Nusantara Bioscience vol. 4, no. 3, November 2012

Tagetes erecta photo by Benary

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

| 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

EDITORIAL BOARD: Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia (sugiyarto_ys@yahoo.com) Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia (jrwitono@yahoo.com) Editorial Advisory Boards: Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia (janung4@yahoo.com.au) Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia (pattiselannofreddy@yahoo.com) Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India (pmkrai@hotmail.com) Biomedical Sciences, Afiono Agung Prasetyo, Sebelas Maret University, Surakarta, Indonesia (afieagp@yahoo.com) Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia (iyahya@uns.ac.id) Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia (cecep_kusmana@ipb.ac.id) Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia (lufehakim@yahoo.com) Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia (nnsutarno@yahoo.com) Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia (gadis@limnologi.lipi.go.id) Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt (shokry_1@yahoo.com) Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia (charis@ukdw.ac.id) Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia (ajamsari@yahoo.com) Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China (xiuyunzh@yahoo.com.cn) Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia (pudjiwi@yahoo.com) Management Boards: Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta (unsjournals@gmail.com) Associated Editor (English Editor), Wiryono, State University of Bengkulu (wiryonogood@yahoo.com) Associated Editor (English Editor), Suranto, Sebelas Maret University Surakarta Technical Editor, Ari Pitoyo, Sebelas Maret University Surakarta (aripitoyo@yahoo.co.id) Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok, Indonesia (nusbiosci@gmail.com)

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 University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: nusbiosci@gmail.com ONLINE: biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 97-100 November 2012

Determination of ethanol in acetic acid-containing samples by a biosensor based on immobilized Gluconobacter cells ANATOLY N. RESHETILOV1,♥, ANNA E. KITOVA1, ALENA V. ARKHIPOVA2, VALENTINA A. KRATASYUK2, MAHENDRA K. RAI3 1

Laboratory 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: anatol@ibpm.pushchino.ru 2 Department of Biophysics, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, 79 Svobodny Prospect, 660041, Russia 3 Department 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 containing samples by a biosensor based on immobilized Gluconobacter cells. Nusantara Bioscience 4: 97-100. A biosensor based on Gluconobacter oxydans VKM B-1280 bacteria was used for detection of ethanol in the presence of acetic acid. It was assumed that this assay 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 signal and the analytical parameters of the sensor (detection range, sensitivity) were investigated. The residual content of ethanol in acetic acid samples was analyzed. The results of the study are important for monitoring the acetic acid production process, as they represent a method 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 mengandung asam asetat dengan biosensor sel Gluconobacter yang diimobilisasi. Nusantara Bioscience 4: 97-100. Sebuah biosensor berdasarkan bakteri Gluconobacter oxydans digunakan untuk mendeteksi etanol pada sampel yang mengandung asam asetat. Pengukuran dilakukan dengan elektroda Clark berdasarkan biosensor amperometrik. Uji ini diharapkan berguna untuk mengendalikan produksi asam asetat dari etanol dan menentukan tahap akhir proses fermentasi. Pengaruh pH pada stabilitas pengukuran dipelajari. Berbagai jenis larutan bufer (sitrat, Tris maleat, natrium fosfat) diuji untuk memilih varian optimal, yang merupakan bufer fosfat dengan pH dalam kisaran 6 sampai 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 asetat dari berbagai produksi dinilai. Hasil dari penelitian ini penting untuk memantau proses produksi asam asetat, karena mereka mewakili metode pelacakan tahapannya.

Kata kunci: Gluconobacter, biosensor, etanol, asam asetat

INTRODUCTION Bacteria of the genus Gluconobacter are widely used in various biotechnological processes, in particular, in production of vinegar and acetic acid from alcoholcontaining products. At the initial stage of the fermentation process, the bioreactor contains a maximum amount of ethanol, which decreases as the content of acetic acid goes up. The decrease of ethanol concentration down to a certain level indicates the completion of the process. A real fermentation process (modeling, measurement and control) of acetic acid production by the repeated batch method using Acetobacter strains is given in Hekmat and Vortmeyer (1992). The fermentation was controlled by such parameters as acetic acid concentration, ethanol concentration and optical density of cell suspension.

According to the data of the work, the uptake of ethanol and accumulation of acetic acid had close-to-linear

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

There are various biosensor approaches to detection of ethanol and acetic acid, based on the use of enzymes or microbial cells (Tkac et al. 2002, Wang et al. 2006). They enable monitoring the formation of acetic acid by the content of ethanol in the fermentation medium. Still, approaches based on microbial biosensors making possible ethanol assays in the presence of acetic acid have not been described.

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

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MATERIALS AND METHODS A microbial biosensor based on cells of Gluconobacter oxydans VKM B-1280 (purchased from All-Russian Collection of Microorganisms) and an enzyme sensor based on alcohol oxidase (isolated from Hansenula polymorpha NCYC 495 ln, activity 14 units/mg) (Ashin et al. 2004) were used for the ethanol assay.

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

In formation of the microbial biosensor, cells were immobilized by their physical sorption on Whatman GF/A glass fibre filters. For this, 1 mg biomass was applied on a filter and dried for 20 min at room temperature (frozen biomass was used; during the preparation to immobilization, cells were preliminarily unfrozen).

Alcohol oxidase was immobilized in a layer of DEAE dextran on nitrocellulose membranes activated with benzoquinone (Zaitsev et al. 2007).

The bioreceptor (enzymes or cells immobilized on membranes) 33 mm2 in size was fixed on the measuring surface of a Clark oxygen electrode (Kronas Ltd, Russia).

The rate of stirring the solutions by a magnetic stirrer was 400 rpm. The measurements were carried out in an open cuvette (volume, 2 mL) by an IPC2L galvanostat/ potentiostat (Kronas Ltd, Russia) connected to a computer.

A 100-µL sample of a required concentration was introduced into the cuvette. The sample was diluted 1:20.

The measurements were done at room temperature. A 30 mM sodium phosphate buffer, pH 6.6, was used as a basic solution. The registered parameter was the maximum rate of signal change (nA/s).

The pH dependences were studied using the following buffer solutions: sodium phosphate buffer, MacIlvaine’s citrate 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 fermentation process were used for the analysis: 10% ethanol, the onset of the process; 5% ethanol in 5% acetic acid, the midpoint of 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 solution Various types of buffers (sodium phosphate, MacIlvaine’s citrate phosphate, Tris maleate) were used. It was assumed that buffer solution components could affect in different ways the stability of cells to pH changes. The concentrations of ethanol and acetic acid in the initial sample were, respectively, 0.1% (22 mM) and 9% (1.5 mM). This content of acetic acid corresponds to a minimum concentration of acetic acid in the fermentation medium, at which the process is terminated. For measurements, the sample was diluted 80-fold.

The effect of the type of buffer on the stability of cells is given in Figure 1. The range of investigated pH values for MacIlvaine’s citrate phosphate buffer solution was 3.27 (curve 1). The bioreceptor enabled measurements without a loss of activity within the pH range of 3.2 to 6.4. At pH 7, the measurement error exceeded 10%.

0.35

1

0.30

Sensor response, nA/s

comparison, on alcohol oxidase. We assumed that this assay could be useful for controlling acetic acid production from ethanol and determining the final stage of the process.

In spite of a significant dilution of the analyzed initial sample containing acetic acid, pH of the basic solution inevitably changes, which can affect the biosensor signal.

A comprehensive theoretical calculation of the model, including pH changes and biosensor responses in this process, appears to be complex, due to which an experimental verification was used. Special attention was paid to the case simulating the final stage of acetic acid production, when the content of ethanol in the sample is low (from 1 down to 0.1%), and the concentration of acetic acid is high (of the order of 10%). The purpose of the work was also to choose the type of buffer solution and its effect on the stability of the bioreceptor, as well as to study the parameters of the sensor when the optimal type of buffer was used. Commercial solutions of acetic acid were taken as an approximation to the applied aspect of the problem.

Publications on the subject have not investigated the issue in this statement of the problem.

2a

0.25

2b

0.20

3 0.15

0.10

0.05

0.00 3

4

5

6

7

рН

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

When using sodium phosphate buffer, the bioreceptor was stable within the pH range 5-7 in assaying ethanol samples (curve 2a). When assaying samples containing ethanol and acetic acid (curve 2b), the sensor responses were stable within the pH range of 6-6.6. When using a buffer solution containing Tris maleate, the range of

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

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

The type of buffer solution affected the magnitude of sensor response. Thus, the highest response was obtained using a citrate buffer solution. However, in studies of sensor stability in citrate buffer the responses decreased in the first 6 h, which was not characteristic of other buffer types. Sodium phosphate buffer was chosen as an optimal basic solution and was used in further experiments.

Study of the major parameters of the sensor The main analytical parameter of a biosensor is its calibration dependence. It was plotted as a function of ethanol concentrations in the measuring cuvette. Figure 2 (curve 1) presents the calibration dependence of a biosensor based on cells of the strain G. oxydans VKM B1280. The range of assayed concentrations was 0.0125-2.00 mM (0.0006-0.0092%) (the final concentration of ethanol in the measuring cuvette is given). Sensor responses were recorded in a 30 mM sodium phosphate buffer solution with pH 6.6. The maximum sensitivity in the linear range was 1.2 (nA/s)/mM; the linear range of detection, 0.01250.5 mM (0.0006-0.023%). Curve 2 is a calibration curve for ethanol samples containing acetic acid as a background concentration (0.11% (18.7 mM), concentration in the cuvette; in the initial sample, the concentration was 9%).

The linear range was 0.0125-1.25 mM (0.0006-0.06%) of ethanol; the sensitivity in the linear range, 1.2 (nA/s)/mM.

1 2 1.5

0.5

0.0

-26 -28 -30 -32 -34

Sample

рН of samples 2.4

responses

on

ethanol

pH of Dilution of Sensor samples in samples response, measuring with buffer nA/s cuvette 80-fold 5.5 0.200±0.005

2.4

800-fold

6.6

0.206±0.004

2.5

4000-fold

6.6

0.202±0.009

5.0

8000-fold

6.6

0.200±0.019

-38 -40 0

200 400 600 800 1000120014001600

Time, s

0.5

Table 1. Dependence of sensor concentrations in a model sample.

-36

-42

0.0

Assay of samples simulating the acetic acid production process with various contents of ethanol in acetic acid solution Information on the ethanol content is vital for the acetic acid production process to be optimal, as its decrease to a certain final level (0.1%) indicates the completion of the process. We analyzed samples simulating acetic acid production at various stages of the process.

The values of sensor responses to samples simulating various fermentation stages are given in Table 1. In the analysis 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 of the initial buffer solution equal to 6.6 decreased by unity.

For subsequent assays, the dilution was increased by an order of magnitude, which in practice had no effect on pH of the buffer solution.

Thus, to assess ethanol in assayed samples they should be diluted 80 times (final dilution in a cuvette) and more.

With this dilution, pH of the basic buffer solution changes insignificantly.

-24

Signal amplitude, nA

Sensor response, nA/s

2.0

1.0

variation was 6%. The measurements were done in a sodium phosphate buffer solution of a 30 mM concentration. The response time was 120 s. The sensor enabled 6 measurements per hour.

0.1% ethanol in 9% acetic acid 1% ethanol in 9% acetic acid 5% ethanol in 5% acetic acid 10% ethanol

2.5

99

1.0

1.5

2.0

2.5

Ethanol, mM

Figure 2. Calibration dependences of a biosensor based on the strain G. oxydans VKM B-1280 for detection of ethanol (1) and ethanol in the presence of acetic acid (2); the dashed line shows the 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 evaluated for the reproducibility of responses to samples containing ethanol and acetic acid (a sample contained 0.025% (22 mM) ethanol and 2.4% (375 mM) acetic acid, in the cuvette, the sample was diluted 20-fold). The coefficient of

Assay of ethanol in real samples We estimated the residual ethanol in samples of vinegar produced by a microbiological method (Table 2). The correlation coefficient of the data obtained using a microbial sensor and enzyme (alcohol oxidase-based) sensor was 0.98.

Table 2. Content of residual ethanol in acetic acid.

Sample Apple vinegar (Egorye) Apple vinegar (Abriko) Wine vinegar (Baltimor)

Ethanol (mM) G. oxydans Alcohol oxidase 12.0±0.7 11.9±1.1 10.9±1.0 11.7±0.8 16.6±1.2 14.9±1.1

High dilutions of the initial sample simulating the composition of the fermentation medium in acetic acid production were shown to practically buffer low levels of

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pH. At these dilutions with the basic buffer solution, ethanol concentrations corresponded to the linear range of the biosensor based on G. oxydans cells. Insignificant pH changes caused by the presence of acetic acid did not affect the measurement results when using this type of biosensor.

The buffer systems studied had different effects on the biosensor parameters, due to which an optimal (sodium phosphate) buffer solution was chosen.

CONCLUSION The optimal conditions for operating a Gluconobacter oxydans-based biosensor in an acetic acid-containing medium were determined. Under these conditions, the biosensor was shown to be capable of assaying the content of ethanol in acetic acid-containing samples within the linear segment (0.0125-0.5 mM) of the calibration dependence. The data obtained indicate a possibility of monitoring the acetic acid production process by both microbial and enzyme biosensors. The enzyme biosensor having a higher selectivity as compared with the microbial

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

REFERENCES Hekmat D, Vortmeyer D. 1992. Measurement, control, and modeling of submerged acetic acid fermentation. J Ferment Bioeng 73 (1): 26-30 Tkac J, Vostiar I, Gemeiner P, Sturdik E (2002) Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity. Bioelectrochem 56: 127-129 Wang YF, Cheng SS, Tsujimura S, Ikeda T, Kano K. 2006. Escherichia coli-catalyzed bioelectrochemical oxidation of acetate in the presence of mediators. Bioelectrochem 69 (1): 74-81 Ashin VV, Toropova IA, Kuvichkina TN, Reshetilov AN. 2004. A comparative characteristic of alcohol oxidase from methylotrophic yeasts Pichia methanolica and Hansenula polymorpha. Abstracts of papers of the 3rd Congress of Russian Biophysicists. Voronezh, Russia Zaitsev MG, Ashin VV, Reshetilov AN. 2007. A novel method of immobilization of methylotrophic cells and alcohol oxidase isolated from them for biosensor detection of lower alcohols. Book of abstracts of the 3rd International School-Conference of Young Scientists “Important aspects of modern microbiology�. Moscow

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 101-104 November 2012

Eco-friendly synthesis and potent antifungal activity of 2substituted coumaran-3-ones PRABHA SOLANKI1,♥, PRACHI SHEKHAWAT2 1

Department of Chemistry, Vidyabharti Mahavidyalaya, C.K. Naidu Road, Amravati 444602, Maharashtra, India. Tel. +91-721-2662740, Fax. +91-721 2662740, email: prabha.solanki@rediffmail.com 2 Department 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-methyl1,3-propanediones (Ia-c) with bromine or sulphuryl chloride in dioxane respectively. These chromones were employed in the synthesis of 2-acetyl-coumaran-3-ones (IVa-f). These were subjected to Knoevenagel condensation to give 2-cinnamoyl coumaran-3-ones. In vitro assay and field trials of these compounds against Fusarium oxysporum were carried out to study the antifungal effect of target compounds. Compound Va was the most effective growth inhibitor of the pathogen, whereas Vc showd 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-metil1,3-propanedion (Ia-c) dengan brom atau belerang klorida dalam dioksan secara berturut-turut. Kromon ini digunakan dalam sintesis 2asetil-coumaran-3-ona (IVa-f). Lalu, dilakukan kondensasi Knoevenagel untuk menghasilkan 2-cinnamoyl coumaran-3-on. Uji in vitro dan uji coba lapangan dari senyawa-senyawa ini terhadap Fusarium oxysporum dilakukan untuk mempelajari efek antifungi senyawa target. Senyawa Va adalah inhibitor pertumbuhan patogen yang paling efektif, sedangkan Vc menunjukkan kecenderungan sedikit dan Vb, Vd, Ve dan Vf tidak menghambat pertumbuhan.

Kata kunci: microwave, siklodehidrasi, kondensasi Knoevenagel

INTRODUCTION Synthons having chromone moiety are associated with various biological activities such as antibacterial (Tanaka et al. 2009), antifungal, antiallergic and diuretic (Abraham and Rotella, 2010). Substitution of halogen in these molecules enhanced the above activities, likewise 2coumaranones i.e. 3H-2-benzofuranones are also proved to be potential synthons for various products extended for agriculture or having physiological effects. Therefore, processes are continuously being sought which allow it to be obtained rapidly and cheaply from inexpensive commercially available products. Researchers have also synthesized coumaranone from cyclohexanone and glyoxalic acid in presence of dehydration catalyst (Vallejos et al. 1997). Process has been also described for preparation of enol lactone 2-oxocyclihexidine acetic acid and to its application to preparation of 2-coumaranone by (Carmona et al. 1998). Benzofuranone derivatives found to possess potential antipsychotic (Aranda et al. 2008), anticancer (Charrier et al. 2009; Mishra et al. 2011), peroxidase activity (Ghadami et al. 2012), cytotoxicity activity (Terasawa et. al,2001),antibacterial activity (Hadj-

Esfandiari et al. 2007) and other biological activities (Li and Chen et al. 2008; Adadiran et al. 2001). With reference to observation and versatility of chromones and coumaranones, attempts have been made to synthesize the compound under microwave irradiation (Goncalo et al.

1999) with a rapid environment benign, cleaner and cheaper work up.

MATERIAL AND METHODS Eco-friendly synthesis All chemicals and solvents were purchased from Sigma Aldrich.Melting points were determined by open capillary methods on a ‘Veego’ VMP-D apparatus and are uncorrected. TLC was done using silica gel G plates using 3x8 cm (Sigma-Aldrich) and visualized in an iodine chamber. The IR spectra (KBr) were determined on "Perkin Elmer 577 spectrometer and the values are expressed in cm-1 and H1NMR (chemical shift in δ ppm) were recorded on Perkin Elmer R-32 and Varian XL-100A high NMR spectrophotometer using TMS as reference either in CDCl3 or DMSO-d6 as solvent. C, H and N analyses were carried

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4 (3): 101-104, November 2012

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

3-bromo-2-methylchromones (IIa-c): 1- (2-hydroxyphenyl)-3-methyl-1-propane diones (Ia-c) (10 mmol) was dissolved in dioxane (0.5 mL) and solution of pure bromine (0.5 mL) in dioxane (15 mL) was added with constant stirring. The reaction mixture was warmed and kept for 30 minutes. After cooling, the reaction mixture was diluted with water and then the crude product was filtered and crystallized using ethanol to get 75-80% yield. Ia-IR in cm1 -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 of 1- (2-Hydroxyphenyl)-3-methyl-1,3-propanediones (Ia-c) (10 mmol) and sulfuryl chloride (10 mmol) in dioxane (25 mL) was irradiated under microwave at 450 W for 45 sec with intermittent heating. It was then diluted and crude product thus obtained was crystallized from ethanol to get 70-75% 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 3halochromones (IIa-c) or (IIIa-c) (1 g) in ethanol (20 mL) was treated with aq. KOH solution separately and the reaction mixture was exposed to MW for 1 minute. Cool and diluted product was acidified with HCl. The crude product thus obtained was crystallized from ethanol to get compound (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,COCH), 7.21-8.00 (m, 3H, Ar-H). UV λmax 345 mm.

2-Cinnamoyl coumaran-3-ones (Va-f): A mixture of 2-acetylcoumaran-3-one (IVa-c) (10 mmol) and aromatic aldehyde (20 mmol) in ethanol (20 mL) and few drops of piperidine (0.5 mL) was exposed to MW for 1 minute with intermittent heating. After cooling the reaction mixture was diluted, filtered and crystallized from ethanol to get 6570% yield. Va-IR in cm-1-2950 (-OH),1600 (C=O). PMR-δ 2.41 (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. 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Entry IIa IIb IIc IIIa IIIb IIIc IVa IVb IVc Va Vb Vc Vd Ve Vf

M.F.

R

R1

R2

C11H9O2Br C10H7O2Br C11H9O2Br C11H9O2Cl C10H7O2Cl C11H10O2Cl C11H10O3 C10H8O3 C11H10O3 C18H13O3 C19H15O4 C17H11O4 C18H13O4 C18H13O3 C19H15O4

CH3 H H CH3 H H CH3 H H CH3 H H H H H

H H CH3 H H CH3 H H CH3 H H H H CH3 CH3

H OCH3 H OCH3 H OCH3

M.W. Sec 45 40 45 60 55 60 60 60 60 65 60 65

M.P. (°C) 132 132 93 124 128 121 126 138 142 119 211 154 134 140 149

Figure 1. Scheme of the eco-friendly synthesis

In vitro assay of target compounds against Fusarium oxysporum Application and utility of heterocycles in agriculture crop to eradicate the alarming diseases has drawn the attention of research scientist. The following samples were tested at the concentration noted against Fusarium oxysporum by using poisoned food technique (Schmitz, 1930). One week old culture of F. oxysporum was grown on potato dextrose agar medium (PDA) in petri plates for assessing efficiency of newly synthesized samples.

Solutions of 100 ppm concentration were taken in 250 mL conical flask containing 100 mL of sterilized and melted potato dextrose agar medium, mixed thoroughly by gentle swirling the flask and poured into a sterile petri disc and allowed to solidify. A 8 mm culture disc pathogen is F. oxysporum was inoculated and the plates were incubated in an inverted position at room temperature. Inoculated PDA medium without sample served as control. Three replications were maintained. The mean radial growth of the colony was measured at 48, 72 and 96 hrs after inoculation respectively. The results were expressed as percent inhibition over control (Table 2). The percent inhibition of the growth was calculated by the formula of Vincent (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 Table 2. Radial growth of F. oxysporum on PDA medium at 100 ppm concentration of samples at 48, 72 and 96 hrs of incubation; and percent growth inhibition of F. oxysporum at 100 ppm after 96 hours.

Mean radii growth (mm) Sample Va Vb Vc Vd Ve Vf Control

48 hrs

72 hrs

96 hrs

0.00 24.33 15.83 25.33 24.33 24.66 25.66

0.00 44.66 26.50 45.16 46.16 45.83 46.33

0.00 68.83 41.83 69.50 69.16 69.33 69.33

% Growth inhibition after 96 hrs 100.00 0.72 39.66 -0.24 0.28 0.00 -

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Table 3. Fungicide effect of newly synthesized compound Va on Cicer arietinum Observations Periofor control dicity A experiment days a b c d a b c 8 20 8 4 80 70 7 5 15 - 30 10 - 80 75 20 30 - 25 10 10 90 25 45 - 25 10 5 102 27 75 - 26 10 125 30 120 - - - 130 35

Replications B d 30 -

a 80 85 -

b 8 74 95 105 115 120

C c 5 20 25 29 22 32

d 20 -

a 75 88 -

b c 8 5 72 25 75 29 98 32 112 35 118 35

d 25 -

Table 4. Fungicide effect of newly synthesized compound Vc on Cicer arietinum.

Among the six samples tested, sample Va was the most effective recorded percent growth inhibition over control after 96 hrs of incubation. Compound Vc also showed 39.66% of inhibition where as samples Vb, Vd, Ve and Vf did not inhibit the growth of F. oxysporum.

Control of fungal diseases in agricultural crops The most general means of controlling plants diseases is in field, green house and sometimes in storage, through the use of chemical compounds that are toxic to the pathogens, such chemical either inhibits germination, growth and multiplication of the pathogen or outright lethal to the pathogen. In this part the synthesized compounds Va and Vc had been tested on Bengal grams (Chickpea or Gram; Cicer aritinum L.) whose growth is generally retarded due to vascular cause by F. Oxysporum (Table 3-4).

Gram is mostly affected by wilt F. oxysporum. It is most important rabi crop grown on large scale in India. So in the present work, in vitro assay of compound I and IV that have shown to possess a remarkable fungicidal activity, had been selected to evaluate the antifungal activity against F. oxysporum, a pathogen of Bengal gram crop. Post culture method is adopted in this experiment.

Studies have been have carried out in triplicate to select the requisite concentration of newly synthesized fungicide for plant growth.

Design of experiment Seven pots of the size 30x20 cm were taken for the three replication to increase the precision of the experiment. Approximately 1.5 kg soil for each pot was autoclaved for complete sterilization. A control pot C was filled up with sick soil 100 g of fungal culture had been mixed with soil in 100: 1 proportion. Three pots for each sample were filled with sick soil and labeled as P1-P3 and Q1-Q3. Pregerminated seeds of Bengal gram were procured from Krishi Vigyan Kendra Durgapur, Amravati for these trials. Aqueous solution of 100 pp, of the sample P and Q were prepared and ten seeds for each replication, soaked in the sample solution and allowed to dry. Treated seeds sowed in pot P1-P3 and Q1-Q3. Seeds soaked in distil water were 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 noted periodically.

Observations Replications Periofor control dicity A B C experiment days a b c d a b c d a b c d a b c d 8 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

RESULTS AND DISCUSSION 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 of IIa shows characteristic peak at 1340 cm-1 (-pyrone) and disappearance of phenolic-OH singlet of 1,3-propanediones. 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 in dioxane under microwave at 450 W for 45 sec gave 3chloro-2-methylchromones (IIIa-c) in 80% yield. IR and PMR showd presence of -pyrone nucleus and absence of signals of phenolic-OH and keto-enol tautomerism.

2-Acetylcoumaranones (IVa-c) have been synthesized from a solution of 3-halochromones (IIa-c) or (IIIa-c) in ethanol in aqueous alkaline medium under microwave exposure for one minute. PMR signal at 6.25 δ is identified as (-COCH) coumaran proton and a acetyl signal at 2.46 δ.

In IR spectra frequency for -pyrone (1340 cm-1) get disappeared, a signal at 3340 cm-1 (-OH) also confirm the structure (IVa-c). These compounds (IVa-c) are subjected to condensation with aromatic aldehyde (0.02 mol) in ethanolic condition in presence of piperidine under microwave radiation. The absence of acetyl signal and appearance of-COCH=CH-dd at 7.19-7.25 range confirms the presence of cinnamoyl group and a singlet at 9.18 δ forOH group. IR frequency at 2950 cm-1 (-OH) and 1600

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

A remarkable antifungal activity was shown by compound Va and Vc. Fungicidal activity of these compounds was found to be excellent in different trials in vitro assay against. F. oxysporum. Compound Va had shown to have better fungicidal effect as compared to compound Vc. In control experiments rate of mortality is found to be very high due to sick soil, which diminished in remarkable extent in three trials where the seeds were treated with synthesized compounds. Thus synthesized heterocycles are found to possess potential antifungal activity

CONCLUSION Microwave assisted organic synthesis has attracted attention in recent years due to enhanced reaction rates, higher yields, improved purity, ease of work up after reaction and eco-friendly reaction conditions compared to the conventional methods. Some of the synthesized compounds possess potent antifungal activities against Fusarium oxysporum. Compound Va was the most effective growth inhibitor of the pathogen, whereas Vc showd a little tendency and Vb, Vd, Ve and Vf hardly inhibits the growth. Candidly, these are few prototype trials taken to root out the overwhelming situation prevailing in biosphere. This work will definitely provide an access to organic chemists synthesizing ever lasting number of compounds of high potent which may serve mankind.

ACKNOWLEDGEMENTS The author expresses their sincere thanks to the Principal of Vidya Bharti Mahavidyalaya, Amravati, Maharashtra, India and the Director of Krishi Vigyan Kendra, Durgapur Badnera, Maharashtra, India for providing necessary laboratory facilities.

REFERENCES Abraham DJ, Rotella DP (eds). 2010. Burger's medicinal chemistry, drug discovery and development, vol. 7, 8th ed. Wiley, New York.

Adadiran SA, Cabaret D, Drouillat B, Pratt RF, Waksalman M. 2001. The synthesis and evaluation of benzofuranones as β-Lactamase substrates, Bio-organic Med Chem 9 (5): 1175-1183.

Aranda R, Villalba K, Raviña E. 2008. Synthesis, binding affinity, and molecular docking analysis of new benzofuranone derivatives as potential antipsychotics. J Med Chem 51 (19): 6085-6094, Carmona N, Carmona L, Perrard A, Vallejos JC. 1998. Process for the preparation of enol lactone of 2-oxocyclohexlidene acetic acid and application to the preparation of 2 coumaranone. U.S. Patent 5773632.

Charrier C, Clarhaut J, Gesson JP, Estiu G, Wiest O, Roche J, Bertrand P. 2009. Synthesis and modeling of new benzofuranone histone deacetylase inhibitors that stimulate tumor suppressor gene expression. J Med Chem 52 (9): 3112-3115.

Ghadami SA, Hosseinpour Z, Khodarahmi R, Ghobadi S, Adibi H. 2012.

Synthesis and in vitro characterization of some benzothiazole-and benzofuranone-derivatives for quantification of fibrillar aggregates and inhibition of amyloid-mediated peroxidase activity. Med Chem Res DOI 10.1007/s00044-012-0012-3.

Goncalo P, Roussel C, Mélot JM, Vébrel J. 1999. Contribution of microwaves in organic synthesis: statement of a methodology for the microwave-induced preparation of benzofuran-2 (3H)-one and its comparison with classical heating. J Chem Soc Perkin Trans 2: 21112115.

Hadj-Esfandiari N, Navidpour L, Shadnia H, Amini M, Samadi N, Faramarzi MA, Shafiee A. 2007. Synthesis, antibacterial activity, and quantitative structure-activity relationships of new (Z)-2(nitroimidazolylmethylene)-3 (2H)-benzofuranone derivatives. Bioorg Med Chem Lett 17 (22): 6354-6363.

Li BZ, Chen WM. 2008. New advances in the synthesis of 2 (5H)furanones. Chinese J Org Chem 28 (1): 29-36.

Mishra RC, Karna P, Gundala SR, Pannu V, Stanton RA, Kumar Gupta K, Robinson MH, Lopus M, Wilson L, Henary M, Aneja R. 2011.

Second generation benzofuranone ring substituted noscapine analogs: Synthesis and biological evaluation. Biochem Pharmacol 82 (2): 110-121.

Tanaka N, Kashiwada Y, Nakano T, Shibata H, Higuchi T, Sekiya M, Ikeshiro Y, Takaishi Y. 2009. Chromone and chromanone glucosides from Hypericum sikokumontanum and their anti-Helicobacter pylori activities. Phytochem 70 (1): 141-146.

Terasawa K, Sugita Y, Yokoe I, Fujisawa S, Sakagami H. 2001. Cytotoxic activity of 2-aminomethylene-3 (2H)-benzofuranones against human oral tumor cell lines. Anticancer Res 21 (5): 3371-5.

Vallejos JC, Perrard A, Chrisditis Y, Gallezot P. 1997. Preparation method of 2 Coumaranone, U.S. Patent 5616 733 AP. Vincent JM. 1927. Distortion of fungal hyphae in the presence of certain inhibitors. Nature 159: 850.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 105-108 November 2012

In vitro rapid multiplication of Stevia rebaudiana: an important natural sweetener herb SHIVAJI DESHMUKH♥, RAVINDRA ADE Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India. Tel: +91-721-2662206 to 8, Fax: +91-7212662135, 2660949, email: shivgajanan@rediffmail.com 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. Nusantara Bioscience 4: 105-108. Stevia rebaudiana Bertoni, belonging to family Asteraceae and natural sweet plant, but due to poor seed viability, fertility and vigor, Stevia cultivation is a challenging task. In the present study in vitro rapid multiplication method was established for S. rebaudiana by inoculating explants on M.S. medium, supplemented with different combination of phytoharmone. The maximum number of shoots (18.3±0.8) was obtained on M.S. medium supplemented with BAP + KIN (1.5 + 0.5 mg/L). The highest rooting percentage (95.25) was observed with (IAA 0.1 mg/L). The rooted plants were successfully established firstly in soil with coco peat (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 Naphthalene acetic 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, namun karena viabilitas, kesuburan dan kekuatan benih yang buruk budidaya Stevia menjadi tugas yang menantang. Dalam penelitian ini metode perbanyakan cepat secara in vitro dilakukan pada S. rebaudiana dari eksplan inokulasi pada media MS, dilengkapi dengan kombinasi fitoharmon yang berbeda. Jumlah maksimum tunas (18,3±0,8) diperoleh pada media MS dengan BAP + KIN (1,5 + 0,5 mg/L). Persentase perakaran tertinggi (95,25) diamati dengan (IAA 0,1 mg/L). Tanaman berakar berhasil ditanam pertama kali pada tanah 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 family Asteraceae, is a perennial herb which can be growing up to 1 meter (Kinghorn et al. 1985; Handro et al. 1989; Tadhani et al. 2005). It is natural sweetener plant called as “sweet weed”, “sweet leaf” and “honey leaf” (Ahmed et al. 2007).

The leaves of Stevia are source of glycoside, viz stevioside and rebaudioside which are 100-300 time sweeter than sucrose (0.4% solution) but zero calories (Mousumi 2008), hence it is important medicinal plant and it has been traditionally used for hundreds of year in Paraguay and Brazil in South America continent to sweeten tea and medicine and also used as a sweet treat (Tiwari 2010). It is recommended for diabetes and has been extensively tested on animal and human with no side effects (Megeji et al.

2005). The crude extracts from leaves have been used few decades to sweeten soft drinks and other foods (Komissarenko et al. 1994).

The stevioside, can be used in tea and coffee, cooked or baked 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 generated much attention with the rise in demand for low carbohydrate, low sugar food alternative. Stevia also has shown promise in medical research for treating obesity and high blood pressure, due to these important medicinal properties the Stevia is being cultivated in Japan, Taiwan, Philippines, Hawaii, Malaysia and overall South America and used in several food and pharmaceutical products (Das et al. 2011).

The main problem in cultivation of Stevia is that the plant is heterozygous. Self incompatible nature of flowers leads to lack of fertilization, poor seed viability and vigor, due to this plant propagation by seed is not efficient.

(Tadhani et al. 2006; Rathi and Arya 2009). Propagation by seeds does not allow the production of homogeneous population, resulting in great variability in important features like sweetening level and composition (Tamura and Nakamura 1984). Due to such difficulties in cultivation of Stevia, tissue culture is the only rapid process for the mass propagation. The present study was carried out to optimize a suitable and efficient protocol for In vitro rapid

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multiplication of S. rebaudiana Bertoni.

MATERIALS AND METHODS Collection and surface sterilization of explants Stevia 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 running tap water, then treated with 0.1% (w/v) Bavistin solution for 5 min to remove superficial dust particle as well as fungal spores. After that the explants were treated with 70% alcohol for 2 min followed by 4-5 time wash by double distilled water to remove bacterial contaminants.

Sodium hypochlordte (0.3%) also used for the decontamination. It was again treated with 0.1% HgCl2 aseptically for 3-4 min and washed with sterile distilled water 4-5 times.

A

D

Inoculation of explants on the multiplication medium The surface sterilized auxiliary bud were aseptically inoculated vertically on MS medium (pH 5.7) supplemented with specific concentration of growth regulators (BAP, KIN and NAA) singly as well as in combination, 0.7 % agar was used as a gelling agent and 30 gm/lit sucrose was used as a carbon source.

Culture conditions All the standard conditions were provided such as photoperiod was 16 hrs light (2000 lux) to 8hrs darkness.

The cultures were maintained at 26¹1°C and 70% humidity. Subculturing was done for every fortnight and well-grown sub-cultured shoots were further inoculated on multiple shoot formation medium. Regenerated multiple shoots were cut and individual shoots were placed in MS medium containing different concentrations of IBA, NAA and IAA for root induction.

B

E

C

F

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 supplemented with IAA (0.1 mg/L). E. Growth of Stevia in plastic pot containing coco peat and soil 1:1. F. In ordinary soil.

DESHMUKH & ADE – In vitro rapid multiplication of Stevia rebaudiana

Hardening of plants In vitro rooted shoots were kept under normal growth room condition for 7-8 days until the induced roots become partially brown. The shoots were taken out from culture bottle carefully and medium attached to the roots were gently washed out with running tap water. The rooted plants treated with 0.1% bavistin (antifungal) for 1-2 min then transferred on soil: coco peat (1:1) for hardening then directly in ordinary soil (Figure 1).

RESULTS AND DISCUSSION Shoot-apex were inoculated on MS medium supplemented 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 BAP with KIN or with NAA (0.5, 1.0mg/L) as shown in Table 1.

This shows different response during the primary establishment period. After 15 days, multiple shoots emerged directly from auxiliary node of cultured explants.

The response of explants to the treatment is presented in Table1. The maximum number of shoots was observed on MS medium containing BAP (1.5 mg/L) among single hormonal set. Similar findings were recorded by (Sivaram and Mukundan 2003). BAP + KIN (1.0 + 0.5 mg/L) and BAP + KIN (1.0 + 1.0 mg/L) also shows significant multiplication. The response was best at BAP + KIN (1.5 + 0.5 mg/L) combination, where highest percentage of explants showing shoot proliferation was found to be 89.25%,whereas highest average number of total shoot was found to be 18.3 + 0.8, with average length 4.8 + 0.8 cm were recorded (Table 1). Similar results have already been reported in Fragaria indica (Bhatt et al. 2000). Patil et al.

(1996) reported that shoot tips and auxiliary buds produce multiple shoots. When the explants were inoculated on MS medium with IAA + BAP (0.5 + 5.0 mg/L) supplemented with 10 mg/L GA. The numbers of multiple shoots were around 20-25 per plant.

Multiple shoot formation requires the presence of cytokinins in the culture medium (Tadhani et al. 2006). In present study the BAP containing medium is better for the shoot formation at lower concentration as compared to KIN. However Tamura et al. (1984) reported that high concentration of KIN (10 mg/L) was required for multiple shoot production in S. rebaudiana . Progressively higher concentration of BAP resulted in decreasing multiple shoot formation in all the explants of Stevia. (Table 1) Although the process of in vitro rooting is a labor intensive in the micropropagation studies of Stevia, but it seems to be an essential step for plant survival. The addition of auxin at certain level enhances the root formation. Micro cuttings taken from in vitro proliferated shoots were implanted on MS medium containing different concentrations 0.1mg/L, 0.3 mg/L, 0.5 mg/L, 0.8 mg/L, 1.0 mg/L, of IAA, NAA and IBA for rooting. Here each treatment consists of four replications and in each replication 10 explants were used.

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

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for rooting with highest length of root (4-5cm), number of roots 12-13 and maximum root induction (95.25%) as shown in Table 2. It was observed that the root induction gradually decreased with increased concentration of auxin.

There was not any satisfactory root induction in another case except IAA. Similar findings were recorded in Chrysanthemum morifolium (Hoque et al. 1995), Pigeon pea (Sivaprakash et al. 1994), Vitex negundo (Thiruvengadam et al. 2000) and Psoralea corylifolia (Jeyakumar et al.

2002). However Tadhani et al. (2005) reported 0.1mg/L IBA was the best concentration for rooting.

In vitro rooted shoots were kept under normal growth room condition for 2 weeks until the induced roots become partially brown. The shoots were taken out from growth room and from the culture bottle carefully and gently washed with running tap water. The rooted plant was treated with 0.1% Bavistin for 1 minute and transferred to soil and coco peat (1:1) for primary hardening followed by ordinary soil in natural environment.

Table 1. Effect of auxin and cytokinin concentration on in vitro multiplication.

BAP 0.5 BAP 1.0 BAP 1.5 BAP 2.0 KIN 0.5 KIN 1.0 KIN 1.5 KIN 2.0

Explants proliferation (%) 49.50 55.25 68.00 57.50 42.00 60.25 58.50 52.25

5.1+ 1.7 4.2 + 1.3 4.1 + 1.4 4.5 + 1.5 3.6 + 0.9 3.2 + 0.9 3.2 + 0.9 3.6 + 0.9

Average length of shoots (cm) 2.6 + 0.5 2.3 + 0.4 3.3 + 0.6 2.4 + 0.5 2.3 + 0.4 3.4 + 0.5 2.4 + 1.0 1.6 + 0.6

BAP+KIN (1.0+0.5) BAP+KIN (1.0+1.0)

67.25 76.50

13.4 + 1.2 14.4 + 1.7

3.5 + 0.5 3.4 + 0.5

BAP+KIN (1.5+0.5) BAP+KIN (1.5+1.0) BAP+KIN (2.0+1.0) BAP+KIN (2.0+0.5) BAP+NAA (1.0+0.5) BAP+NAA (1.0+1.0) BAP+NAA (1.5+0.5)

89.25 57.50 56.25 59.50 35.25 34.50 37.40

18.3 + 0.8 12.8 + 0.9 11.6 + 0.8 11.3 + 0.6 7.4 + 0.5 4.3 + 0.4 12.8 + 0.9

4.8 + 0.4 4.4 + 0.5 3.4 + 0.5 1.6 + 0.6 1.6 + 0.5 3.3 + 0.4 2.6 + 0.5

BAP+NAA (1.5+1.0) BAP+NAA (2.0+0.5) BAP+NAA (2.0+1.0) NAA +KIN (1.0+0.5) NAA +KIN (1.0+1.0) NAA +KIN (1.5+0.5) NAA +KIN (1.5+1.0) NAA+KIN (2.0.+0.5) NAA +KIN (2.0+1.0)

44.50 42.00 52.50 26.00 45.25 56.50 35.25 42.00 48.25

11.3 + 0.6 11.5 + 0.5 13.9 + 0.8 5.7 + 0.8 5.0 + 0.8 5.7 + 0.8 2.5 + 0.5 3.7 + 0.8 12.8 + 0.9

2.7 + 0.4 3.5 + 0.5 1.6 + 0.5 1.7 + 0.4 1.5 + 0.5 1.4 + 0.5 3.6 + 0.5 2.8 + 0.6 1.4 + 0.5

Growth regulator (mg/L)

Average no. of shoots

Note: * Each treatment consists of three replication and in each replication 10 explants were used.

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Table 2. Effect of different type of auxin on adventitious root formation.

Root Root Conc. initiation initiation (mg/L) (days) (%) IAA 0.1 6-8 95.25 0.3 10-11 92.0 0.5 7-8 85.0 0.8 10-12 65.50 1.0 9-10 67.75 NAA 0.1 7-8 70.35 0.3 7-8 65.25 0.5 7-8 58.50 0.8 7-8 62.00 1.0 7-8 51.50 IBA 0.1 8-10 65.75 0.3 10-12 60.25 0.5 10-12 45.50 0.8 8-9 48.00 1.0 8-9 42.75 Note: *Each treatment consisted of 3 replication 10 explants were used.

Auxin

Average no. of roots 12.8 + 0.6 10.7 + 0.8 9.5 + 0.5 7.4 + 0.5 5.1 + 0.9 5.2 + 0.4 4.6 + 0.5 4.7 + 0.4 4.5 + 0.5 3.5 + 0.5 7.4 + 0.5 6.5 + 0.5 4.5 + 0.5 4.3 + 0.4 4.6 + 0.5 replications

Average length of roots (cm) 4.7 + 0.4 2.4 + 1.0 1.4 + 0.5 3.5 + 0.5 2.4 + 0.5 2.3 + 0.4 2.7 + 0.4 1.4 + 0.5 2.4 + 1.0 2.7+ 0.6 1.9 + 0.8 2.4 + 1.0 1.5 + 0.5 2.4 + 0.5 3.6 + 0.5 and in each

CONCLUSION Stevia is an important sweetner herb. In normal propagation it is very difficult to regenerate due to various reasons like heterozygous nature, self incompatibility of flowers, lack of efficient fertilization and most importantly poor seed viability, hence there is urgent need to develop efficient protocol for rapid multiplication and thus conservation, because till date no appropriate and efficient method is available for its regeneration. Since tissue culture technology is the only process for the mass propagation, this rapid and efficient regeneration protocol provides a good platform for the multiplication and effective conservation of this important plant.

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Tamura Y, Nakamura S, Fukui H, Tabata M. 1984. Comparision of Stevia plants grown from seeds, cuttings and stem tip cultures for growth and sweet diterpene glycoside. Plant Cell Rep 3: 180-182.

Thiruvengadam M, Jayabalan N. 2000. Mass Propagation of Vitex negundo L. In vitro J Plant Biotech 2 (3): 151-155.

Tiwari S. 2010. In vitro propagation of Stevia rebaudiana Bertoni: Review. Int J Pharm Life Sci 1 (5): 274-277.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, Pp. 109-112 November 2012

Tagetes erecta mediated phytosynthesis of silver nanoparticles: an ecofriendly approach UMESH P. DHULDHAJ1,2, SHIVAJI D. DESHMUKH1, ANIKET K. GADE1, MADHU YASHPAL2, MAHENDRA K. RAI1,♼ 1

Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India. Tel: +91-721-2662206 to 8, Fax: +91-7212662135, 2660949, ď‚Šemail: mkrai123@rediffmail.com 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 the various fields like medicine, pharmacy, engineering and biotechnology. An important step in nanotechnology is to develop simple and eco-friendly method for the nanomaterial synthesis. Here we describe simple and eco-friendly method for synthesis of silver nanoparticles by extract of Tagetes erecta plant leaves. The phytosynthesis (synthesis by plant) of silver nanoparticles was detected by color change from light-green to dark-brown. Synthesis of silver nanoparticles was confirmed by UV-Vis spectrophotometry, further characterization includes nanoparticle tracking analysis system (NTA) (LM20) and transmission electron microscopy (TEM). TEM analysis 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 oleh Tagetes erecta: pendekatan ramah lingkungan. Nusantara Bioscience 4: 109-112. Nanoteknologi merupakan bidang multidisiplin yang dapat diaplikasikan pada berbagai bidang seperti kedokteran, farmasi, teknik, dan bioteknologi. Sebuah tahap penting pada nanoteknologi adalah mengembangkan metode yang sederhana dan ramah lingkungan untuk menyintesis nanomaterial. Dalam penelitian ini, digambarkan metode sederhana dan ramah lingkungan untuk sintesis nanopartikel perak oleh ekstrak daun tanaman kenikir Tagetes erecta. Fotosintesis (sintesis oleh tanaman) dari nanopartikel perak terdeteksi oleh perubahan warna dari hijau-terang ke gelap-coklat. Sintesis nanopartikel perak dikonfirmasi dengan spektrofotometer UV-Vis, lebih lanjut karakterisasi meliputi sistem analisis pelacakan nanopartikel (NTA) (LM20) dan mikroskop elektron transmisi (TEM). Analisis TEM menegaskan terjadinya sintesis bola 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 with the synthesis of nanomaterials and nanoparticles of size 1100 nm and their technological applications in various fields (Tenover 2006). The combination of the nanotechnology and biology presents the new branch of nanotechnology called Nanobiotechnology. It deals with the application of biotechnological principle for the development of devices, and systems at nano level (Hassellov et al 2008; Kholoud et al. 2010).

There are different methods for nanoparticles synthesis as chemical, physical and a recently developed biological method. Biological method is cost-effective and ecofriendly 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 of nanoparticles. Biosynthesis of inorganic materials, especially metal nanoparticles using microorganisms (Mandal et al. 2006, Gade et al. 2010) and plants (GardeaTorresdey et al. 2003) were carried out. Various plants and

fungi having the potential of metal nanoparticle synthesis, proving that biological synthesis of metal nanoparticles is simple and cost effective (Rai et al. 2008). Among the biological 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 silver nanoparticles.

Use of plant extracts for the nanoparticles synthesis is rapid, eco-friendly and simple alternative to bacterial and fungal system as leaf extract is easily prepared and there is no need of isolation, culture and maintenance of bacterial and fungal culture. Plant extracts have more potential of reducing metal ions than microbes (Rai et al. 2008). The successful examples of synthesis of nanoparticles either intracellularly or extracellularly include geranium leafextract (Shivshankar et al. 2003, 2004, 2005) sun dried leaves of Cinnamomum (Huang et al. 2007), Azadirachta indica (neem) (Shankar et al. 2004), Aloe vera (Chandran et al. 2006), Capsicum annuum (Li et al. 2007), Carica papaya (Mude et al. 2009), Opuntia ficus-indica (Gade et al. 2010), and Murraya koenigii (Bonde et al. 2012).

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

MATERIALS AND METHODS Synthesis of silver nanoparticles Leaves of the Tagetes erecta were collected from the garden of S.G.B. Amravati University, Amravati. After several washes with tap water, leaves were surface sterilized with HgCl2 (0.1%) for 1-2 min to remove microbial contamination, if any. These leaves were cut into small pieces and crushed with mortar and pestle by adding little amount of double sterilized distilled water. The extract obtained was filtered with Whatman filter paper No. 42, the volume was adjusted to 100 ml by double sterilized distilled water. The extract was challenged with the silver nitrate (1mM) solution, and incubated at room temperature.

Only leaf extract without silver nitrate (1mM) treatment was used as the control and the experiments were carried out in triplicate.

Detection and characterization of silver nanoparticles Visual observation Synthesis of silver nanoparticles was detected by change of color from light-green to dark-brown of the filtrate challenged with silver nitrate.

By UV-visible spectrophotometer The synthesis was confirmed with the help of UV-Vis spectrophotometer (Perkin-Elmer, Lambda 25) by scanning the absorbance spectra in the range of 200-800 nm wavelengths.

Nanoparticle tracking and analysis system (NTA) (LM 20) The nanoparticles tracking analysis was carried out by using the liquid sample of silver nanoparticles prepared by diluting with the nuclease free water and 0.5 ml of diluted sample was injected onto the sample chamber and observed through LM 20. Nanoparticles present within the laser beam path were observed by optical instrument (LM-20, NanoSight Pvt. Ltd., UK) having CCD camera and size of the nanoparticles was measured on the basis of Brownian motion of the particles.

nitrate (1 mM AgNO3) at room temperature. The color change was due to the reduction of silver ions to silver nanoparticles i.e. Ag+ to Ag0 and appearance of brown color indicates Silver nanoparticle synthesis (Figure 1). The conformation of the silver nanoparticle synthesis was done

by UV-visible spectrophotometer analysis, which shows the absorbance peak at 438 nm (Figure 2). These observations are similar to those reported by many researchers (Ingle et al. 2008, Raheman et al. 2011).

The dispersion characteristics i.e. average size and particle size distribution were measured with NTA by NanoSight LM-20. NTA allows individual nanoparticles in a suspension to be microscopically visualized and on the basis of the Brownian motion the size distribution and average size of the particle was obtained. It shows that the average size of nanoparticles are 40 nm most repeated nanoparticles was of 37 nm, the concentration of the nanoparticles was 1.47x 108 particles/mL-1 The particle size distribution histogram and 3-D plot of particle size distribution were shown in Figure 3 and 4, respectively.

Particle size and intensity distribution of nanoparticles was shown in Figure 4 which was found to be similar to the results obtained by Montes-Burgos et al., (2010) and Raheman et al. (2011).

Finally, the synthesis of spherical and polydispersive silver nanoparticles by the leaf extract was confirmed by TEM analysis and it was found that the particles are in the range of 20-50 nm having 30 nm as the average diameter (Figure 5). Even the size predicted by TEM analysis was smaller than predicted by NTA analysis. Generally, NTA analysis resulted in larger particle sizes measurements compared to TEM, which is in agreement with previous work (Farkas et al. 2010, 2011). The differences can be mainly explained by the bias from each specific method (Hassellov et al. 2008). For example, TEM is a number based method has a bias towards the smaller particle sizes compared to NTA which is a number based method but fails to probe the weakly scattering particles (below 10-20 nm) and is consequently mainly measuring the NP aggregates which may explain some of the differences in size measurements.

TEM analysis The synthesized silver nanoparticles were also characterized by TEM (Philips, CM 12), on conventional carbon coated copper grids (400 meshes, Plano Gmbh, Germany). For TEM analysis, 5 ÂľL of silver nanoparticles sample was taken, and three image of each sample were selected for the clarification of the composition.

A

B

RESULTS AND DISCUSSIONS The indication of the silver nanoparticles synthesis was marked by the rapid change in the color of plant extract from light-green to dark-brown after treatment with silver

Figure 1. Synthesis of silver nanoparticles from leaf extract of Tagetes erecta (A) Leaf extract before treatment with AgNO3 (control) and (B) Leaf extract after treatment with AgNO3 (experimental)

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

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For the development of green synthesis, Raveendran et al. (2003) suggested three main factors in nanoparticle synthesis should be considered i.e. solvent choice, the use of an environmentally benign reducing agent, and the use of a non-toxic material for nanoparticle stabilization. In the present study water is used as an environmentally benign solvent, replacing toxic organic solvents from chemical methods and biomolecules from leaf extract of Tagetes erecta was used as both reducing and stabilizing agents for green synthesis.

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

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

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

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

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

Bawaskar M, Gaikwad S, Ingle A, Rathod D, Gade A, Duran N, Marcato PD, Rai M, 2010. A New Report on Mycosynthesis of Silver Nanoparticles by Fusarium culmorum. Curr. Nanosci 6: 376-380.

Bonde SR, Rathod DP, Ingle AP, Ade RB, Gade AK, Rai MK. 2012.

Murraya koenigii Mediated Synthesis of Silver Nanoparticles and Its Activity against Three Human Pathogenic Bacteria. Nanosci Methods 1: 25-36.

Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M. 2006.

Synthesis of gold nanotriangles and silver nanotriangles using Aloe vera plant extract. Biotech Prog 22: 577-579 Farkas J, Christian P, Gallego-Urrea JA, Roos N, Hassellov M, Tollefsen KE, Thomas KV. 2010. Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat Toxicol 96: 44-52.

Farkas J, Christian P, Gallego-Urrea JA, Roos N, Hassellov M, Tollefsen KE, Thomas, KV. 2011. Uptake and effects of manufactured silver nanoparticles in rainbow trout (Oncorhynchus mykiss) gill cells.

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Gade AK, Ingle AP, WhiteleyC, Rai MK. 2010. Mycogenic metal nanoparticles: progress and applications. BiotechnolLett 32 (5): 593600.

Gade AK, Bonde P, Ingle AP, Marcato PD, Duran N, Rai MK. 2008.

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Gade AK, Gaikwad SC, Tiwari V, Yadav A, Ingle AP, Rai MK. 2010.

Biofabrication of silver nanoparticles by Opuntia ficus-indica: In vitro antibacterial activity and study of the mechanism involved in the synthesis. Curr Nanosci 6: 370-375.

Gardea-Torresdey J, Gomez E, Jose-Yacaman M, Parsons J, Peralta-Videa J, Tioani H. 2003. Alfalfa sprouts: A natural source for the synthesis of silver nanoparticles. Langmuir 19: 1357-61.

Hassellov M, Readman JW, Ranville JF, Tiede K. 2008. Nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles. Ecotoxicology 17: 344-361.

Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X,Wang H, Wang Y, Shao W, He N, Hong J, Chen C. 2007. Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf.

Nanotech18: 11-20.

Ingle A, Gade A, Pierrat S, Sonnichsen C, Rai M. 2008. Mycosynthesis of silver nanoparticles using the fungus Fusarium acuminatum and its activity against some human pathogenic bacteria. Curr Nanosci 4: 141-44.

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Ingle I, Gade A, Bawaskar M, Rai M. 2009. Fusarium solani: a novel biological agent for the extracellular synthesis of silver nanoparticles.

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Kholoud MM, El-Nour A, Eftaiha A, Abdulrhman AQ, Ammar AA. 2010.

Synthesis and applications of silver nanoparticles. Arab JChem 3: 135-140.

Li S, Qui L, Shen Y, Xie A, Yu X, Zhang L, Zhang Q. 2007. Green synthesis of silver nanoparticles using Capsicum annum L. extract.

Green Chem 9: 852-858.

Mandal D, Bolander M, Mukhopadhyay D, Sarkar S, Mukherjee P. 2006.

The use of microorganisms for the formation of metal nanoparticles and their application. ApplMicrobiolBiotechnol 69: 485-492.

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Characterisation of nanoparticle size and state prior to nanotoxicological studies. J. NanopartRes12: 47-53.

Mude N, Ingle A, Gade A, Rai M. 2009. Synthesis of silver nanoparticles using callus extract of Carica papaya-A first report. J. Plant Biochem Biotech 18: 83-86.

Raheman F, Deshmukh S, Ingle A, Gade A, Rai M. 2011. Silver Nanoparticles: Novel Antimicrobial Agent Synthesized from an

Endophytic Fungus Pestalotia sp. Isolated from Leaves of Syzygium cumini (L) Nano Biomed Eng 3 (3): 174-178 Rai M, Yadav A, Gade A. 2008. Current trends in phytosynthesis of metal nanoparticles. Crit. Rev Biotechnol 28 (4):277-284.

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Shivshankar S, Rai A, Ahmad A, Sastry M. 2004. Rapid synthesis of Au, Ag, and bimetallic Au core-Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci 275: 496-502.

Shivshankar S, Rai A, Ahmad A, Sastry M. 2005. Controlling the optical properties of lemon grass extract synthesized gold nanotriangles and potential application in infrared-absorbing optical coatings. Chem Mater 17: 566-572.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 113-117 November 2012

Seed viability of Jatropha curcas in different fruit maturity stages after storage BAMBANG BUDI SANTOSO1,♼, ARIS BUDIANTO2, IGP MULIARTA ARYANA1 1

Energy Crops Centre, Faculty of Agriculture, University of Mataram, Jl. Majapahit No. 62 Mataram 83125, West Nusa Tenggara, Indonesia. Tel. +620370 621435, Fax. +62-0370 640189, ď‚Šemail: bbs_jatropha@yahoo.com 2 Faculty 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 were investigated. Seed samples of West Lombok, West Nusa Tenggara genotype of Jatropha curcas were collected from a stand of two-year-old trees at an experimental 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 fruit maturity and storage period had an influence on the seed viability of J. curcas. The best fruit maturity stage for seed viability including seed oil content was found in yellow fruit and brownies fruit. For germination to be preserved, seeds could be stored in the ambient room storage for at least five months. For the purpose of oil extraction, seed should preferably be stored not more than four months under 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 setelah penyimpanan. Nusantara Bioscience 4: 113-117. Pengaruh tahapan kematangan dan periode penyimpanan terhadap viabilitas biji telah diteliti. 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 (buah kuning), kematangan berlebih (buah kecoklatan) dan tua (buah kering hitam). Hasilnya menunjukkan bahwa kematangan dan periode penyimpanan memiliki pengaruh pada viabilitas biji J. curcas. Tingkat kematangan terbaik untuk biabilitas biji ditemukan pada buah kuning dan kecoklatan. Untuk .mempertahankan perkecembahan, biji harus disimpan paling tidak lima bulan di kondisi ruangan penyimpanan. 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 many attributes and considerable potential. It is a tropical plant that can be grown in low to high rainfall areas and can be planted in reclaimed land as a fence or commercial crop.

The seed of this plant produces oil. Because Jatropha oil can be used in place of kerosene and diesel fuel, it has been promoted to make rural areas self sufficient in fuel for cooking, lighting, and motive power (Openshaw 2000).

Then, J. curcas is expected to be a highly potential energy crop in Indonesia (Nazir and Setyaningsih 2010).

Despite these numerous benefits and potential, the production and development program of J. curcas in Indonesia has been faced with a number of challanges. One of the constraints is the lack of good seeds in quality and quantity owing to some problems in seed multiplication.

Little information is available on quality seed production and postharvest handling.

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

(ISTA 1999). Simic et al. (2007) also viewed seed quality as a multiple criterion that encompasses several important seed attributes such genetic and chemical composition, germination and vigour, seed water content, and also the presence of seed-borne pathogen. Moreover, poor germination can be resulted from the use of immature seeds (Batin 2011) and storage duration and condition (Dharmaputra et al. 2009; Akowuah et al. 2012). Crop productivity can be increased by increasing the germination rate which is possible by optimizing important parameteres which are crucial for germination (Cheema et al. 2010). In the same manner, successful plantation activities for J. curcas provides viable seeds for the production of quality seedlings. Seed germination and seedling establishment are the most critical stages for survival during the life cycle of the individual J. curcas plant. To date, the potential of this plant is still constrained by the lack of technical information particularly in selecting the best fruit maturity color that could give the most excellent seed germination and seedling growth performance.

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As Cheema et al. (2010) state, that seed should be produced in proper condition as seed from harsh environment is not good for further crop production. So , proper selection of fruit maturity of J. curcas based on color must be ascertained in order to produce quality planting stocks to meet the increasing demand for J. curcas as rehabilitation species and source of oil. Hence, the documentation of fruit color indicating maturation stages of J. curcas is necessary to address issues on poor germination and growth. This article described the effect of fruit maturity stages at different time of seed storage periode on seed viability.

MATERIALS AND METHODS Plant materials Seed sampels of West Lombok, West Nusa Tenggara genotype of J. curcas were collected from a stand of twoyear-old trees at an experimental field. The seed samples were obtained in four different stages of maturity involving early maturity (green fruit), physiological maturity (yellow fruit), over maturity (brown fruit), and senescence (black and dry fruit) as shown in Fig.1. The harvested was done in February-March 2010 and the seed storage was done during April-September 2010.

Procedures Collection, packaging, and storing of seeds Seeds of J. curcas were sun-dried for two days and the moisture content was determained using standard hot air oven method at 105Âą 1OC for 24 hours (Pradhan et al.

2009). Then two kg of sun-dried J. curcas seed was placed in a sac of polypropilen (PP) plastic and then stored in the room condition for duration of 6 months. Three replicates were used for each seed maturity stages. The ambient temperature and relative humidity of the storage room were recorded using a thermohygrometer.

Samples handling Each sample (100-150 g of seeds) derived from each seed sack (replication) was taken monthly for the determinations of seed water content, seed oil content, seed weight, and seed viability (number of germinate seed and germination rate).

Determination of seed viability Viability (percentage of germination and germination rate) of seeds from each sample was determined by growing 100 seeds in plastic container containing sand media under green house conditions. Daily germination counts were taken and recorded up to 15 days (time after which no seed was observed to germinate). The results were calculated as percentage of normal seedlings.

Determination of seed water content Water contents of seeds (based on wet basis) were determined every month based on oven method (gravimetry method). Two samples were used for each replicate(sack).

Determination of seed oil (lipid) content Seed (kernel) oil (lipid) contents were determined based on Soxchlet extraction method (AOAC 1999) with hexane as the solvent. The extracted lipid was obtained by filtrating the solvent using a rotary evaporator apparatus at 40 OC followed by heating in an oven at 105 OC for three hours to evaporate any remaining solvent and water.

Statistical analysis The data were statistically analysed using mean and standard deviation. Analysis of Variance was applied to test the variation between different stages of fruit maturaty through seed viability, seed moisture content, seed oil content, and other characteristics. Least significant difference (LSD at 5% level) was also subjected on significant findings.

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

SANTOSO et al. – Seed viability of Jatropha curcas

RESULTS AND DISCUSSION Results The range of ambient temperature and relative humidity of the storage room is presented in Table 1. The temperature ranged between 26.1 and 29.6 OC, whereas the humidity ranged between 71.4 and 83.4 %. The condition of storage room was relatively steady state during the storage periode.

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

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

Temperature ( OC) 26.5 - 28.3 26.1 - 28.9 26.3 - 29.4 26.7 - 29.5 26.4 - 29.6 26.3 - 29.4

Relative humidity (%) 72.2 - 80.5 75.1 - 83.4 75.5 - 81.9 73.4 - 80.8 72.6 - 80.8 71.4 - 80.7

Variations and significantly difference of fresh fruit and seed characteristic among fruit maturity are given in Table 2. Fruit weight was found maximum (14.7±1.37 g) in green fruit and minimum (5.8±0.78 g) in black-dry fruit of fruit maturity. Not only weight of fruit but also seed moisture content, weight of fruit shell, and weight of seed were found maximum in green fruit and minimum in black-dry fruit.

Table 2. Characteristics of fresh fruit and seed after harvest at different fruit maturity stages Seed Weight of Weight of moisture Weight of fruit fruit shell content seed (g) (g) (g) (%) Green fruit 40.6 ±1.64 14.7 ±1.37 11.8 ±1.02 2.7 ±0.33 Yellow fruit 32.7 ±1.42 12.4 ±1.02 9.4 ±0.87 2.5 ±0.21 Brown fruit 29.8 ±1.11 9.7 ±0.82 7.9 ±0.83 2.1 ±0.11 Black-dry fruit 21.9 ±0.95 5.8 ±0.78 4.1 ±0.82 1.8 ±0.06 LSD 5% 6.6 3.8 4.5 0.7 Note: ±: value of standard deviation. Means differ significantly at P<0.05.

Fruit maturity (fruit color)

Percentage of seed germination and germination rate of J. curcas seeds in this study differ significantly among fruits maturity stages in the storage period of six months (Table 3 and Table 4). Seed taken from yellow fruit and brown fruit had the highest percentage of seed germination during six months of storage period. For germination rate, it was seed taken from yellow fruit, brown fruit, and blackdry fruit had higher rate that of green fruit.

Weight of 100 of J. curcas seeds in this study differ significantly among fruit maturity stages in the storage period until six months (Table 5). Decrease of seed weight during storage as consequency of decrease in their moisture content (Table 6). It was observed that there was significant difference in the moisture content of the seeds at room

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

Oil analysis of seeds were carried out after six months of storage. Seed oil content was influenced by storage period and maturity stage of fruit (Table 7.). The oil content of seeds varied from minimum of 32.3% (green fruit) to maximum which ranged between 35.8 to 36.9% (yellow, brownies, and blck-dry fruit) at the beginning of storage period. After six months of storage, oil content of seeds varied from minimum 8.9% (green fruit) to maximum 23.3% (yellow fruit). Therefore, seed oil content of J. curcas seeds harvested at yellow maturity was not diffrent from that at brownies to black-dry maturityfruit.

Those phenomena existed during first three months of storage period. At the period of three to six month of storage the seed oil content harvested at yellow fruit was no different from that of brownies fruit.

Discussion Seed germination is affected by two factors, i.e. internal and external factors. Internal factor consists of the level of seed maturity, seed size, dormancy, and germination inhibitor. In this study, fruit maturity stages had siginificant effect on germination of seed, germination rate, and also on the seed weight, water content, and oil content of storaged J. curcas seeds (one to six months of storage). In present study, the highest percentage of seed germination was observed in seeds taken from yellow fruit and brownies fruit from the beginning of storage until six months of storage.

Seed taken from young fruit (green fruit) produced immature seed, therefore, resulting in low and delayed germination. This proved the claim of Basra, (2006) and Batin (2011) that seed viability include J. curcas seed, is higher at the mature stage and decreases at early or late harvest or maturity. Harvesting J. curcas fruit too early (green fruit) results in more immature seeds with lower germination of seed and germination rate.

The fresh weight of fruits, shells, and seeds changed during maturation, ripening and senescence. Fruits, shells, and seeds fresh weight increased significantly when the fruits were ripe (fully yellow) but reduced when they started to senesce. Biomass of J. curcas fruits were significantly different according to their maturity stage due to high water content at physiological maturity stage and low water content at senescence stage (Gunaseelan 2009).

Germination capacity as seed viability increases during seed maturation. In this J. curcas, maximum seed viability coincided with the attaiment of maximum seed dry weight or physiological maturity (yellow to brownies fruit color) and decline therafter. According to Adikadarsih and

116 Hastono (2007) , the lowest percentage of J. curcas seed germination (7%) was found in seeds derived from green fruits while the highest percentage (91.6%) was found in seeds derived from yellow fruit.

Moreover, Wellbaum and Bradford (1990)found that germination capacity generally increased progressively and coordinatelly during seed maturation.

Probert and Hay (2000) state that maximum seed quality, normally harvested as dry seeds, was attained at or close to physiological maturity eventhough for others continue to increase well into the post-abscission phase.

Hard seed coat preventes oxygen and moisture entering the seed and prevents autoxidation of linoleic and linolenic acid which are responsible for degradation of cellular organelles (Cantliffe 1998).

Therefore, as the time of seed storage increases, so does cellular damage.

Gingwal et al. (2004) also reported that germination of J. curcas seed fell below 50% within 15 month of storage. In the same manner, Ellis et al. (1990) and Ghasemnezhad and Honemeier (2009) say that melon and sunflower seeds are difficult to store because germination and vigour deteriorate quickly in storage due to the high oil content in the seed. This study also was in agreement with that of Kumari et al. (2011), that the Indias J. curcas seeds looses its viability upon stirage. In addition, Cheema et al. (2010) state, that there was a decrease in rate of germination of castor seeds due to low availability of moisture (seed water content). Therefore, the seeds of J. curcas for seedling should be derived from the yellow and brown fruits with storage period not more than four month within room condition.

As storage period increased in present study, oil content of seed decreased. Then this study is in agreement with that of Akowuah et al. (2012) which showed that percentage of seed oil content of J. curcas gradually decreased with increasing storage time. According to Ahmadhan and Shahidi (2000) and Morello et al. (2004), it was due to the development of rancidity or deterioration of lipids in vegetable oil during storage. As Akowuah et al. (2012) state that aging process naturally affects the quality of seeds during storage at various conditions, especially oil content which is sensitive to deterioration as result of the reaction between unsaturated fatty acid and oxygen. This might be a reason

4 (3): 113-117, November 2012 Table 3. Seed germination at different fruit maturity stages after storage Duration of storage (month) 2 3 4 5 6 % Green fruit 90.3 a 86.6 a 57.3 a 41.5 a 29.6 a 11.9 a 8.6 a Yellow fruit 97.5 bc 95.4 b 81.6 b 79.9 c 68.7 c 59.3 c 56.9 c Brown fruit 98.7 c 96.1 b 79.9 b 70.1 c 61.3 c 53.4 c 48.4 c Black-dry fruit 92.1 ab 87.1 a 60.1 a 52.2 b 45.8 b 32.9 b 25.5 b LSD 5% 6.2 5.7 5.8 10.4 9.8 8.5 8.9 Note: numbers in the column with the same letter did not differ significantly at P<0.05.

Fruit maturity (fruit color)

0

1

Table 4. Rate of seed germination at different fruit maturity stages after storage Fruit maturity (fruit color)

Duration of storage (month) 2 3 4 5 6 day Green fruit 9.3 b 7.8 10.6 b 14.2 b 20.5 b 26.1 b 33.2 b Yellow fruit 6.6 a 6.9 7.4 a 7.6 a 7.9 a 9.6 a 9.9 a Brown fruit 6.7 a 6.8 7.4 a 7.5 a 7.8 a 9.8 a 10.2 a Black-dry fruit 5.8 a 6.5 6.8 a 7.3 a 7.6 a 8.7 a 10.8 a LSD 5% 2.5 ns 2.6 3.1 4.5 4.7 5.2 Note: numbers in the column with the same letter did not differ significantly at P<0.05, ns: not significant 0

1

Table 5. Weight of 100 seeds at different fruit maturity stages after storage Duration of storage (month) 2 3 4 5 6 g Green fruit 108.3 c 98.7 c 68.2 b 63.4 a 61.9 a 52.1 a 47.7 a Yellow fruit 90.7 b 87.8 b 85.6 b 84.3 b 83.1 b 82.7 c 80.3 c Brown fruit 81.6 b 78.4 b 76.7 b 75.2 b 74.8 b 74.4 c 74.1 c Black-dry fruit 69.8 a 67.2 a 66.5 a 65.1 a 64.7 a 63.2 b 62.8 b LSD 5% 11.5 10.2 9.8 9.4 10.3 10.1 12.2 Note: numbers in the column with the same letter did not differ significantly at P<0.05.

Fruit maturity (fruit color)

0

1

Table 6. Seed water content at different fruit maturity stages after storage Duration of storage (month) 2 3 4 5 6 % Green fruit 12.4 b 10.2 b 8.9 b 7.8 7.2 5.6 4.4 a Yellow fruit 8.2 a 7.9 a 7.5 ab 7.3 7.1 6.8 6.4 b Brown fruit 7.4 a 7.2 a 7.1 ab 7.1 6.7 6.6 6.3 b Black-dry fruit 6.7 a 6.5 a 6.4 a 6.1 5.8 5.2 4.9 a LSD 5% 2.1 1.9 2.2 ns ns ns 1.7 Note: numbers in the column with the same letter did not differ significantly at P<0.05, ns: not significant Fruit maturity (fruit color)

0

1

Table 7. Seed oil content at different fruit maturity stages after storage Fruit Duration of storage (month) maturity 0 1 2 3 4 5 6 % (fruit color) Green fruit 32.3 a 31.9 a 26.6 a 23.7 a 18.3 a 14.2 a 8.9 a Yellow fruit 36.9 b 37.2 b 36.5 b 34.6 c 31.1 b 29.7 c 23.3 c Brown fruit 36.2 b 36.8 b 36.1 b 34.2 bc 30.8 b 29.1 bc 22.8 bc Black-dry fruit 35.8 b 36.1 b 35.3 b 32.1 b 29.7 b 27.5 b 20.1 b LSD 5% 3.3.

3.6 4.2 2.1 2.6 2.0 2.7 Note: numbers in the column with the same letter did not differ significantly at P<0.05.

SANTOSO et al. â&#x20AC;&#x201C; Seed viability of Jatropha curcas

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

CONCLUSION Fruit maturity and storage period had an influence on the seed viability of J. curcas. The best fruit maturity stage for good seed viability including seed oil content was found in yellow fruit and brown fruit. Germination percentage and germination rate were statistically the same when the fruits were harvested at yellow and brown ripe.

For germination to be or preserved, seeds could be stored in the ambient room storage for at least five months. In additon, for the purpose of oil extraction, seed should preferably be stored not more than four months under ambient room conditions.

REFERENCES Adikadarsih S, Hastono J. 2007. The effect of fruit maturity on the quality of physic nut seeds in Indonesian. Proceeding of Workshop II: Technology State of Jatropha curcas plant. Bogor, Indonesia, 29 Nov. 2006.

Ahmadhan M, Shahidi F. 2000. Oxidative stability of stripped and no stripped borage and evening primrose oils and their emulsions in water. J Amer Oil Chem Soc 77: 963-968.

Akowuah JO, Addo A, Kemausuor F. 2012. Influence of storage duration of 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. Official methods of the Association of Agricultural Analytical Chemist.

Association of Analytical Chemist, Inc., Arlington Basra AS. 2006. Handbook of seed science and technology. Haworth Press, New York.

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

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

Cantliffe JD. 1998. Seed germination for transplants. Hort Technol 8: 414.

Cheema NM, Malik MA, Qadir G, Rafique MZ, Nawaz N. 2010.

Influence of temperature and osmotic stress on germination induction of different castor bean cultivars. Pakistan J Bot 42: 4035-4041.

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

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

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

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

Gunaseelan NV. 2009. Biomass estimates, characteristics, biochemical methane potential, kinetics and energy flow Jatropha curcas on dry lands. Biomass Bioen 33: 589-596.

ISTA [International Seed Testing Association]. 1999. International rules for seed testing. Seed Science and Technology. International Seed Testing Association, Bassersdorf, Switzerland Kumari A, Joshi PK, Arya MC, Ahmed Z. 2011. Enhancing seed germination of Jatropha curcas L. under Central-Western Himalayas of Uttrakhand, India. Plant Arch 11: 871-874.

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

Nazir N, Setyaningsih D. 2010. Life cycle assessment of biodiesel production from palm oil and jatropha oil in Indonesia. 7th Biomass Asia Workshop. Jakarta, Indonesia.

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

Pradhan RC, Naik SN, Bhatnagar N, Vijay VK. 2009. Moisture-dependent physical 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 Academic Press, 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, soybean and 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 development and germination in muskmelon (C. melo L.). V. Water relation of imbibition and germination. Plant Physiol 92: 1046-1052.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 118-123 November 2012

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: imannrc@yahoo.com 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 and chelated 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 with chelated zinc (100, 200 and 300 mg/L) and stigmasterol (50, 100 and 150 mg/L) on growth and chemical constituents of moringa plants.

The results indicated that treatment of plants with 300 mg/L chelated zinc or 150 mg/L stigmasterol significantly influenced the vegetative growth of moringa plants. The same treatments also significantly increased total sugars%, total protein%, total phosphorous and microelements contents in the leaves. The changes in the pattern of protein electrophoresis (SDS-PAGE) extracted from the newly formed leaves of moringa plants treated with different concentrations of chelated Zinc (Zn) or stigmasterol showed beneficial 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 stigmasterol dan 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 penyemprotan daun dengan kelat seng (100, 200, 300 mg/L) dan stigmasterol (50, 100 , 150 mg/L) terhadap pertumbuhan dan kandungan kimia tanaman kelor. Hasil penelitian menunjukkan bahwa perlakuan tanaman kelor dengan 300 mg/L kelat seng atau 150 mg/L stigmasterol berpengaruh signifikan terhadap pertumbuhan vegetatif tanaman. Perlakuan yang sama juga secara signifikan meningkat 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 dan kuantitas daun.

Kata kunci: Moringa oleifera, stigmasterol, kelat seng

INTRODUCTION The search for new drugs of plant origin has yielded fruitful result in the past. Today it is possible to use molecular biology techniques for detection of genetic variability and tagged desired traits as well as culling out duplicates in accessions. The availability of high throughout screen has made the possibility of covering ‘hits’ into lead compounds in comparatively short time. Drug development from plant sources using gene/molecular techniques is becoming increasingly important. Although few people have ever heard of Moringa oleifera tree today, Moringa could soon become one of the world's most valuable plants, at least in humanitarian terms. Perhaps the fastest-growing of all trees, it commonly reaches three meters in height just 10 months after the seed is planted. Furthermore, it has more than 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 extraction yielded a number of amino acids viz, aspartic acid, glutamic acid, serine, glycine, threonine, β-alanine, valine, leucine, iso-leucine, histidine, lysine, arginine, phenylalanine, tryptophan, cystine and methionine. The ether extract of leaves yielded α-and β-carotene. Also, 9 amino acids in the flowers, 8 in the fruits and 7 each in the protein hydrolysate of flowers and fruits of M. oleifera were identified. 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 as the fruits, and lycine occurred only in the flowers. The flowers contained both sucrose and d-glucose, whereas the fruits showed the presence of sucrose only (Ram 1994).

The juice from the leaves and stem bark of M. oleifera inhibited Staphylococcus aureus but not Escherichia coli.

The 50% ethanolic extract of root bark of M. oleifera showed antiviral activity against vaccinia virus, but was inactive against Ranikhet disease virus. M. oleifera root extract (50% ethanolic) at a dose of 200 mg/kg led to fetal

EL-MOURSI et al. â&#x20AC;&#x201C; Effect of stigmasterol and chelated zinc on Moringa oleifera

resorption in 60% female pregnant rats. Ethanolic extract (50%) of M. oleifera (whole plant excluding roots) showed anticancer activity against human epidermoid carcinoma of nasopharynx in tissue culture and P388 lymphocytic leukemia in mice (Javayardhanan et al. 1994).

The roots are carminative, stomachic, abortifacient, cardiac tonic and also used in paralytic conditions and intermittent fever; also useful as rubefacient in rheumatism, in spasmodic affections of the bowels, hysteria and flatulence as well as in epilepsy. Root bark is used as fermentation to relieve spasm. Bark is considered to be an abortifacient. The fruit is recommended in diseases of liver and spleen, in tetanus and paralysis. Flowers are stimulant and aphrodisiac. Seed oil is applied externally in rheumatism. Leaves are emetic and their juice with black pepper is used in headache. The poultice of leaves is used in reducing glandular swellings. The gum is given in dental caries with sesamum oil and also for relief of otalgia and it is applied with milk on the temples in headache. Seeds are used in veneral affections and to relieve the pain of gout and acute rheumatism (Eilert et al. 1981; Pal et al.1995).

Stigmasterol is a structural component of the lipid core of cell membranes and is the precursor of numerous secondary metabolites, including plant steroid hormones, or as carriers in acyl, sugar and protein transport (Genus 1978).

Brassinosteroids (BR) are known as a group of naturally occurring polyhydroxysteroids. All brassinosteroids isolated from plants are characterized as 5-ď&#x201A;ľ-cholestane derivatives that classified as C27, C28, C29 steroids as revealed by (Yokota et al. 1982). BR has the same biological action like gibberellins and auxins. The pollen grains of plant flowers contained the highest values of BR compared with the other plant organs (Horgan et al. 1984). Brassinosteroids have been found to evoke both cell elongation and cell division resulting in elongation, swelling, curvature and splitting of the internode (Mandava 1998). Physiological functions proposed for Brassinosteroids included cell elongation, cell division, leaf bending, vascular differentiation, proton pumpmediated membrane polarization, sink/source regulation responses (Sasse 1999). In addition, brassinosteroids caused changes in enzymatic activities, membrane potential, DNA, RNA, protein synthesis, photosynthetic activity and changes in the balance of endogenous phytohormones (Steven and Jeneth 1998). Particular interest in sterols was elicited by enhanced growth characters and yield of chamomile plant (Abdel-Wahed and Gamal El-Din 2004). Recently, these studies provided strong evidence that sterols could be essential for normal plant growth and development (Ozedemir et al. 2004).

In recent years, zinc is one of the most important elements for the growth and flowering of some plants as reported by Chandler (1982). Abou-Leila et al. (1994) found that foliar application of Ocimum basilicum L with Zn at 75 mg/l gave the highest values of herbal yield, carbohydrate and oil contents. The effect of Zn on enzyme system responsible for biosynthesis of carbohydrates was reported

by Sandmann and Boger (1983). The necessity of Zn for most crops was emphasized by Singh and Ganguar (1973).

They mentioned that Zn participates in the production of IAA which resulted in an increase in growth and sugar

119

production in sugar beet. Adding Zn fertilizers as foliar application was suggested in Egyptian alkaline soils, where the availability of Zn and other microelements for plant roots becomes relatively low (El-Sayed 1971).

MATERIAL AND METHODS Plant materials. Moringa oleifera L. seeds secured from the Institute of Horticulture Research, Ministry of agriculture, Giza, Egypt. 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 with chelated zinc (100, 200 and 300 mg/L) and stigmasterol (50, 100 and 150 mg/L) on growth and chemical constituents of moringa plants. On 15th October 2009 and on 19th October 2010, the seeds were sown in pots (35 cm in diameter), each pot contained 12 kg clay loamy soil. Treatments were distributed in complete randomized block design with five replications, five pots each. Fifteen days after sowing, the seedlings were thinned to the most three uniform plants in each pot. Each pot received equal and adequate amounts of water and fertilizers. Phosphorous as calcium superphosphate was mixed with the soil before sowing at the rate of 4.0 g/pot.

Three g of nitrogen as ammonium sulphate were added in three applications (one g each) with intervals of two weeks started 30 days after sowing. Also, two g of potassium sulphate were added as soil application. Other agricultural processes were performed according to normal practice. M. oleifera plants were sprayed with stigmasterol and chelated zinc solutions 45 days after sowing. The volume of the spraying solution was maintained just to cover completely the plant foliage till drip. Distilled water was sprayed in the same previous manner on untreated plants (control plants).

The first sample (vegetative stage) was taken 10 days after treatment. The second sample (full vegetative stage) was taken 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 of leaves and stems (g/plant) were determined.

Chemical analysis. Represented samples of the leaves of each treatment were subjected to the following different chemical analyses. Determination of total sugars were carried out according to Dubois et al. (1956). Total nitrogen (modified micro-Kjeldahl) was determined as described by Jackson (1973) and from which protein was calculated. Potassium, calcium and phosphorous were determined according to the procedure described by Brown and Lilliand (1946) and Troug and Meyer (1939), respectively. Iron was determined by atomic absorption spectrophoto-meter (Chapman and Pratt 1961).

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

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4 (3): 118-123, November 2012

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

Data also indicated that all treatments of chelated zinc and stigmasterol resulted in significant increases in calcium content and the highest values were recorded in plants treated with 300 mg/L chelated zinc and 150 mg/L stigmasterol which recorded 133.35 and 130.38 mg/100 g dry matter compared to 87.10 mg/100 g dry matter of untreated control.

Data presented in Table 3 reveal that foliar spray of chelated zinc and stigmasterol significantly increased iron content except in plants treated with 100 mg/L chelated zinc which was not significantly affected. Application of 300 mg/L chelated zinc recorded the highest value of iron content (83.67 mg/100 g dry matter) followed by treatment with 150 mg/L stigmasterol which recorded 82.00 mg/ 100 g dry matter, while untreated control recorded (63.50 mg/g dry matter).

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

Treatment of moringa plant with chelated zinc and

RESULTS AND DISCUSSION

Effect on vegetative growth Table 1 clearly revealed that foliar spray of stigmasterol and chelated zinc significantly increased plant height at all treatments. The most effective treatments in this respect was that of 300 mg/L Zn and 150 mg/L stigmasterol which recorded the highest values of plant height (35 cm and 37.33 cm) compared to 27.25 cm of untreated control.

Number of leaves/plant showed the same trend of plant height which recorded 44.17 and 40.50 at treatments 300 mg/L Zn and 150 mg/L stigmasterol, respectively compared to (28.75) of untreated plants. The same treatments resulted in the highest values of fresh and dry weights of leaves which recorded 7.34 and 9.17 g/plant compared to 3.75 g/plant of untreated plants as fresh weights and 1.58 and1.88 g/plant compared 0.93 g/plant of untreated plants as dry weights. The least increases were obtained at treatments 100 mg/L Zn and 50 mg/L stigmasterol related to slight increases in total sugars% and total protein% in the same treatments.

Data presented in Table 2 indicated that foliar application of stigmasterol and chelated zinc significantly increased plant height, number of leaves, fresh and dry weights of branches/plant, fresh and dry weights of leaves/plant. The most effective treatments in this concern was that of 300 mg/L Zn and 150 mg/L stigmasterol. On the other hand, number of branches/plant was not significantly Table 1. Physiological effect of chelated zinc affected. growth of moringa plants.

The positive effect of stigmasterol and chelated zinc on plant growth was previously reported on wheat plant (AbdelTreatments Plant height Number of (mg/L) (cm) leaves Wahed et al. 2000), sugar beet plant (AbdelWahed and Ali 2001), and Tagetes erecta L Zn 100 30.67 32.00 plant (Balbaa et al. 2008), geranium plant Zn 200 31.33 35.00 (Ayad et al. 2009), flax plant (El-Lethy et al.

Zn 300 35.00 44.17 2010; Hashem et al. 2011), Matthiola incana Stigma 50 31.00 31.00 Stigma100 33.00 38.00 plant (Mahgoub et al. 2011), basil plant Stigma150 37.33 40.50 (Youssef et al. 2004) and fenugreek plant control 27.25 28.75 (Gamal El-Din 2005).

LSD (5%) 3.07 3.18 The pronounced increases of vegetative growth of moringa plants when treated with stigmasterol could be attributed to its role in cell elongation and division (Mandava 1988; Table 2. Physiological effect of chelated zinc Clouse and Sasse 1998). The favourable moringa plants at full vegetative stage. action of chelated zinc might be attributed to its role in the synthesis of tryptophan (the Fresh precursor of IAA) which in turn affected Plant weight Treatments No. of No. of several plant phenomena as reported by height of (mg/L) leaves branches (cm) branches Valke and Wecker (1970).

Effect on chemical constituents Data presented in Table 3 also indicate that foliar application of chelated zinc and stigmasterol to moringa plants significantly increased potassium (K) content in the leaves at treatments 100 mg/L and 150 mg/L stigmasterol, but treatments 100, 200, 300 mg/L chelated zinc and 50 mg/L

Zn 100 Zn 200 Zn 300 Stigma 50 Stigma100 Stigma150 control LSD (5%)

106.67 117.67 143.33 119.00 122.00 122.33 99.33 6.87

12.33 14.67 17.00 14.92 16.00 14.00 10.00 1.84

6. 33 7.00 7.00 6.00 7.00 7.33 6.00 N.S.

(g/plant) 54.35 60.10 89.61 57.81 62.92 64.70 32.97 9.48

and stigmasterol on vegetative

Fresh wt of leaves (g/plant) 5.54 6.20 7.34 6.12 6.15 9.17 3.75 0.94

Dry wt of leaves (g/plant) 1.44 1.48 1.58 1.30 1.32 1.88 0.93 0.32

and stigmasterol on growth of

Dry Fresh Dry weight of weight of weight of branches leaves leaves (g/plant) (g/plant) (g/plant) 16.04 19.74 28.41 17.38 22.00 24.26 10.06 3.03

58.08 65.95 74.94 65.79 73. 05 73. 13 50.01 5.61

15.74 16.94 18.11 14.63 16.90 17.72 17.67 3.97

EL-MOURSI et al. â&#x20AC;&#x201C; Effect of stigmasterol and chelated zinc on Moringa oleifera

121

stigmasterol led to significant increases in total sugars% in bands ranging between 11.86-108.57 kDa. It is evident that all treatments, except that treatment 50 mg/L stigmasterol this treatment induced the appearance of 2 new protein was non-significant. Treatments 300 mg/L chelated zinc bands having molecular weights of 66.03 and 31.87 kDa. and 150 mg/L stigmasterol recorded the highest values of These two new bands having protein intensity 6.05 and total sugars% (3.80 and 3.46%, respectively) compared 7.39%, respectively. with 2.68% in control plants (Table 3).

Application of chelated Zn at 300 mg/L showed the Data presented in Table 3 also indicate that foliar existence of 6 protein bands with the molecular weights spraying moringa plants with chelated zinc or stigmasterol ranging between11.86-108.57 kDa as compared to the significantly increased total protein%, except treatments 50 protein bands obtained from the control plants. The data and 100 mg/L stigmasterol which were not significant. show that this treatment induced the appearance of 1 newly Treatments 300 mg/L chelated zinc and 150 mg/L protein band having the molecular weight of 31.87 kDa and stigmasterol recorded the highest values of total protein% protein intensity 7.84%. Meanwhile, one protein band (3.95 and 3.50%, respectively), compared to 2.45% in disappeared having molecular weight of 18.40 kDa. control plants.

The positive effect of chelated Table 3. Physiological effect of chelated zinc and stigmasterol on chemical constituents zinc and stigmasterol on chemical of moringa plants. constituents was previously reported on lemon grass plant (Gamal El-Din K Fe P et al. 1997), sugar beet plants (AbdelTreatCa (mg/100 g (mg/100 g (mg/100 g Total Total Wahed and Ali 2001), fenugreek ments (mg/100 g dry dry dry dry sugars% protein% Mg/L matter) plant (Gamal El-Din 2005), Tagetes matter) matter) matter) erecta L. Plant (Blabaa et al. 2008), Zn 100 43.00 105.68 69.00 98.67 3.47 3.32 geranium (Ayad et al. 2009), flax Zn 200 43.67 120.66 74.33 110.00 3.70 3.48 plant (El-Lethy et al. 2010) and Zn 300 45.00 133.35 83.67 113.33 3.80 3.95 Matthiola incana plants (Mahgoub et Stigma 50 44.00 108.10 72.50 101.00 2. 80 2.80 al. 2011).

Stigma100 45.33 124.11 76.00 103.00 3.35 2.95 Protein pattern Table 4 reveals the changes in the pattern of protein electrophoresis (SDS-PAGE) extracted from the newly formed leaves of moringa plants treated with different concentrations of chelated zinc (Zn) or stigmasterol (stigma.). The molecular weights of the proteins ranged between 9.82-108.57 kDa and exhibited a maximum number of 17 bands. The scanning profile of such detected protein bands revealed that the band number 9 having the molecular weight of 31.87 kDa produced the highest intensity of protein which recorded 19.36% in plants treated with chelated Zn at concentration equal 100 mg/L. Treatment of moringa plants with chelated Zn at 100 mg/L led to the appearance of 6 protein bands ranging between 11.86-108.57 kDa. Comparing with the features of protein banding pattern obtained from the untreated plants, it is evident that such treatment induced the appearance of 1 newly protein band having the molecular weights 31.87 kDa. The electrophoretic pattern of the plants treated with 200 mg/L chelated Zn showed the presence of 7 protein

Stigma150 Control LSD (5%)

51.00 41.00 4.24

130.38 87.10 11.06

82.00 63.50 5.54

111.17 100.00 5.66

3.46 2. 68 0.55

3.50 2.45 0.82

Table 4. Comparative analysis of relative area (%) of each band of the coomassie bluestained gels of moringa plants treated with different concentrations of chelated Zn and Stigmasterol.

Mol. Zn at Zn at Stigma Band Zn at 300 weight Control 100 200 at 50 number mg/L (kDa) mg/L mg/L mg/L 1 108.57 7.13 10.12 8.88 7.03 7.42 2 101.88 3 87..81 3.13 4.63 5.43 3.16 4.10 4 74.23 5 66.03 6.05* 8.36* 9.36* 6 49.95 7 42.92 5.13 9.20 6.40 4.74 5.74 8 34.62 9 31.87 19.36* 7.39* 7.84* 7.09* 10 28.51 11 22.96 8.10* 12 20.83 13 18.40 10.43 11.60 11.77 14 16.61 15 14.50 16 11.86 6.87 12.01 3.95 10.80 11.80 17 9.82 Total no.

5 6 7 6 7 of bands New 1 2 2 3 bands Disappear 1 1 bands Note: Zn = chelated Zn, Stigma = stigmasterol, *= new bands

Stigma at 100 mg/L 7.09

Stigma at 150 mg/L 8.98

3.71

4.47

7.832*

8.81*

7.23

7.44

6.31*

10.32*

-

-

9.51 6

11.55 7.83* 7

2

3

-

-

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4 (3): 118-123, November 2012

The electrophoretic banding pattern of proteins resulted from the application of stigmasterol at 50 mg/L on moringa plants showed the appearance of 7 protein bands with molecular weights ranging from11.86-108.57 kDa. Three new bands were shown to be induced as a result of this treatment having molecular weights of 66.03, 31.87 and 22.96 kDa. The three new bands having protein intensity 9.36, 7.09 and 8.10, respectively. Meanwhile, one protein band disappeared having molecular weight of 18.40 kDa. Spraying the plants with 100 mg/L stigmasterol resulted in the induction of two new protein band having molecular weights of 66.03 and 31.87 kDa. This treatment also resulted in the disappearance of one protein bands of molecular weight of 18.40 kDa. Application of stigmasterol at 150 mg/L on moringa plants led to the appearance of 17 protein bands ranging between 9.82-108.57 kDa and induced the appearance of 3 newly protein bands having the molecular weights of 66.03, 31.87 and 9,82 kDa, respectively. It is necessary to mention here that, this treatment led to appearance of 1 new band having molecular weight 9.82 kDa which didnâ&#x20AC;&#x2122;t appear in the control and all other treatments with protein intensity 7.83%. Meanwhile, one protein band disappeared having molecular weights of 18.40 kDa. The outcome of the obtained results clearly indicate that spraying moringa plants with different concentrations of chelated Zn or stigmasterol led to the appearance of new protein bands which varied according to the applied concentration. The existence of such newly formed protein bands in treated moringa plants might be explained basing on the potentiality of chelated Zn and stigmasterol to trigger the expression of specific genes along DNA molecule in the target cells, a process which appears to play a key role in regulating a cascade of biochemical reactions which might determine the ultimate appearance of growth patterns and yield of the produced plants. This might be accompanied by a persistent effect carrying over to the progeny via alteration of DNA-binding protein receptors mechanism which might amplify the signaltransduction pathway, this suggestion is reinforced by the findings of Jacobsen and Beach (1985) and Abdel-Hamid (2002).

Bekheta (2004) showed that application of pacloputrazol accombined with gibberellic acid (GA3) on wheat plants changed the electrophoretic profile of protein patterns. In addition, Bekheta and Talaat (2009) showed changes in the pattern of protein electrophoresis (SDSPAGE) extracted from the newly formed leaves of mung bean plants treated with different concentrations of salicylic acid, glutahione or pacloputraszol. The molecular weights of the proteins ranged between 8.094-2455.534 kDa and exhibited a maximum number of 21 bands. The scanning profile of such detected protein bands revealed that the band number 20 having the molecular weight of 8.291 KDa produced the highest intensity of protein which recorded 42.13% in plants treated with 100 mg/L glutathione.

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

beneficial influences for improving plant growth, leaves quality and quantity.

CONCLUSION From the obtained data, it could be concluded that stigmasterol or chelated zinc might play an important role in plant phytochemical mechanism through its effect on the electrophoretic pattern of protein electrophoresis and/or on the mineral ions content, but further studies are needed to learn more about these mechanisms.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 124-133 November 2012

Review: Biological fertilization and its effect on medicinal and aromatic plants KHALID ALI KHALID Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., 12311 Dokki, Cairo, Egypt. Tel. +202-3366-9948, +20233669955, Fax: +202-3337-0931, e-mail: ahmed490@gmail.com 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 dramatic expansion of their population. However, this is also associated many cases with a reduction of the areas of arable land which leaves no opinion for farmers but to increase the yield per unit area through the use of improved the crop varieties, irrigation and fertilization. The major problem facing the farmer is that he cannot afford the cost of these goods, particularly that of chemical fertilizers. Moreover, in countries where fertilizer production relies on imported raw materials, the costs are even higher for farmer and for the country. Besides this, chemical fertilizers production and utilization are considered as air, soil and water polluting operations. The utilization of biofertilizers is considered today by many scientists as a promising alternative, particularly for developing countries. Bio-fertilization is generally based on altering the rhizosphere flora, by seed or soil inoculation with certain organisms, capable of inducing beneficial effects on a compatible host. Bio-fertilizers mainly comprise nitrogen fixes (Rhizobium, Azotobacter, Azospirellum, Azolla or blue green algae), phosphate dissolvers or vesicular-arbuscular mycorrhizas and silicate bacteria. These organisms may affect their host plant by one or more mechanisms such as nitrogen fixation, production of growth promoting substances or organic acids, enhancing nutrient uptake or protection against plant pathogens. Growth characters, yield, essential oil and its constituents, fixed oil, carbohydrates, soluble sugars and nutrients contents of medicinal and aromatic plants were significantly affected by adding the biological fertilizers compared with 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 Bioscience 4: 124-133. Peningkatan produksi pangan menjadi tujuan utama sebagian besar negara-negara berkembang karena pertambahan jumlah penduduk yang dramatis. Namun, hal ini terhambat oleh berkurangnya lahan garapan, yang tidak memberi pilihan kepada petani, kecuali meningkatkan hasil panen per satuan luas melalui penggunaan varietas unggul, irigasi dan pemupukan. Namun banyak petani yang tidak mampu membayar biaya-biaya tersebut, terutama pupuk kimia. Di negara-negara yang mengandalkan bahan baku impor untuk produksi pupuk, biaya yang ditanggung petani dan negara bahkan lebih tinggi lagi. Di samping itu, produksi pupuk kimia dan pemanfaatannya dianggap sebagai penyebab pencemaran udara, tanah dan air. Pemanfaatan pupuk hayati dianggap oleh banyak ilmuwan sebagai alternatif yang menjanjikan, khususnya pada negara-negara berkembang. Pupuk hayati umumnya didasarkan pada perubahan flora rhizosfer, dengan biji atau inokulasi tanah yang mengandung organisme tertentu, yang mampu merangsang efek menguntungkan pada inang sasaran. Pupuk hayati terutama terdiri dari organisme yang memfiksasi nitrogen (Rhizobium, Azotobacter, Azospirellum, Azolla atau ganggang biru hijau), pelarut fosfat atau mikoriza vesikular-arbuskular dan bakteri silikat. Organisme-organisme tersebut dapat mempengaruhi tanaman inang dengan satu atau beberapa mekanisme seperti fiksasi nitrogen, produksi zat pengatur tumbuh atau asamasam 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 signifikan lebih 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 substance which contains living microorganisms, when applied to seed, plant surfaces, or soil colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant (Vessey 2003). Bio-fertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the

synthesis of growth-promoting substances. Bio-fertilizers can be expected to reduce the use of chemical fertilizers and pesticides. The microorganisms in bio-fertilizers restore the soil's natural nutrient cycle and build soil organic matter. Through the use of bio-fertilizers, healthy plants can be grown, while enhancing the sustainability and the health of the soil. Since they play several roles, a preferred scientific term for such beneficial bacteria is plant-growth promoting rhizobacteria (PGPR). Therefore, they are extremely advantageous in enriching soil fertility

KHALID â&#x20AC;&#x201C; Biological fertilization of medicinal and aromatic plants

and fulfilling plant nutrient requirements by supplying the organic nutrients through microorganism and their byproducts. Hence, bio-fertilizers do not contain any chemicals which are harmful to the living soil. Biofertilizers are eco-friendly organic agro-input and more cost-effective than chemical fertilizers. Bio-fertilizers such as Rhizobium, Azotobacter, Azospirillum and blue green algae (BGA) have been in use a long time. Rhizobium inoculantsâ&#x20AC;&#x2122; is used for leguminous crops. Azotobacter can be used with crops like wheat, maize, mustard, cotton, potato and other vegetable crops. Azospirillum inoculations are recommended mainly for sorghum, millets, maize, sugarcane and wheat. Blue green algae belongings general cyanobacteria genus, Nostoc, Anabaena, Tolypothrix or Aulosira, fix atmospheric nitrogen and are used as inoculations for paddy crop grown both under upland and low-land conditions. Anabaena in association with water fern Azolla contributes nitrogen up to 60 kg/ha/season and also enriches soils with organic matter (Vessey 2003).

Other types of bacteria, so-called phosphate-solubilizing bacteria, such as Pantoea agglomerans strain P5 or Pseudomonas putida strain P13 are able to solubilize the insoluble phosphate from organic and inorganic phosphate sources (Subba Rao 1984). In fact, due to immobilization of phosphate by mineral ions such as Fe, Al and Ca or organic acids, the rate of available phosphate (Pi) in soil is well below plant needs. In addition, chemical Pi fertilizers are also immobilized in the soil, immediately, so that less than 20 percent of added fertilizer is absorbed by plants.

Therefore, reduction in Pi resources, on one hand, and environmental pollutions resulting from both production and applications of chemical Pi fertilizer, on the other hand, have already demanded the use of new generation of phosphate fertilizers globally known as phosphatesolubilizing bacteria or phosphate bio-fertilizers. At present, however, the yield of many crops in the world has reached a plateau. Moreover, the negative effects of heavy applications of chemical inputs are becoming apparent, in terms of both production and the environment, especially in the case of medicinal and aromatic plants. Physiological disturbance of plant metabolism is common, due to the accumulation of excess plant nutrients in the soil. The spread of soil-borne diseases is a threat to medicinal and aromatic plants production, especially where monoculture is prevailing. Pollution of underground and surface water by nitrates is sometimes reported from medicinal and aromatic plants producing areas. Quality deterioration, in terms of a decrease in the content of vitamins, sugars active principals, is becoming a subject of concern. All these factors are giving farmers an interest in the function and utilization of soil microorganisms, as a way of repairing the damage from the overuse of chemical inputs. Many farmers in world are showing a strong interest in the utilization of microorganisms to help stimulate plant nutrient uptake; provide biological control of soil-borne diseases; hasten the decomposition of straw and other organic wastes; improve soil structure; and promote the production of physiologically active substances in the rhizosphere or in organic matter. The main incentive for farmers to use microorganisms seems to be that they hope to increase the

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yield or quality of their crops at a relatively low cost, without a large investment of money and labor. Although many microbial materials are sold commercially, most of them are not microbiologically defined, i.e. the microorganisms contained in the products are not identified, and the microbial composition is not fixed.

Many of these commercial products are advertised as if they could solve any problem a farmer is likely to encounter. Because most extension advisors lack any knowledge of microbial products, confusion and trouble frequently occur.

The main objectives of this manuscript are: (i) Types of biological fertilization, i.e. nitrogen fixation, phosphate solubilizing microorganisms, sulphur oxidizing bacteria, silicate bacteria, plant growth promoting rhizobacteria (PGPR), mycorrhizal fungi, decomposition, and (ii) Effect of biological fertilization on medicinal and aromatic plants.

This manuscript concentrates on biological fertilization its effects on medicinal and aromatic plants because of there is no review article was published before concentrated on these items.

TYPES OF BIOLOGICAL FERTILIZATION Nitrogen fixation Nitrogen can be found in many forms in our environment. Nitrogen is also very important for plants to live. The earth's atmosphere is made up of 78 percent nitrogen in the form of a colorless, odorless, nontoxic gas.

The same nitrogen gas found in the atmosphere can be found in spaces between soil particles. However, plants are unable to use this form of nitrogen. Certain microorganisms found in the soil are able to convert atmospheric nitrogen into forms plants can use. This is called biological nitrogen fixation (Subba Rao 1984). One of the most interesting forms of biological nitrogen fixation is that which takes place by microorganisms living in very small nodules on the roots of certain plants such as legumes. This is called symbiotic nitrogen fixation. A symbiotic relationship is an association or relationship where both organisms mutually benefit. In this case, microorganisms obtain food and energy from the root of the plant while producing nitrogen the plant can use for growth and development. The form of nitrogen produced is the same form of nitrogen that is found in several types of commercial nitrogen fertilizers (Subba Rao 1984). The microorganism's ability to fix atmospheric nitrogen is often discussed in terms of the plant's ability to fix nitrogen. The amount of fixation that takes place is strongly influenced by soil conditions. Factors such as moisture, temperature, oxygen supply and fertility in the soil can influence fixation. Diseases and insects can also affect the degree of nitrogen fixation (Subba Rao 1984). One of the most common groups of plants that fix nitrogen is legumes. Of the total nitrogen required by legumes, generally about half is nitrogen fixed from the atmosphere, with the remainder being taken up from residual nitrate in the soil. This means that where legumes are grown, outside applications of manure or fertilizer nitrogen are not needed. Different

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

Phosphate solubilizing microorganisms Phosphorous is added to cultivated soil in different forms as mineral phosphate fertilizers or organic manure, the soluble P in these fertilizers is quickly turns into unavailable form for plant nutrition, this problem is well known in Egyptian soils specially those rich in Calcium Carbonate (El-Gamal 1996), phosphorous is commonly deficient in most natural soils, since it is fixed as calcium phosphates in alkaline soils (Cunningham and Kuiack 1992; Goldstein 1995). Faisal and El-Dawwy 1999 indicated that inadequate phosphorous is a widespread problem in crop production in Egypt and elsewhere. In Egyptâ&#x20AC;&#x2122;s alkaline soils, low solubility Calcium triphosphate are formed following the application of P fertilizer, soluble phosphate ions also are adsorbed on solid calcium carbonate surfaces. Fortunately, soil microorganisms known as phosphate solubilizing microorganisms play a fundamental role in converting P fixed form to be soluble ready available for plant nutrition. The microbial breakdown of soil organic matter is associated with an increase organic, inorganic acids and CO2 production with possibly increases the solubility of soil phosphate (Quastel 1965, Taha et al. 1969, Mishustin et al. 1972, Alexander 1977, Pamela and Hayasaka 1982, Subba Rao 1984 and Curl and Truelove 1985). However Cunningham and Kuiack 1992 and Goldstein 1995 reported that, insoluble calcium phosphate can be dissolved and made available to plants by rhizosphere microorganisms via a mechanism that thought to involve the release of organic acids. Most studies of phosphate solubilizing microorganisms involve inoculating the soils (Mishustin et al. 1972). Inoculation with phosphate-dissolvers is claimed to 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 reports have examined the ability of different bacterial species to solubilize insoluble inorganic phosphate compounds, such as tricalcium phosphate, dicalcium phosphate, hydroxyapatite, and rock phosphate (Goldstein 1986). Among the bacterial genera with this capacity are Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Microccocus, Aereobacter, Flavobacterium and Erwinia. There are considerable populations of phosphate-solubilizing bacteria in soil and in plant rhizospheres (Sperberg 1958; Katznelson et al. 1962; Raghu and MacRae 1962; Alexander 1977).

These include both aerobic and anaerobic strains, with a prevalence of aerobic strains in submerged soils (Raghu and MacRae 1962). A considerably higher concentration of phosphate solubilizing bacteria is commonly found in the rhizosphere in comparison with nonrhizosphere soil (Katznelson et al. 1952; Raghu and MacRae 1962).

Sulphur oxidizing bacteria Sulphur is one of the essential plant nutrients and it contributes to yield and quality of crops. Sulphur occurs in a wide variety of organic and inorganic combinations. The transfer of sulphur between the inorganic and organic pool

is entirely caused by the activity of the soil biota, particularly the soil microbial biomass, which has greatest potential for both mineralization and also for subsequent transformation of the oxidation state of sulphur. Thiobacilli play an important role in sulphur oxidation in soil. Sulphur oxidation is the most important step of sulphur cycle, which improves soil fertility. It results in the formation of sulphate, which can be used by the plants, while the acidity produced by oxidation helps to solubilize plant nutrients and improves alkali soils (Wainwright 1984). The sulphur oxidizing microorganisms are primarily the gram negative bacteria currently classified as species of Thiobacillus, Thiomicrospira and Thiosphaera, but heterotrophs, such as some species of Paracoccus, Xanthobacter, Alcaligens and Pseudomonas can also exhibit chemolithotrophic growth on inorganic sulphur compounds (Kuenen and Beudeker 1982). Two clear metabolic types exist in this group: The obligate chemolithotrophs, which can only grow when supplied with oxidizable sulphur compounds (and CO2 as the source of metabolic carbon) and heterotrophs that can also use the chemolithoautotrophic mode of growth. The obligate chemolithotrophs include Thiobacillus thioparus, T. neapolitanus, T. denitrificans (facultative denitrifier), Thiobacillus thiooxidans (extreme acidophile), Thiobacillus ferrooxidans (acidophilic ferrous iron-oxidizer), Thiobacillus halophilus (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 pantotroph and Thiomicrospira thyasirae.

Several Thiobacillus species are able to utilize mixtures of inorganic and organic compounds simultaneously, often referred to as mixotrophic growth (Kuenen and Beudeker 1982). Depending on the ratio of inorganic and organic substrates, CO2 may serve as an additional carbon source.

Of the 13 species of the genus Thiobacillus recognized, occurring in diverse habitats, only five species are important in sulphur oxidation in soil (Starkey 1966). Four of these Thiobacillus thiooxidans, T. ferrooxidans, T. thioparus and T. denitrificans are obligate chemoautotrophs while T. novellus is considered a facultative chemoautotroph (Taylor and Hoare 1969). Also fungi are capable of oxidizing elemental sulphur and thiosulphate, which include, Alternaria tenius, 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 by various species of microorganisms and fungi, Waksman (1932) pointed out Thiobacilli as the most characteristic group of microorganism performing the oxidative part of sulphur transformation in soil. Studies on the distribution of Thiobacillus thioxidans and T. thioparus showed that these bacteria are found in an active state mainly in soils fertilized with sulphur. The distribution of T. thioparus in soils was studied by Starkey (1934) who demonstrated the almost ubiquitous presence of bacteria in alkaline and neutral soils and their absence in strongly acid soils fertilized with sulphur. The widespread occurrence of

KHALID â&#x20AC;&#x201C; Biological fertilization of medicinal and aromatic plants

Thiobacilli in soils fertilized with sulphur or in soils in which accumulation of sulphur compounds occurs under natural conditions (marshes and peats) indicates that these bacteria play an important role in the oxidation of sulphur and its compounds in soils. Inoculation of Thiobacilli generally increases the rate of sulphur oxidation (Kapoor, and Mishra 1989). Kapoor, and Mishra (1989) observed that sulphur was rapidly oxidized in a field soil of pH 8.0 and the rate of oxidation could be enhanced by inoculation with T. thiooxidans.

Silicate bacteria Silicate bacteria or biological potassium fertilizer can activate the fixed potassium for plant nutrition, as well as prevention and control of plant diseases; also it can effectively prevent crops from early aging and have a strong resistance to drought and cold (Subba Rao 1984).

Zahra et al. (1984) reported that, silicate-dissolving bacteria played a pronounced role in the biological weathering of soil minerals and it can promote K and Si releasing from feldspar. Sheng et al. 2003 showed that, silicate-dissolving bacteria could activate soil P, K, and micronutrients reserves and promote plant growth. Styriakova et al 2003 reported that, the activity of silicate dissolving played a pronounced role in release of Si, Fe and K from feldspar and Fe oxyhydroxides.

Plant growth promoting rhizobacteria (PGPR) Plant growth promoting rhizobacteria (PGPR) comprise a diverse group of rhizosphere-colonizing bacteria and diazotrophic microorganisms which, when grown in association with a plant, stimulate growth of the host. PGPR can affect plant growth and development indirectly or directly (Glick 1995; Vessey 2003). In indirect promotion, the bacteria decrease or eliminate certain deleterious effects of a pathogenic organism through various mechanisms, including induction of host resistance to the pathogen (Van Loon 2007). direct promotion, the bacteria may provide the host plant with synthesized compounds; facilitate uptake of nutrients; fix atmospheric nitrogen; solubilize minerals such as phosphorus; produce siderophores, which solubilize and sequester iron; synthesize phytohormones, including auxins, cytokinins, and gibberellins, which enhance various stages of plant growth; or synthesize enzymes that modulate plant growth and development (Lucy et al. 2004; Gray and Smith 2005). radyrhizobium and Sinorhizobium, of the bacterial family Rhizobiaceae, are known for their ability to fix atmospheric nitrogen while living symbiotically on and nodulating the roots of leguminous plants. However, members of this family also display non-specific associative interactions with roots of other plants, without forming nodules (Van Loon 2007). These rhizobial strains are presumed to produce plant growth regulators, and are classified as PGPR (Vessey 2003).

Mycorrhizal fungi Mycorrhizal types Of the many types of mycorrhizal association (Harley and Smith 1983), two are of major economic and ecological

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importance: ectomycorrhizal associations, and the endomycorrhizal association of the vesicular-arbuscular (VA) type. In ectomycorrhizal associations, the fungi invade the cortical region of the host root without penetrating cortical cells. The main diagnostic features of this type of mycorrhiza are (i) the formation within the root of a hyphal network known as the Hartig net around cortical cells and (ii) a thick layer of hyphal mat on the root surface known as sheath or mantle, which covers feeder roots. Infection of host plants by ectomycorrhizal fungi often leads to changes in feeder roots that are visible to the naked eye. Feeder roots colonized by the fungi are thicker and more branched than uncolonized roots; ectomycorrhizal feeder roots also tend to be colored differently.

In endomycorrhizal associations of the VA type, the fungi penetrate the cortical cells and form clusters of finely divided hyphae known as arbuscules in the cortex. They also form vesicles, which are membrane-bound organelles of varying shapes, inside or outside the cortical cells.

Arbuscules are believed to be the sites where materials are exchanged between the host plant and the fungi. Vesicles generally serve as storage structures, and when they are old they can serve as reproductive structures. Vesicles and arbuscules, together with large spores, constitute the diagnostic features of the VA mycorrhizas. Roots have to be cleared and stained in specific ways and examined under a microscope to see that they are colonized by VA mycorrhizal fungi. Because vesicles are not always found in these types of mycorrhizal associations, some researchers now prefer the designation arbuscular mycorrhiza (AM) over the term vesicular-arbuscular (VA) mycorrhiza. Both AM fungi and ectomycorrhizal fungi extend hyphae from the root into the soil, and these external (or extraradical) hyphae are responsible for translocating nutrients from the soil to the root.

Most ectomycorrhizal fungi belong to several genera within the class basidiomycetes, while some belong to the zygosporic zygomycetes and ascomycetes. On the other hand, AM fungi belong to six genera within the azygosporous zygomycetes.

Host specificity AM associations occur in a wide spectrum of tropical and temperate tree species. They are known not to occur only in a few plants, namely members of the families Amaranthaceae, Pinaceae, Betulaceae, Cruciferae, Chenopodiaceae, Cyperaceae, Juncaceae, Proteaceae, and Polygonaceae. The ectomycorrhizas, on the other hand, occur primarily in temperate forest species, although they have been reported to colonize a limited number of tropical tree species.

Functions of mycorrhizal fungi Results 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 Gildon 1983). However, the most prominent and consistent nutritional effect of AM fungi is in the improved uptake of immobile nutrients, particularly P, Cu, and Zn (Pacovsky 1986; Manjunath and Habte 1988). The fungi enhance immobile nutrient uptake

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by increasing the absorptive surfaces of the root. The supply of immobile nutrients to roots is largely determined by the rate of diffusion. In soils not adequately supplied with nutrients, uptake of nutrients by plants far exceeds the rate at which the nutrients diffuse into the root zone, resulting in a zone around the roots depleted of the nutrients. Mycorrhizal fungi help overcome this problem by extending their external hyphae to areas of soil beyond the depletion zone, thereby exploring a greater volume of the soil than is accessible to the unaided root. Enhanced nutrient uptake by AM fungi is often associated with dramatic increase in dry matter yield, typically amounting to several-fold increases for plant species having high dependency on mycorrhiza.

AM fungi may have biochemical capabilities for increasing the supply of available P and other immobile nutrients. These capabilities may involve increases in root phosphatase activity, excretion of chelating agents, and rhizosphere acidification. However, these mechanisms do not appear to explain the very pronounced effect the fungi have on plant growth (Habte and Manjunth 1991).

AM fungi are often implicated in functions which may or may not be related to enhance nutrient uptake. For example, they have been associated with enhanced chlorophyll levels in leaves and improved plant tolerance of diseases, parasites; water stress, salinity, and heavy metal toxicity (Bethlenfalvay and Gabor 1992). Moreover, there is increasing evidence that hyphal networks of AM fungi contribute significantly to the development of soil aggregates, and hence to soil conservation (Miller and Jastrow 1992).

Charcoal application The amount of nutrients (N, P, and K) absorbed by the shoots showed a trend similar to that of the shoot fresh weight. The amount of N fixed by the nodules and transported to the shoots was calculated by subtracting the N content of the shoots of the plants not inoculated with rhizobia from the N content of the inoculated plants. The addition of charcoal increased this amount of N 2.8-4.0 times. Added charcoal also increased the nodule weight by 2.3 times. Significant correlation was observed between the increments of P and N, suggesting that the stimulation of nitrogen fixation by charcoal addition may be due to the stimulation of P uptake. Charcoal may stimulate the growth of AMF by the following mechanism. Charcoal particles have a large number of continuous pores with a diameter of more than 100µm. They do not contain any organic nutrients, because of the carbonization process. The large pores in the charcoal may offer a new microhabitat to the AMF, which can obtain organic nutrients through mycelia extended from roots. This may enable the AMF to extend their mycelia far out from the roots, thus collecting a larger amount of available phosphate (Nishio and Okano 1991).

Decomposition Organic matter decomposition serves two functions for the microorganisms, providing energy for growth and supplying carbon for the formation of new cells. Soil organic matter (SOM) is composed of the “living”

(microorganisms), the “dead” (fresh residues), and the “very dead” (humus) fractions. The “very dead” or humus is the long-term SOM fraction that is thousands of years old and is resistant to decomposition. Soil organic matter has two components called the active (35%) and the passive (65%) SOM. Active SOM is composed of the “living” and “dead” fresh plant or animal material which is food for microbes and is composed of easily digested sugars and proteins. The passive SOM is resistant to decomposition by microbes and is higher in lignin.

Microbes need regular supplies of active SOM in the soil to survive in the soil. Long-term no-tilled soils have significantly greater levels of microbes, more active carbon, more SOM, and more stored carbon than conventional tilled soils. A majority of the microbes in the soil exist under starvation conditions and thus they tend to be in a dormant state, especially in tilled soils. Dead plant residues and plant nutrients become food for the microbes in the soil. Soil organic matter (SOM) is basically all the organic substances (anything with carbon) in the soil, both living and dead. SOM includes plants, blue green algae, microorganisms (bacteria, fungi, protozoa, nematodes, beetles, springtails, etc.) and the fresh and decomposing organic matter from plants, animals, and microorganisms (Hoorman and Islam 2010).

Climate, Temterature, and pH effects on SOM SOM is affected by climate and temperature. Microbial populations double with every 10 degree Fahrenheit change in temperature. If we compare the tropics to colder arctic regions, we find most of the carbon is tied up in trees and vegetation above ground. In the tropics, the topsoil has very little SOM because high temperatures and moisture quickly decompose SOM. Moving north or south from the equator, SOM increases in the soil. The tundra near the Arctic Circle has a large amount of SOM because of cold temperatures. Freezing temperatures change the soil so that more SOM is decomposed then in soils not subject to freezing.

Moisture, pH, soil depth, and particle size affect SOM decomposition. Hot, humid regions store less organic carbon in the soil than dry, cold regions due to increased microbial decomposition. The rate of SOM decomposition increases when the soil is exposed to cycles of drying and wetting compared to soils that are continuously wet or dry.

Other factors being equal, soils that are neutral to slightly alkaline in pH decompose SOM quicker than acid soils; therefore, liming the soil enhances SOM decomposition and carbon dioxide evolution. Decomposition is also greatest near the soil surface where the highest concentration of plant residues occur. At greater depths there is less SOM decomposition, which parallels a drop in organic carbon levels due to less plant residues. Small particle sizes are more readily degraded by soil microbes than large particles because the overall surface area is larger with small particles so that the microbes can attack the residue.

A difference in soil formation also occurs traveling east to west across the United States. In the east, hardwood forests dominated and tree tap roots were high in lignin, and deciduous trees left large amounts of leaf litter on the

KHALID – Biological fertilization of medicinal and aromatic plants

soil surface. Hardwood tree roots do not turn over quickly so organic matter levels in the subsoil are fairly low. In forest soils, most of the SOM is distributed in the top few inches. As you move west, tall grassland prairies dominated the landscape and topsoil formed from deep fibrous grass root systems. Fifty percent of a grass root dies and is replaced every year and grass roots are high in sugars and protein (higher active organic matter) and lower in lignin. So soils that formed under tall grass prairies are high in SOM throughout the soil profile. These prime soils are highly productive because they have higher percentage of SOM (especially active carbon), hold more nutrients, contain more microbes, and have better soil structure due to larger fungal populations.

Carbon to Nitrogen(C:N) ratio Low nitrogen content or a wide C:N ratio is associated with slow SOM decay. Immature or young plants have higher nitrogen content, lower C:N ratios and faster SOM decay. For good composting, a C:N ratio less than 20 allows the organic materials to decompose quickly (4 to 8 weeks) while a C:N ratio greater than 20 requires additional N and slows down decomposition.

The C:N ratio of most soils is around 10:1 indicating that N is available to the plant. The C:N ratio of most plant residues 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 SOM rises as decomposition progresses. The 10:1 C:N ratio of most soils reflects an equilibrium value associated with most soil microbes (bacteria 3:1 to 10:1, fungus 10:1 C:N ratio).

Bacteria are the first microbes to digest new organic plant and animal residues in the soil. Bacteria typically can reproduce in 30 minutes and have high N content in their cells (3 to 10 carbon atoms to 1 nitrogen atom or 10 to 30% nitrogen). Under the right conditions of heat, moisture, and a food source, they can reproduce very quickly. Bacteria are generally less efficient at converting organic carbon to new cells. Aerobic bacteria assimilate about 5 to 10 percent of the carbon while anaerobic bacteria only assimilate 2 to 5 percent, leaving behind many waste carbon compounds and inefficiently using energy stored in the SOM. Fungus generally release less carbon dioxide into the atmosphere and are more efficient at converting carbon to form new cells. The fungus generally captures more energy from the SOM as they decompose it, assimilating 40 to 55 percent of the carbon. Most fungi consume organic matter higher in cellulose and lignin, which is slower and tougher to decompose. The lignin content of most plant residues may be of greater importance in predicting decomposition velocity than the C:N ratio.

Mycorrhizal fungi live in the soil on the surface of or within plant roots. The fungi have a large surface area and help in the transport of mineral nutrients and water to the plants. The fungus life cycle is more complex and longer than bacteria. Fungi are not as hardy as bacteria, requiring a more constant source of food. Fungi population levels tend to decline with conventional tillage. Fungi have a higher carbon to nitrogen ratio (10:1 carbon to nitrogen or 10%

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nitrogen) but are more efficient at converting carbon to soil organic matter. With high C:N organic residues, bacteria and fungus take nitrogen out of the soil.

Protozoa and nematodes consume other microbes.

Protozoa can reproduce in 6-8 hours while nematodes take from 3 days to 3 years with an average of 30 days to reproduce. After the protozoa and nematodes consume the bacteria 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 back and forth in the soil. Plants absorb ammonia and soil nitrates for food with the help of the fungi mycorrhizal network.

Microorganism populations change rapidly in the soil as SOM products are added, consumed, and recycled. The amount, the type, and availability of the organic matter will determine the microbial population and how it evolves.

Each individual organism (bacteria, fungus, and protozoa) has certain enzymes and complex chemical reactions that help that organism assimilate carbon. As waste products are generated and the original organic residues are decomposed, new microorganisms may take over, feeding on the waste products, the new flourishing microbial community (generally bacteria), or the more resistant SOM. The early decomposers generally attack the easily digested sugars and proteins followed by microorganisms that attack the more resistant residues.

It can be summarized that (i) Microorganisms abound in the soil and are critical to decomposing organic residues and recycling soil nutrients. Bacteria are the smallest and most hardy microbe in the soil and can survive under harsh conditions like tillage. Bacteria are only 20-30% efficient at recycling carbon, have high nitrogen content (3 to 10 carbon atoms to nitrogen atom or 10 to 30% nitrogen), lower carbon content, and a short life span. Carbon use efficiency is 40-55% for mycorrhizal fungi so they store and recycle more carbon (10:1 carbon to nitrogen ratio) and less nitrogen (10%) in their cells than bacteria. Fungi are more specialized but need a constant food source and grow better 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 or animal material which is food for microbes and is composed of easily digested sugars and proteins. The passive SOM is resistant to decomposition by microbes (higher in lignin). Active SOM improves soil structure and holds plant available nutrients. Every 1% SOM contains 1,000 pounds of nitrogen, 100 pounds of phosphorus, 100 pounds of potassium, and 100 pounds of sulfur along with other essential plant nutrients. Tillage destroys SOM by oxidizing the SOM, allowing bacteria and other microbes to quickly decompose organic residues. Higher temperatures and moisture increase the destruction of SOM by increasing microbial populations in the soil. Organic residues with a low carbon to nitrogen (C:N) ratio (less than 20) are easily decomposed and nutrients are quickly released (4 to 8 weeks), while organic residue with a high C:N ratio (greater than 20) decompose slowly and the microbes will tie up soil nitrogen to decompose the

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residues. Protozoa and nematodes consume other microbes in the soil and release the nitrogen as ammonia, which becomes available to other microorganisms or is absorbed by plant roots.

EFFECT OF BIOOGICAL FERTILIZATION ON MEDICINAL AND AROMATIC PLANTS Growth characters of rosemary, K, N content essential oil constituents (alpha-pinene, β-pinene, limonene,1,8cineole, linalool, campor, β-terpineol, borneol, terpinen 4-ol, carvone, thymol, carvacrol, linalylacetate, geranylacetate, β-caryophyllene, caryophyllene oxide) were significantly increased under biofertilizer (Azotobacter vinelandii) treatments (Leithy and El-Meseiry 2006). The effects of biofertilization on growth, fruit yield, and oil composition of fennel plants were investigated by Mahfouz and SharafEldin (2007). Application of biofertilizer, which was a mixture of Azotobacter chroococcum, Azospirillum liboferum, and Bacillus megatherium applied with chemical fertilizers (only 50% of the recommended dosage of NPK) increased vegetative growth (plant height, number of branches, and herb fresh and dry weight per plant) compared to chemical fertilizer treatments only. The tallest plants, the highest number of branches per plant, and the highest fresh and dry weights of plants were obtained from the treatment of biofertilizer plus a half dose of chemical fertilizer (357 kg ammonium sulphate + 238 kg calcium super phosphate + 60 kg potassium sulphate ha-1). The lowest fresh and dry weights of plants occurred with the 50% NPK. Also, addition of biofertilizer with the chemical fertilizer increased these characters more than the half dose of chemical fertilizer alone. Total carbohydrates in the dry plant material were influenced by the biofertilizer. The highest values of total carbohydrates were found in the treatment with biofertilizer plus a half dose of nitrogen and phosphorus. Nitrogen, phosphorus, and potassium levels in the plant tissue increased when soil was inoculated by nitrogen-fixing bacteria, phosphate dissolving bacteria, and a mixture of all strains, respectively. The least amount of N, P and Kin the plant tissue occurred with the half dose of chemical fertilizer. Essential oil content in the fennel fruits was increased due to inoculation compared to the half dose of chemical fertilizer. The highest oil yield per plant was observed with the treatment of biofertilizer plus a half dose of nitrogen and phosphorus. The lowest amount of essential oil yield was obtained with the half dose of chemical fertilizer. Oxygenated compounds were increased as a result of using biofertilizer. The highest anethol (trans-1-methoxy4-(prop-1-enyl) benzeen; C10H12O) in fennel essential oil occurred with the half dose of N, P, and K and inoculation with Bacillus megatherium. The effect of compost and biofertilizers on the growth, yield and essential oil constituents of marjoram (Majorana hortensis L.) was investigated by Gharib et al. (2008). Forty five days old seedling were transplanted in soil treated with 15 and 30% aqueous extracts of compost and/or biofertilizers (mixture of Azospirillum brasilienses, Azotobacter chroococcum, Bacillus polymyxa and B. circulans) in addition to the recommended nitrogen,

phosphorus and potassium (NPK) doses as control. Use of combined treatment of bio-fertilizers gave better results for all studied traits than those obtained from either nitrogen fixers (Azos. brasiliense, Azot. chroococcum, B. polymyxa) or B. circulans alone). The essential oil percentage and yield per plant for three cuttings was almost two fold higher on fresh weight basis as a result of aqueous extracts of compost at low level + bio-fertilizers compared with control, indicating that combinations of low input system of integrated nutrient management could be beneficial to obtain relatively good yields of essential oil. Essential oil composition using GC/MS revealed that marjoram belongs to the cis-sabinene hydrate/terpinene-4-ol chemotype. The chemical composition of marjoram essential oil did not change due to the fertilization type or level; rather the relative percentages of certain 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 microorganisms involve inoculating the soils (Mishustin et al. 1972).

Inoculation with phosphate-dissolvers is claimed to 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). Gomaa (1989) demonstrated that, some effects of phosphate solubilizing microorganism’s inoculation have been observed in terms of increasing the amounts of available P and plant growth of crop production. Hauka et al. (1990) stated that, phosphate solubilizing microorganisms significantly increased dry yields and protein content of Barley and Tomato plant. ElGamal (1996) revealed that, P level or inoculation with phosphorene (phosphate solubilizing microorganisms) significantly increased soil available P, tuber N and P contents and their uptake, foliage dry weight, foliage P content and P uptake, total P uptake and dry matter yield of Potato plants. Applying phosphorene improved growth and P uptake by the Olive seedlings in comparison to the phosphate fertilizer alone (Faisal and El-Dawwy 1999).

Applying phosphate solubilizing microorganisms with calcium superphosphate to mustard (Sinapis alba L.) plants improved growth, seed yield, lipids content, N, P and their uptake, and protein content but total carbohydrates, soluble sugars and insoluble sugars were decreased, also saturated and unsaturated fatty acids content were changed compared with applying calcium superphosphate fertilizer alone. It recommended that using phosphate solubilizing microorganisms because it increases the production (quantity and quality) and medicinal properties. Also it is very cheap and expressed cash money improving the income of farmer, in addition, uses biofertilizer (phosphate solubilizing microorganisms) is safe for human health (Khalid 2004).

Awad and Khalil (2003) reported that, the biofertilizer (Thiobacillus thioxidans) and sulphur significantly increased the growth of squash and raised their nutrient content than Sulphur fertilizer alone. Treated celery (Apium graveolens L cv. dulce) plants with different levels of sulphur and sulphur-oxidizing bacteria resulted in a

KHALID â&#x20AC;&#x201C; Biological fertilization of medicinal and aromatic plants

significant 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 comparison with control plants. Khalid (2005) states that chemical composition analysis of treated plants showed an increase in 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 main components 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 of some important elements such as rock phosphate as a source of phosphorous and feldspar mica as a source of potassium with biological potassium phosphorous fertilizers or biological potassium fertilizers (Silicate bacterium) at different levels (0.0, 25, 50 and 100 g/L) on Ruta graveolens L. plant instead of the chemical fertilizes were investigated by Khalid et al. (2007). Adding biological fertilizer with feldspar or rock phosphate improved vegetative growth characters such as plant height (cm), branches number/plant, fresh and dry weights of different plant parts i.e. leaves, stems and roots (g/plant), in addition to some chemical constituents as essential oil, total flavonoides, P, K, Fe, Zn and Cu content. On the other hand, the main constituents of essential oil and N content were decreased compared with adding recommended chemical fertilizers.

According to Banchio et al. (2008) the effects of root colonization by plant growth promoting rhizobacteria (PGPR) on biomass, and qualitative and quantitative composition of essential oils were determined in the aromatic crop Origanum majorana L. (sweet marjoram).

PGPR strains evaluated were Pseudomonas fluorescens, Bacillus subtilis, Sinorhizobium meliloti, and Bradyrhizobium 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, in comparison to control plants or plants treated with other PGPR. Essential oil yield was also significantly increased relative to non-inoculated plants, without alteration of oil composition. P. fluorescens has clear commercial potential for economic cultivation of O. majorana.

In studies on Coriandrum sativum, Anethum graveolens and Foeniculum vulgare, it was shown that AMF root colonization enhances the essential oil quality by altering essential oil components (Kapoor et al. 2002 and 2004). Four organic 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) were evaluated for their effect on the herbage yield, essential oil content and inoculum potential (IP) of native arbuscular mycorrhizal fungi (AMF) on three varieties of Java citronella, 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 dry matter yield significantly. Bio-13 performed better and produced the highest herbage, essential oil and dry matter yield. The type and dose of the various organic amendments also significantly influenced the indigenous AMF infectious propagules in soil. Highest number of

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AMF propagules were recorded in the LC amended plots in all the three varieties. Amongst the varieties, highest native mycorrhizal inoculum was recorded in the Bio-13. Least number of AM infectious propagules was recorded in the Mandakini plants grown in 40 t ha-1 SSL (Tanu 2004).

Khaosaad et al. (2006) observed that essential oil levels in Origanum species are increased in the presence of arbuscular mycorrhizal fungi. A field experiment was conducted to study and compare the effectiveness of two arbuscular mycorrhizal fungi (AMF), Glomus macrocarpum (GM) and Glomus fasciculatum (GF) on three accessions of Artemisia annua. The AM inoculation significantly increased the production of herbage, dry weight of shoot, nutrient status (P, Zn and Fe) of shoot, concentration of essential oil and artemisinin in leaves as compared to non-inoculated plants. The extent of growth, nutrient concentration and production of secondary plant metabolites varied with the fungus-plant accession combination. The mycorrhizal dependency of the three accessions was related to the shoot: root ratio. Comparing the two fungal inoculants in regard to increase in essential oil concentration in shoot, the effectiveness of GF was more than that of GM. While in two accessions, GM was more effective in enhancing artemisinin concentration than GF. Increase in concentration of essential oil was found to be positively correlated to P-status of the plant conversely (Chaudhary et al. 2008).

Five strains of bacteria (1. Azotobacter chroococcum, 2. Azospirillum lipoferum, 3. Bacillus polymyxa, 4. Bacillus megatherium and 5. Pseudomonas fluorescens) were mixed in equal parts and used as biofertilizer in this experiment.

The biofertilizer treatment was applied alone or in combination with 1/3, 2/3 or full recommended dose of mineral nitrogen fertilizer. The results indicated that applying biofertilizer treatment alone or in combination with chemical N fertilizer increased the growth, yield and chemical constituents of dill plant compared to the untreated control. The highest values of vegetative growth, oil yield, chlorophyll content and NPK percentages were recorded by the treatment of bio-fertilizer plus two third of recommended dose of nitrogen fertilizer. The lowest values in this respect were obtained by control plants during two seasons. The GC analysis of volatile oil indicated that the main components were carvone, limonene and apiol. These components were affected by biofertilization and chemical N treatments. Partial substitution of mineral nitrogen fertilizer by bio-fertilizer was recommended to increase the yield as well as the quality of dill plant (Hellal et al. 2011).

Bio-fertilizer treatments increased the growth characters and essential oil composition of coriander compared with the chemical fertilizers treatments (Hassan et al. 2012).

Bio-fertilizer treatments (mycorrhizal and phosphate bacteria) increased the seed yield and essential oil of fennel plants compared with vermicompost treatments (Darzi 2012). Application of phosphate bio-fertilizer and phosphorus were significant on the vegetative growth characters of Tagetes erecta L. plants (Hashemabad et al. 2012).

Adding dry yeast at the rate of 6 g/L. was the most effective on growth parameters and oil-percent of Borago officinalis plant (Ezz El-Din and Hendawy 2010). An

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Iranian investigation revealed that inoculation of Ocimum basilicum roots with plant growth-promoting rhizobacteria (PGPR) improved growth and accumulation of essential oils. Treatments were Pseudomonas putida strain 41, Azotobacter chroococcum strain-5 and Azosprillum lipoferum strain. In comparison to the control treatment, all factors were increased by PGPR treatments. The maximum Root fresh weight (3.96 g/plant), N content (4.72%) and essential oil yield (0.82%) were observed in the Pseudomonas + Azotobacter + Azosprillum treatment. All factors were higher in the Pseudomonas + Azotobacter + Azosprillum and Azotobacter + Azosprillum treatments (Ordookhani et al. 2011).

CONCLUSION What is the key behind specificity of certain Nitrogen fixing microorganisms to selected plants? Why not other Non-Nitrogen fixing microorganisms acquire the property of nitrogen fixation? Can we evolve nitrogen fixing plants?

The search for new microorganisms capable of fixing nitrogen. Exploiting other plant microorganisms associations.

Proper utilization of fertilizer nitrogen by means of slow release nitrogen fertilizer. Domestication and cultivation of promising nodulated legume species. Recycling of wastes for elements; microorganisms abound in the soil and are critical to decomposing organic residues and recycling soil nutrients. The above questions and statements are an outlook of future research.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 3, pp. 134-137 November 2012

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: surendrasingh.bhu@gmail.com 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 and protein content of an Antarctic Cyanobacterium Nostoc commune. Nusantara Bioscience 4: 134-137. Effect of temperature variation on biomass accumulation, pigment composition and protein content were studied for the cyanobacterium Nostoc commune, isolated from Antarctica. Results confirmed the psychrotrophic behavior (optimum growth temperature 250C) of the cyanobacterium. Low temperature increased the duration of lag phase and exponential growth phase. Maximum increase in biomass was recorded on 24th day at 250C and on 12th day at 50C. The downshift from 25 to 50C had almost negligible effect on chl a content. Maximal protein content was recorded for cultures growing at 50C on 12th day. The carotenoids/chl a ratio was maximum (2.48) at 50C on 9th day. It remained almost constant for cultures growing at 5 and 350C. There was an induction in protein synthesis following downshift in temperature from 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 dan kandungan protein cyanobacterium dari Antartika Nostoc commune. Bioscience Nusantara 4: 134-137. Pengaruh variasi suhu terhadap akumulasi biomassa, komposisi pigmen dan kandungan protein dipelajari pada cyanobacterium Nostoc komune, yang diisolasi dari Antartika. Hasil penelitian menegaskan perilaku psikrotrofik (suhu pertumbuhan optimum 25◦C) dari cyanobacterium tersebut. Suhu rendah meningkatkan durasi fase lambat dan fase pertumbuhan eksponensial. Peningkatan biomassa maksimal tercatat hari ke-24 pada 25◦C dan hari ke-12 pada 5◦C. Penurunan suhu dari 25 ke 5◦C hampir tidak berpengaruh pada kandungan chl a. Kandungan protein tertinggi 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. Hal ini hampir selalu konstan untuk kultur yang tumbuh pada 5-35◦ C. Terdapat induksi dalam sintesis protein mengikuti penurunan suhu dari 25 ke 5◦C. Kata kunci: cyanobacterium, suhu rendah, tingkat pertumbuhan, phycobiliproteins, pigmen

Cold-stress is often lethal to living organisms. For growth to occur in low temperature environments, cellular components such as membranes, proteins and nucleic acids have to adapt to the cold (Cavicchioli et al. 2002).

Microbial diversity of the Antarctica is composed of either psychrophilic (optimum growth temperature <150C) or psychrotrophic (optimum growth temperature >150C) (Morita 1975; Veerapaneni 2009). Psychrotrophs constitute the bulk of continental Antarctica microflora. Adaptive responses of the Antarctic microbes growing in the permanently cold environments, especially at 0-40C are little studied (Chattopadhyay 2006). Majority of Antarctic cyanobacteria grow in a wide range of temperature (Nadeau and Castenholz 2000; Nadeau et al. 2001). The temperature-growth response suggests their close relationship with moderate regions of the Antarctic (Seaberg 1981).

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

Singh, a member of the Seventeenth Indian Expedition to Antarctic (Pandey and Upreti 2000). The cyanobacterium was isolated, purified using standard microbiological techniques, and is maintained in nitrogen free BG-11medium in a culture room set at 25±10C, illuminated with day-light fluorescent tubes having the photon fluence rate of 35E m-2 s-1 at the surface of vessels. Here, we studied the biomass accumulation (in terms of fresh weight and dry weight ml-1 volume of liquid culture harvested at 3 day intervals) and pigment composition of N. commune, an Antarctic cyanobacterial to different temperatures (i.e., 5, 15, 25, 350C).

Growth of N. commune was estimated in terms of chlorophyll a (chl a) content and expressed in terms of specific growth rate computed as per the method of Myers and Kratz (1955). Growth rate (µ) of N. commune at each temperature was estimated as changes in biomass over time from the log-linear portion of the curve using non linear curve fit method with Boltzmann constant. Chl a was

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

1.0 50C 150C 250C 350C

0.8

Biomass -1 (dry weight g ml )

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 and quantitatively) were analysed by recording the absorption spectra and absorbance at various wavelengths in a UVVIS spectrophotometer (Varian, Cary100-Bio, USA) with a 1 cm light path. Fluorescence excitation and emission spectra were recorded in a fluorescence spectrophotometer (Hitachi F-2500, Japan). The amounts of different PBPs namely, phycocyanin (PC, Amax 565 nm), allophycocynain (APC, Amax 620 nm) and phycoerythrin (PE, Amax 650 nm) were determined according to the equations given by Tandeau and Houmard (1993). Protein was estimated following the method of Lowry et al. (1951) using lysozyme as the standard.

The specific growth rate (K) and log10 specific growth rate (log10 K) of N. commune growing at different temperatures (5 to 350C) are shown in Table 1. The optimum temperature (Topt) for growth of N. commune ranged in between 15 and 25oC with less difference in µ max values. The specific growth rate of N. commune was maximal at 250C. Maximum growth rate was 1.9590 day-1 at 150C. The Q10 value ranged from 2-3. Results suggest psychrotrophic behavior of N. commune.

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

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

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

5 15 25 35

0.48 1.0072 1.48 0.48

0.5972 1.9590 1.2900 0.4800

Curve fitting results suggest that N. commune grew exponentially from 3 to 9 day at 250C, and 9 to 15 day at 50C (Figure 1). After a downshift in temperature from 25 to 50C, exponential phase of the cyanobacterium moved from 3 to 9 day. Duration of lag phase also increased following the downshift. The stationary growth phase started from 15th day for cultures growing at 50C, and continued with increase in incubation period. This indicates the adaptability of N. commune to low temperature (50C), and its preference for 250C. Maximum increase in biomass was recorded at 24th day at 250C and on 12th day at 50C (Figure 2).

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

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

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

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4 (3): 134-137, November 2012

PC/chl a ratio was highest on 21st day at 350C. At 50C, the ratio of PC/chl a increased upto 6th day, decreased thereafter. A minimum increase in PC/chl a ratio (0.0148 and 0.0126) was recorded on day 15th (also 18 day) at 50C. The maximum increase in PC/chl a ratio (0.1876) was recorded on 21 day at 250C (Figure 5A). PE/chl a ratio was maximum on 21st day for cultures growing at 350C. However, cultures growing at 5 and 150C exhibited minimum PE/chl a ratio on 15th and 18th day, respectively.

The ratio increased upto 12th day at 50C and declined thereafter (Figure 5B). APC/chl a ratio was found to be highest on 24th day at 150C. Its value for cultures growing at 50C increased from 12th day (Figure 5C).

3.0 0 5 C

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

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

B

C Figure 5. Effect of temperature on N. commune: A. PC/chl a ratio, 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 cultures growing at 5 and 350C (Figure 6). Protein/chl a ratio increased upto 24th day at 50C. There was an induction in protein synthesis following downshift in temperature from 25 to 50C (Figure 7).

The growth and survival of cyanobacteria inhabiting Antarctic environments are less understood. N. commune grew within a range of 10-300C, suggesting eurythermal (broad tolerance range) nature of the cyanobacterium.

Reduced growth at low temperature (e.g. ≤ 5 0C) and responsiveness to the temperature changes suggest that in Antarctica cyanobacteria could accumulate biomass during the brief periods of summer.

The increase in carotenoids/chl a ratio at low temperature could help the cyanobacterium from protection against photooxidation, resulting at low temperature (Young 1991; Chalifour and Juneau 2011). The consistent decrease in phycobilliproteins/chl a ratio with increasing temperature suggests that all pigment ratios (including carotenoids/chl a) are controlled primarily by cellular chl a content (biosynthesis).

The algal communities in the polar environments are exposed to continuous high irradiance during the summer.

This, in combination with the low temperature, it increases the chances of photoinhibition (Bascuñán-Godoy et al.

2012). It was reported that carbon fixation limits growth and photosynthesis at low temperature, and algae tend to direct resources away from the synthesis of light-harvesting components at low temperature (Alves et al. 2002)

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

ACKNOWLEDGEMENTS We thank the Head, Department of Botany, Banaras Hindu University, Varanasi, UP, India for providing necessary facilities. This manuscript is dedicated to late Prof. S. N. Tripathi.

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Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. 2002. Low temperature extremophiles and their applications. Curr Op Biotechnol 13: 253-261.

Chalifour A, Juneau P. 2011. Temperature-dependent sensitivity of growth and photosynthesis of Scenedesmus obliquus, Navicula pelliculosa and two strains of Microcystis aeruginosa to the herbicide atrazine. Aqua Toxicol 103: 9–17.

Chattopadhyay MK. 2006. Mechanism of bacterial adaptation to low temperature. J. Biosci. 31(1): 157–165.

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Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the folin-phenol reagent. J Biol Chem 193: 265275.

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Myers J, Kratz WA. 1955. Relationship between pigment content and photosynthetic characteristics in blue-green algae. J Gen Physiol 39: 11-21.

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Nadeau TL, Mibrandt EC, Castenholz RW. 2001. Evolutionary relationships of cultivated Antarctic Oscillatiorians (cyanobacteria). J Phycol 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.

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

Tandeau de Marsac N, Houmard J. 1993. Adaptation of cyanobacteria to environmental stimuli: new steps towards molecular mechanisms.

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

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A-1

Authors Index Ade R Al-Hammady MAM Ammar MSA Arkhipova AV Aryana IGPM Balbaa LK Bekheta MA Bonde S Budianto A Dar M Deshmukh S Dhuldhaj UP Dhuldhaj UP El-Moursi A Farid F Gade A Gaikwad S Gamal El-Din K Ghodile NG Hajizadeh G Hedayati A Hushare VJ Irnidayanti Y Isyadinyati NF Kavosi MR Khalid KA Kitova AE

105 62 62 97 113 11 118 45 113 86 105,109 109 134 11, 118 76 45, 86, 109 45 118 81 27, 76 6 81 1 57 27, 76 36, 124 97

Kon K Kratasyuk VA Laily AN Malpani MO Obuid-Allah AH Rai M Rajput PR Rathod D Reshetilov AN Sable N Santoso BB Sedayu A Shadi A Shekhawat P Sigit DV Singh S Solanki P Sugiyarto Sulistyarsi A Supriyadi Suranto Talaat IM Tarkhani R Tripathi R Varma A Yaherwandi Yashpal M

50 97 16 81 62 45, 50, 86, 97, 109 81 86 97 45 113 57 6 101 57 134 101 16 32 32 16, 32 11, 118 6 134 86 22 109

A-2

Subject Index 3-alkoylchromanone 3-alkoylchromone 3-aroylflavones acetic acid Acropora humilis Ag-NPs antioxidant capacity antioxidant polyphenols Arceuthobium oxycedri aromatic biogeography Biological fertilizers biosensor Brassicaceae Carica pubescens chelated zinc chlorosubstituted 3aroylflavanones combinations community structure corals curcuminoids.

Cyanobacterium cyclodehydration deltamethrin diazinon disinfestation dispersal dwarf mistletoe eco-friendly egg masses epiphyte Escherichia coli essential oils ethanol

fertility fish follicle cells FTIR fungal diversity

81 81 81 33, 41, 65, 83, 97, 98, 99, 100, 101, 105 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 45, 46, 47, 48 16, 17, 18, 19, 21 11, 14, 15 76, 77, 78, 79, 11, 36, 40, 81, 92, 102, 103, 124, 125, 130, 131 57, 59 124, 131 97, 98, 99, 100 22, 23, 24, 25 16, 17, 18, 19, 20, 21, 118, 119, 120, 121, 122 81 50, 51, 52, 54, 55, 126, 130 22 62, 63, 64, 65, 71, 72, 73 11, 12, 13, 14, 15 36, 134, 135 101, 103 6, 7, 8, 9 6, 7, 8, 9 36, 37, 42 22, 57, 58, 60 76, 77, 78, 79, 48, 62, 73, 86, 87, 90, 101, 102, 104, 109, 111, 125 27, 29, 30 76 50, 51, 52, 53, 54, 55, 118 50, 51, 52, 53, 54, 131, 132 12, 17, 32, 33, 65, 81, 82, 82, 83, 97, 98, 99, 100, 102, 103, 118, 119 2, 62, 63, 64, 65, 69, 70, 79, 105, 124, 125, 126 6, 7, 8, 9, 62, 69, 72, 73, 1, 4 45, 46, 47, 48 86

germination rate Gluconobacter growth rate guppy herbicide Hydrilla in vitro integrated management isoxazoles Jatropha curcas Knoevenagel condensation landscape LC50 Leydig cells low-temperature Lymantria dispar mangrove mating disruption mechanical method medicinal medicinal plants micropropagation microwave Moringa oleifera morphological characters mulches multiplication mycoendophyte nanoparticles Nephotettix virescens non-crop vegetation parasitoid Hymenoptera pheromone traps phycobiliproteins phytofabrication phytosynthesis pigments plants

113, 114, 115, 117 97, 98, 100, 134, 135 6, 7, 8, 9 76, 77, 78, 79 45, 46, 47, 48 50, 89, 91, 92, 101, 102, 103, 104, 105, 106, 107 27, 29, 30 81, 82, 83, 84 113, 114, 115, 116, 117 101 22, 23, 24, 25, 129 6, 7, 8, 9 1, 2, 3, 4 16, 18, 134, 135, 136 27, 28, 29, 57, 58, 59, 60, 91 27, 29, 30 27, 29 11, 36, 40, 86, 87, 88, 89, 90, 91, 92, 124, 125, 130 86, 87, 88, 90, 91, 92, 105 105, 107 101, 102, 103, 104 118, 119, 121 16, 17, 18, 19, 21 36, 37, 38, 39 103, 105, 106, 107, 108, 113 86, 87, 89, 90, 91, 92, 94 45, 46, 48, 109, 110, 111 32, 33, 35 22, 23 22, 23, 24, 25 27, 28, 29, 30 134 45, 46 45, 109 38, 134, 135, 136 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

plastic pollution primordial follicle propagules protein banding pattern protein banding.

Red Sea rice room condition secondary metabolites seed oil content seed quality SEM semi-parasitic plant

111, 113, 114, 118, 119, 120, 121, 122, 124, 125, 26, 27, 28, 29, 30, 31, 132 23, 36, 39, 40, 65, 106, 114 6, 22, 36, 39, 62, 63, 64, 69, 72, 73, 79, 125 1, 4 57, 60, 131 16, 17, 20, 34, 35, 121, 16, 17, 20, 32, 34, 35, 121 62, 63, 64, 65, 72, 73 23, 24, 25, 32, 33, 34, 35, 87 107, 113, 114, 115, 116, 117 1, 86, 87, 89, 90, 91, 119 113, 114, 115, 116, 117 113, 116 45, 46, 47, 48 76, 77, 78, 79

soil borne diseases solarization Staphylococcus aureus Stevia rebaudiana stigmasterol Stylophora pistillata sweet marjoram sweetener Tagetes erecta TEM Thymus vulgaris toxicity tungro uterus zearalenone

36, 37, 125 36, 37, 38, 39, 40, 41, 42 50, 51, 52, 53, 54, 55, 118 105, 106, 107 118, 119, 120, 121, 122 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 11, 12, 13, 14, 131 105 109, 110, 111, 120, 121, 131 109, 110 50, 51, 53, 54, 55 6, 7, 9, 54, 87.91, 92, 94, 101, 128 32, 33, 34, 35 1, 2, 3, 4 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, West Java, Indonesia

Ahmad Dwi Setyawan

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

Alka Karwa Jajoo

Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati 444602, 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, Amravati 444602, Maharashtra, India

Bogdan Costea

Faculty of Horticulture and Forestry, University of Agricultural Science and Veterinary Medicine of the Banat Timişoara, Timisoara, Romania

Eddy Nurtjahja

Department of Biology, Faculty of Agriculture, Fisheries and Biology, State University of 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 Medical University, 61022 Pr. Lenina, 4, Kharkiv, Ukraine

Mahendra Rai

Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati 444602, Maharashtra, India

Malcolm R. Clark

National Institute of Water and Atmospheric Research (NIWA), Wellington 6021, New Zealand.

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, Sleman 55281, Yogyakarta, Indonesia

Sugiyarto

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

Suranto

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

Wiryono

Department of Forestry, Faculty of Agriculture, University of Bengkulu. Bengkulu 38371A, 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 reproductive organs of mice YULIA IRNIDAYANTI Toxicity response of Poecilia reticulata Peters 1859 (Cyprinodontiformes: Poeciliidae) to some agricultural pesticides ALIAKBAR HEDAYATI, REZA TARKHANI, AHMAD SHADI

1-5

6-10

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

11-15

Characterization of Carica pubescens in Dieng Plateau, Central Java based on morphological characters, antioxidant capacity, and protein banding pattern AINUN NIKMATI LAILY, SURANTO, SUGIYARTO

16-21

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

22-26

Evaluation of the effectiveness of integrated management and mating disruption in controlling gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) populations GOODARZ 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 virus ANI SULISTYARSI, SURANTO, SUPRIYADI

32-35

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

36-44

Vol. 4, No. 2, Pp. 45-96, July 2012 Phytofabrication of silver nanoparticles by using aquatic plant Hydrilla verticilata NEILESH 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 oils KATERYNA KON, MAHENDRA RAI

50-56

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

57-61

Patterns of fertility in the two Red Sea Corals Stylophora pistillata and Acropora humilis MOHAMMED 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 oxycedri MOHAMMAD REZA KAVOSI, FERIDON FARIDI, GOODARZ HAJIZADEH

76-80

Synthesis, characterization and physiological activity of some novel isoxazoles VINAYSINGH J. HUSHARE, PRITHVIRAJSINGH R. RAJPUT, MANOJKUMAR O. MALPANI, NITIN G. GHODILE Review: Mycoendophytes in medicinal plants: Diversity and bioactivities MAHENDRA RAI, ANIKET GADE, DNYANESHWAR RATHOD, MUDASIR DAR, AJIT VARMA

81-85

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 Gluconobacter cells ANATOLY 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-ones PRABHA SOLANKI, PRACHI SHEKHAWAT

101-104

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

105-108

Tagetes erecta mediated phytosynthesis of silver nanoparticles: an eco friendly approach UMESH P. DHULDHAJ, SHIVAJI D. DESHMUKH, ANIKET K. GADE, MADHU YASHPAL MAHENDRA K. RAI

109-112

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

113-117

Physiological response of Moringa oliefera to stigmasterol and chelated zinc ABDALLA 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 plants KHALID ALI KHALID

124-133

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

134-137

Guidance for Authors Aims and Scope Nusantara Bioscience (Nus Biosci) is an official publication of the Society for Indonesian Biodiversity (SIB). The journal encourages submission of manuscripts dealing with all aspects of biological sciences that emphasize issues germane to biological and nature conservation, including agriculture, animal science, biochemistry and pharmacology, biomedical science, ecology and environmental science, ethnobiology, genetics and evolutionary biology, hydrobiology, microbiology, molecular biology, physiology, and plant science. Manuscripts with relevance to conservation that transcend the particular ecosystem, species, genetic, or situation described will be prioritized for publication.

Article The journal seeks original full-length research papers, short research papers (short communication), reviews, monograph and letters to the editor about material previously published; especially for the research conducted in the Islands of the Southeast Asian reign or Nusantara, but also from around the world.

Acceptance The acceptance of a paper implies that it has been reviewed and recommended by at least two reviewers, one of whom is from the Editorial Advisory Board. Authors will generally be notified of acceptance, rejection, or need for revision within 2 to 3 months of receipt.

Manuscript is rejected if the content is not in line with the journal scope, dishonest, does not meet the requiredquality, written in inappropriate format, has incorrect grammar, or ignores correspondence in three months.

The primary criteria for publication are scientific quality and biological or natural conservation significance. The accepted papers will be published in a chronological order.

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Authors or others are allowed to multiply article as long as not for commercial purposes. For the new invention, authors are suggested to manage its patent before published.

Submission The journal only accepts online submission, through e-mail to the managing editor at unsjournals@gmail.com. The manuscript must be accompanied with a cover letter containing the article title, the first name and last name of all the authors, a paragraph describing the claimed novelty of the findings versus current knowledge, and a list of five suggested international reviewers (title, name, postal address, email address). Reviewers must not be subject to a conflict of interest involving the author(s) or manuscript(s). The editor is not obligated to use any reviewer suggested by the author(s).

Preparing the Manuscript Please make sure before submitting that: The manuscript is proofread several times by the author (s); and is criticized by some colleagues. The language is revised by a professional science editor or a native English speaker. The structure of the manuscript follows the guidelines (sections, references, quality of the figures, etc). Abstract provides a clear view of the content of the paper and attracts potential citers. The number of cited references complies with the limits set by Nus Biosci (around 20 for research papers).

Microsoft Word files are required for all manuscripts. The manuscript should be as short as possible, and no longer than 7000 words (except for review), with the abstract < 300 words. For research paper, the manuscript should be arranged in the following sections and appear in order: Title, Abstract, Key words (arranged from A to Z), Running title (heading), Introduction, Materials and Methods, Results and Discussion, Conclusion, Acknowledgements, and References. All manuscripts must be written in clear and grammatically correct English (U.S.). Scientific language, nomenclature and standard international units should be used. The title page should include: title of the article, full name, institution(s) and address(es) of author(s); the corresponding authors detailed postal and email addresses, and phone and fax numbers.

References Author-year citations are required. In the text give the authors name followed by the year of publication and arrange from oldest to newest and from A to Z. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and

Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should be avoided. Reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., Rifai MA 2007, personal communication; Setyawan AD 2007, unpublished data). In the reference list, the references should be listed in an alphabetical order. Names of journals should be abbreviated. Always use the standard abbreviation of a journal’s name according to the ISSN List of Title Word Abbreviations (www.issn.org/222661-LTWA-online.php). The following examples are for guidance.

Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study 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 tree communities. In: Carson W, Schnitzer S (eds) Tropical forest community ecology. Wiley-Blackwell, New York.

Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007.

Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island. 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, Q u a k e S R , Y o u L . 2 0 0 8 . A s yn t h e t i c E s c h e r i c h i a c o l i p r e d a t o r - p r e y e c o s y s t e m . M o l S ys t B i o l 4 : 1 8 7 . www.molecularsystemsbiology.com Tables should be numbered consecutively and accompanied by a title at the top. Illustrations Do not use figures that duplicate matter in tables.

Figures can be supplied in digital format, or photographs and drawings, which can be ready for reproduction. Label each figure with figure number consecutively.

Uncorrection proofs will be sent to the corresponding author by e-mail as .doc or .docx files for checking and correcting of typographical errors. To avoid delay in publication, proofs should be returned in 7 days.

A charge The cost of each manuscript is IDR 250,000,- plus postal cost or IDR 150,000,- for SIB members. There is free of charge for non Indonesian author(s), but need to pay postal cost for hardcopy.

Reprints Two copies of journal will be supplied to authors; reprint is only available with special request. Additional copies may be purchased by order when sending back the uncorrection proofs by e-mail.

Disclaimer No responsibility is assumed by publisher and co-publishers, nor by the editors for any injury and/or damage to persons or property as a result of any actual or alleged libelous statements, infringement of intellectual property or privacy rights, or products liability, whether resulting from negligence or otherwise, or from any use or operation of any ideas, instructions, procedures, products or methods contained in the material therein.

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

| 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

Determination of ethanol in acetic acid-containing samples by a biosensor based on immobilized Gluconobacter cells ANATOLY 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-ones PRABHA SOLANKI, PRACHI SHEKHAWAT

101-104

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

105-108

Tagetes erecta mediated phytosynthesis of silver nanoparticles: an eco friendly approach UMESH P. DHULDHAJ, SHIVAJI D. DESHMUKH, ANIKET K. GADE, MADHU YASHPAL MAHENDRA RAI

109-112

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

113-117

Physiological response of Moringa oleifera to stigmasterol and chelated zinc ABDALLA 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 plants KHALID ALI KHALID

124-133

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

134-137

Society for Indonesian Biodiversity

Sebelas Maret University Surakarta

Published three times in one year PRINTED IN INDONESIA

ISSN 2087-3948

E-ISSN 2087-3956