudiana: an important natural sweetener herbSHIVAJI DESHMUKH, RAVINDRA ADE

105-108

Tagetes erecta mediated phytosynthesis of silver nanoparticles: an eco friendly approachUMESH P. DHULDHAJ, SHIVAJI D. DESHMUKH, ANIKET K. GADE, MADHU YASHPALMAHENDRA K. RAI

109-112

Seed viability of Jatropha curcas in different fruit maturity stages after storageBAMBANG BUDI SANTOSO, ARIS BUDIANTO, IGP MULIARTA ARYANA

113-117

Physiological response of Moringa oliefera to stigmasterol and chelated zincABDALLA EL-MOURSI, IMAN MAHMOUD TALAAT, MOHAMED ABDEL-GHANY BEKHETA,KARIMA GAMAL EL-DIN

118-123

Review: Biological fertilization and its effect on medicinal and aromatic plantsKHALID ALI KHALID

124-133

Short Communication: Effects of temperature on growth, pigment composition and protein content of an AntarcticCyanobacterium Nostoc communeRANJANA TRIPATHI, UMESH P. DHULDHAJ, SURENDRA SINGH

134-137

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Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation asshown with word “cit” should be avoided. Reference to unpublished dataand personal communication should not appear in the list but should becited in the text only (e.g., Rifai MA 2007, personal communication;Setyawan AD 2007, unpublished data). In the reference list, the referencesshould be listed in an alphabetical order. Names of journals should beabbreviated. Always use the standard abbreviation of a journal’s nameaccording to the ISSN List of Title Word Abbreviations (www.issn.org/2-22661-LTWA-online.php). The following examples are for guidance.

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change at hemic peat natural regeneration following burning; a casestudy in Pelalawan, Riau Province. Biodiversitas 7: 154-158.

The usage of “et al” in long author lists will also be accepted:Smith J, Jones M Jr, Houghton L et al. 1999. Future of health insurance. N

Engl J Med 965: 325–329

Article by DOI:Slifka MK, Whitton JL. 2000. Clinical implications of dysregulated

cytokine production. J Mol Med. Doi:10.1007/s001090000086

Book:Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.

Elsevier, Amsterdam.

Book Chapter:Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization,

biogeography, and the phylogenetic structure of rainforest treecommunities. In: Carson W, Schnitzer S (eds) Tropical forestcommunity ecology. Wiley-Blackwell, New York.

Abstract:Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th

annual symposium of the International Association for VegetationScience, Swansea, UK, 23-27 July 2007.

Proceeding:Alikodra HS. 2000. Biodiversity for development of local autonomous

government. In: Setyawan AD, Sutarno (eds) Toward mount Lawunational park; proceeding of national seminary and workshop onbiodiversity conservation to protect and save germplasm in Javaisland. Sebelas Maret University, Surakarta, 17-20 July 2000.[Indonesia]

Thesis, Dissertation:Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping

plants productivity in agroforestry system based on sengon.[Dissertation]. Brawijaya University, Malang. [Indonesia]

Online document:Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH,

Qu ak e SR , You L. 2 0 0 8 . A s yn th e t i c Esch er i ch ia co l ip red a to r -p rey ec os ys t em. Mol S ys t B i o l 4 : 1 8 7 .www.molecularsystemsbiology.com

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NOTIFICATION: All communications are strongly recommended to be undertaken through email.

Determination of ethanol in acetic acid-containing samples by a biosensor based onimmobilized Gluconobacter cellsANATOLY N. RESHETILOV, ANNA E. KITOVA, ALENA V. ARKHIPOVA, VALENTINA A.KRATASYUK, MAHENDRA K. RAI

97-100

Eco-friendly synthesis and potent antifungal activity of 2-substituted coumaran-3-onesPRABHA SOLANKI, PRACHI SHEKHAWAT

101-104

In vitro rapid multiplication of Stevia rebaudiana: an important natural sweetener herbSHIVAJI DESHMUKH, RAVINDRA ADE

105-108

Tagetes erecta mediated phytosynthesis of silver nanoparticles: an eco friendly approachUMESH P. DHULDHAJ, SHIVAJI D. DESHMUKH, ANIKET K. GADE, MADHU YASHPALMAHENDRA RAI

109-112

Seed viability of Jatropha curcas in different fruit maturity stages after storageBAMBANG BUDI SANTOSO, ARIS BUDIANTO, IGP MULIARTA ARYANA

113-117

Physiological response of Moringa oleifera to stigmasterol and chelated zincABDALLA EL-MOURSI, IMAN MAHMOUD TALAAT, MOHAMED ABDEL-GHANY BEKHETA,KARIMA GAMAL EL-DIN

118-123

Review: Biological fertilization and its effect on medicinal and aromatic plantsKHALID ALI KHALID

124-133

Short Communication: Effects of temperature on growth, pigment composition and proteincontent of an Antarctic Cyanobacterium Nostoc communeRANJANA TRIPATHI, UMESH P. DHULDHAJ, SURENDRA SINGH

134-137

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| Nus Biosci | vol. 4 | no. 3 | pp. 97-137 | November 2012|| ISSN 2087-3948 | E-ISSN 2087-3956 |

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