Nusantara Bioscience vol. 3, no. 3, November 2011

Mangifera indica photo by Enrique Brana

| Nus Biosci | vol. 3 | no. 3 | pp. 105‐150| November 2011 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

| Nus Biosci | vol. 3 | no. 3 | pp. 105‐150 | November 2011 | | 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, Alfiono, 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), 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 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 105-111 November 2011

Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica)

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DILRUBA ASHRAFUN NAHAR MAJUMDER1,♥, LUTFUL HASSAN2, MOHAMMAD ABDUR RAHIM3, MOHAMMAD AHSANUL KABIR4 Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh; Tel. +8801714261388; Fax. +88029261424; ♥ email: dilrubamajumder@yahoo.com 2 Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh 3 Department of Horticulture, Bangladesh Agricultural University, Mymensingh, Bangladesh 4 Department of Horticulture, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh Manuscript received: 1 November 2011. Revision accepted: 26 November 2011.

Abstract. Majumder DAN, Hassan L, Rahim MA, Kabir MA. 2011. Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica). Nusantara Bioscience 3: 105-111. A simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels has been described here. In the present study, three kinds of plant DNA extraction protocols were studied and the target was to establish the water-saturated ether (WSE) with 1.25 M NaCl method as the most efficient protocol for removing the highly concentrated polysaccharides from genomic DNA of woody fruit crops. This method involves the modified CTAB or SDS procedure employing a purification step to remove polysaccharides using the WSE method. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet became easily dissolved in TE buffer. Using these three methods, DNA was extracted from samples of 60 mango genotypes, including young, mature, old, frosted old and withered old leaves. Compared with the three studied DNA extraction protocols of mango, it was found that the WSE method with NaCl had the highest value of average percentage (85.44%) in DNA content of the mango genotypes. The average yield of DNA ranged from 5.05 µg/µL to11.28 µg/µL. DNA was suitable for PCR and RAPD analyses and long-term storage for further use.

Key words: DNA extraction, fruit crops, polysaccharides, RAPD, water- saturated ether.

Abbreviations: CTAB: hexadecyltrimethylammonium bromide; RAPD: Random Amplified Polymorphic DNA; RFLP: Restriction Fragment Length Polymorphism; SSR: Simple Sequence Repeats; RT: Room temperature; WSE: Water: saturated ether.

Abstrak. Majumder DAN, Hassan L, Rahim MA, Kabir MA. 2011. Pengembangan protokol ekstraksi DNA genom mangga (Mangifera indica) yang efisien. Nusantara Bioscience 3: 105-111. Sebuah metode sederhana dan efisien untuk ekstraksi DNA genom tanaman buah berkayu yang mengandung banyak polisakarida telah dilakukan. Dalam penelitian ini, tiga protokol ekstraksi DNA tumbuhan dipelajari; dan tujuannya adalah menetapkan metoda ether jenuh air (WSE) dengan NaCl 1.25 M sebagai protokol yang paling efisien dalam mengeluarkan polisakarida yang sangat melimpah pada DNA genom tanaman buah berkayu. Penelitian ini mencakup CTAB yang dimodifikasi dan prosedur SDS sebagai langkah pemurnian untuk menghilangkan polisakarida, serta penggunaan metode WSE. Presipitasi dengan isopropanol yang sama volumenya menyebabkan pelet DNA terbentuk. Setelah dicuci dengan etil alkohol 70%, pelet menjadi mudah larut dalam buffer TE. Menggunakan tiga metode di atas, DNA diekstraksi dari sampel 60 genotipe mangga, termasuk daun muda, daun dewasa, daun tua, daun kering-beku dan daun kering. Perbandingan tiga protokol ekstraksi DNA mangga, menunjukkan bahwa metode WSE dengan NaCl menghasilkan nilai persentase rata-rata (85,44%) kandungan DNA genotipe mangga yang tertinggi. Hasil rata-rata DNA berkisar antara 5,05 µg/mL hingga 11,28 µg/mL. DNA cocok untuk analisis PCR dan RAPD dan memungkinkan penyimpanan jangka panjang untuk digunakan lebih lanjut.

Kata kunci: ekstraksi DNA, tanaman buah, polisakarida, RAPD, eter jenuh air.

INTRODUCTION Several tropical or subtropical fruit crops like Mangifera indica, Citrus spp. and others are perennial woody plants. In those crops the polysaccharide contents, even in young tissues are higher than those of field crops.

Isolation of high quality DNA is essential for molecular research. Polysaccharide contamination is a common problem in the DNA extraction of higher plant. DNA samples of higher plants often contain melicera colloidal hyalosome, which is almost insolvable in water or TE

buffer, and inhibits enzyme reactions (Fang et al. 1992; Porebski et al. 1997; Schlink and Reski 2002), and hinder the downstream work in molecular biology research. DNA samples are also unstable for long term storage (Lodi et al.

1994; Sharma et al. 2002). Several plant DNA extraction protocols for removing polysaccharides have been reported (Porebski et al. 1997; Schlink and Reski 2002). Moreover, some woody fruit crops like mango (Mangifera indica L.) citrus (Citrus spp.), litchi (Litchi chinensis S.), custard apple (Annona squasoma L.), guava (Pisidium guajava L.), banana (Musa spp.), pomegranate (Punica granatum L.),

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jujube (Zizypus mauritiana M.), papaya (Carica papaya L.) and pineapple (Ananas comosus L.) also contain high polysaccharide levels, the protocols could only be used on vigorous tissue (Luro et al. 1995; .Porebski et al. 1997) and the quality of DNA isolated was not high enough to use in PCR, RAPD, RFLP and SSR analyses. In the present study, a modified protocol was applied utilizing the watersaturated ether and 1.25-1.3 M NaCl. Residual phenols and most polysaccharides were removed and DNA was precipitated selectively in the presence of high salt (Fang et al. 1992; Moller et al. 1992).

The target of the present study was to establish the water-saturated ether with 1.25-1.3 M NaCl method as an efficient protocol for removing the high concentration polysaccharides from the genomic DNA of woody fruit crops.

MATERIALS AND METHODS Plant materials Sixty mango genotypes including land races, as well as exotic and cultivated varieties were used as the plant materials for genomic DNA extraction. Leaves were collected at different developmental stages (i.e. young, recently matured, old, frosted and withered).

Isolation of genomic DNA Total genomic DNA was isolated from the mango leaves following three different methods: Sodium Dodecyl Sulphate (SDS) method, CTAB (hexadecyltrimethylammonoum bromied) method, and water–saturated ether and 1.25 M NaCl method.

SDS method Reagents: a) Extraction buffer: 50 m M Tris-HCl, 25mM EDTA (Ethylenediaminetetraacetic Acid) and 300 mM NaCl, pH=8.0 and 1% SDS (sodium dodecy1 sulfate) b) Phenol: chloroform: isoamy1 alcohol (P: C: I): 25:24:1, equilibrated to pH near 8.0 c) TE buffer: Tris-HCl 10mM, 1mM EDTA, pH=8.0 d) Sodium acetate (3M), pH=5.2 e) Absolute ethanol (100%) f) Ethanol (70%) Protocol of genomic DNA isolation: Genomic DNA was isolated from fully expanded young, recently matured, old, frosted, and withered leaves following Doyle and Doyle (1990) method with a few modifications. Approximately 300 mg of clean leaf tissue was cut into small pieces and poured into eppendrof tube.

The tissue was grounded with 800 µL extraction buffer, vortexed for 20 seconds and incubated at 65oC for 5 minutes in a hot water bath. The extract was centrifuged for 10 minutes at 14000 rpm to allow precipitation of the cell debris. About 600 µL of upper aqueous phase was transferred to another tube; about 600 µL of phenol: chloroform: isoamy1 alcohol (v: v: v= 25:24:1) was added to it and mixed gently. Then the solution was centrifuged

for 10 minutes at 14000 rpm. The upper aqueous layer was carefully transferred to another eppendrof tube without disturbing the lower portion. For precipitation of DNA, about 800 µL of absolute alcohol (100%) was added to the aqueous solution and centrifuged for 3 minutes at 14000 rpm to form pellet. After discarding the liquid completely, the DNA solution was reprecipitated by adding 400 µL of 70% ethanol with 20 µL 3 M sodium acetate and again pelleted by centrifugaing for 3 minutes at 14000 rpm. Then the liquid was removed completely, the pellet was air dried and resuspended in 50 µL of TE buffer and samples were stored at -20oC for use.

CTAB method Reagents: a) Extraction buffer: 100m M Tris-HCl, 20 mM EDTA (ethylenediaminetetraacetic acid) and 1.4M NaCl, pH=8.0 and 2% CATB (wv-1 hexadecyltrimethylammonium bromide), 2% (vv-1) 2-mercaptoethanol, 1% PVP (polyvinylpyrollidone) equilibrated to pH near 8.0 b) 20% SDS (Sodium Dodecyl Sulphate) c) Chloroform: isoamy1 alcohol (C:I): 24:1(v/v), equilibrated to pH near 8.0 d) TE buffer: Tris-HCl 10mM, 1mM EDTA, pH=8.0 e) Sodium acetate (3M), pH=5.2 f) Absolute ethanol (100%) g) Ethanol (70%) Protocol of genomic DNA isolation: The CTAB method as described by Saghai-Maroof et al. (1984) with few modifications was followed for DNA isolation. Healthy leaves of each genotype were taken and washed with distilled water to avoid any spore of microorganisms and wiped dry with paper towels; approximately 300 mg of leaf tissue was cut into small pieces (as small as possible to facilitate grinding) and grounded using pre –cooled (-20oC) mortar and pastel and poured into a 2 mL eppendrof tube. 670 µL extraction buffer and 50 µL SDS (20%) were added with the grinding tissue for digestion and mixed well. The samples were then vortexed for 20 seconds for proper mixing and incubated at 65oC for 10 minutes in hot a water bath. 100 µL NaCl and 100 µL CTAB were added and mixed well. The samples were again incubated at the same temperature for approximately 10 minutes. 900 µL chloroform (chloroform: isoamy1 alcohol: 24:1, v/v) was added and mixed well by shaking.

Then to allow the precipitation of cell debris the extract was centrifuged for 10 minutes at 14000 rpm with a micro centrifuge. About 600 µL of upper aqueous phase was transferred to another tube, and then about 600 µL of ice cooled isopropanal was added to it and mixed gently. At this stage, DNA became visible as white strands by flicking the tube several times with fingerings. The solution was centrifuged for 10 minutes at 14000 rpm. The supernatant was decanted and pelletes were washed with adding 70% ethanol (200 µL), and centrifuged for 5 minutes at 1400 rpm. Then the liquid was removed completely, the pellet was air dried and re-suspended in 50 µL of TE buffer.

Finally the DNA samples were stored at -20oC.

MAJUMDER et al. – DNA extraction protocol

Water-saturated ether and 1.25M NaCl method Reagents: a) Liquid nitrogen b) Extraction buffer: 100 mM Tris-HCl (pH 8), 1.5 mM NaCl, 50 mM EDTA (pH 8), 0.5% 2-mercaptoethanol, 4 % (w/v) CTAB (added just before use), 1% PVPP (polyvinyl polypyrollidone) 0.5% 2- mercaptoethanol c) Chloroform-isoamyl alcohol (24:1) d) Phenol-chloroform-isoamyl alcohol (25:24:1) e) TE buffer (pH 8): 10 mM Tris-HCl, 1 mM EDTA f) 10 mg/mL RNase A (free of DNase) g) Water-saturated ether h) Ethanol i) 5 M NaCl j) 70% ethanol Protocol of genomic DNA isolation: Total genomic DNA was extracted using the hexadecyltrimethylammonium bromide (CTAB) method as described by Saghai-Maroof et al. (1984) by employing a purification step to remove polysaccharides utilizing watersaturated ether and 1.25 M NaCl (Cheng et al. 2003). Fresh leaves (300mg) were grounded to a fine powder in liquid nitrogen, followed by the addition of 900µL extraction buffer (CTAB 2x 1.4 M NaCl, 20 mM EDTA, 100nM TrisHCl pH 8.0, polyvinylpolypyrrolidone and 0.2% 2mercaptoethanol), which was pre-heated to 650C. The mixture was incubated at 650C for one hour with an intermittent gentle vortexing. The homogenate was cooled to room temperature and 600 µL chloroform: iso-amyl alcohol (24:1) solution was added and mixed well. The mixture was then centrifuged at 10000 rpm for 20 munites at 40C and the supernatant was collected. After that, 20µL of 5 M NaCl (final concentration of 1.25-1.3 M) and 60 µL water-saturated ether were added with the top aqueous solution and mixed well by using gentle inversion and then centrifuged at 10000rpm for 10 minutes at 40C. The top ether layer was discarded and the bottom aqueous layer was poured from the slot into a new eppendrof tube. Equal volume (approximately 150 µL) cold isopropanol (-200C) was added with the DNA solution to precipitate the DNA. The mixture was frozen at-200C for 30 minutes to accentuate the precipitation of DNA. Then it was spun at 8000rpm for 20 minutes at 40C to pellet the DNA and washed with 70% alcohol. After having been washed, dried and treated with RNAse (10µg/ ml), the DNA pellet was dissolved in 50 µL of TE (Tris- HCl 10ml and EDTA 1mM pH8.0) buffer and stored in -200C. Notes (i) With this treatment, polysaccharides were concentrated in the interphase layer while the DNA was still dissolved in the bottom aqueous phase. Most polysaccharides could be removed by discarding the gel-like interphase. (ii) To prevent contamination of the bottom aqueous layer by the interphase, the mass should be handled carefully (iii) Ether is highly flammable and can cause drowsiness.

All manipulations involving ether should be performed in a well-ventilated fume hood.

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(vi) High concentration of NaCl may inhibit enzyme activity; thus, the DNA solution purified by this method should be deposited and washed with 70% ethanol to remove residual salt.

RAPD analysis The DNA was amplified using the RAPD primers kits A, B, C and E (Operon Technologies, Inc., Boulevard CA, UAS), following the protocol of Williams et al. (1990) with a few modifications. The amplification reactions were accomplished using a final volume of 13 µL, containing Tris-HCl 20mM (pH 8.0), KCl 50 mM, MgCl2 1.5 mM, BSA 1 mg, 300 mM dNTP (dATP, dCTP, dGTP and dTTP), 22.5 ng primer, 0.2 µL Taq DNA polymerase and approximately 10ng genomic DNA. A 50 µL mineral oil was added to this volume after placing the samples into the thermocycler plates. DNA Ladder 100 bp was used as the standard DNA. Amplification reactions were allowed to perform in a DNA thermocycler (MJ Research) for 40 cycles after an initial denaturation at 920C for 2 minute. In each cycle denaturation for 1 minute at 940C, annealing for 1minute at 350C and elongation by Taq DNA polymerase at 720C for 2 minutes was performed with a final extension step at 720C for 5 minutes after the 40 cycles. Negative control was used initially to check the fidelity of the PCR reaction. Negative control without template sometimes resulted in nonspecific bands which disappeared after adding the template. For further reactions negative controls were not used. The amplified DNA fragments were separated by electrophoresis in 1.5% agarose gels in 1xTBE (Tris- borate EDTA, pH 8.0) buffer, stained with 90 µL ethidium bromide. EDTA was used for electrophoresis and for preparing gels. Wells were loaded with 13 µL of reaction volume and 2.5 µL of loading buffer (sucrose and bromo-cresol green dye) together. Electrophoresis was conducted approximately 4 hours at 90 volts, and at the end, the gels were visualized and photographed on an ultraviolet light transluminator.

RESULTS AND DISCUSSION Very young leaves were not useful for isolation of DNA as those were burnt due to use of various extraction chemicals or on drying. Similarly, highly matured leaves were not useful either as those were highly fibrous and rich in phenols and polysaccharides. The protocol using recently matured leaves resulted in dull white translucent DNA pellets, which were easily dissolved in TE buffer.

Prakash et al. (2002) found the similar results from isolating genomic DNA of Pisidium guajava L. The purified DNA using the WSE protocol was homogenous and not degraded. It was successfully amplifiable using Taq DNA polymerase.

There were no fragments in the “no template” control while in the “positive control”, the similar pattern of fragments were amplified in every PCR reaction indicating contamination free PCR ingredients and a consistent protocol (Figure 6). The strategy to obtain reproducible fragment profiles of mango DNA involved reactions in which various components of the reaction mixture were varied. Large

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cchanges in cooncentrations (i.e. in orderr of magnitudde) of t template DNA A did affect thhe amplification, too little DNA r resulted in eiither reducedd or no ampllification of small f fragments.

Ass the DNA cooncentration was w increasedd, the n number of fraagments appeaaring on the gels g was increeased, w while too mucch DNA eitherr produced a smear s effect or o did n amplify anny fragments (Figures not ( 2, 3 and a 5).

M Cottony mass of DN NA

Figure 1. Whitte DNA pellet of fresh maturre leaf of Mangifera F indica formed inn isopropanol after a polysacchaarides were rem moved.

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9 10 11 12 13 144 15

Figure 2. Qualiity test of DNA F A samples of 15 Mangifera sppp. on 1 agarose gel. Lane M – λ DNA; 1% D Lane 1-115: Fresh maturre leaf s samples (DNA A extracted by using CTAB B Method). Saamples s serious smear and a DNA conccentrated on thee well and apppeared b bright glow wheen placed underr the UV light.

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1

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3

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10 11 12 13

Figure 3. Qualiity test of DNA F A samples of 13 Mangifera indiica on 1 agarose gel. Lane M – λ DNA; 1% D Lane 1-113: Fresh maturre leaf s samples (DNA extracted by ussing SDS Methhod). Samples serious s smear and DNA A concentratedd on the well and appeared bright g glow when placced under the UV light.

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Figure 4. Quality teest of DNA sam mples of 12 Man ngifera indica on o 1% agarose a gel. Lanne M – λ DNA A; Lane 1-10: Fresh F mature leaaf samp ples; Lane 11: Withered old lleaf; Lane 12: Frosted old leaf (DNA A extracted by using u Water-Satuurated Ether with h NaCl method).

Compared C witth two protoccols (SDS an nd CATB), thhe WSE E method rem moved polysaaccharides effficiently beforre DNA A precipitationn. White DNA A pellets form med (Figure 1) 1 and were quickly soluble in TE buffer. Thee A260/280 ratioos ranged from 1.7 to1.9, and thhe A260/230 rattio was greateer than 2. The absennce of a peaak at 270 nm indicated thaat resid dual phenols were removved. DNA saamples can be b storeed at 40C for 1.5 years. Reesults of the agorase a gel test and PCR or RAPD D analysis indicated that polysaccharide p es had been efficienntly removed and the DNA A quantity haad been n enhanced (Fiigures 2-6).

Two T classic DNA D extractioon methods: CTAB C (SaghaaiMaro oof et al. 19844) and SDS (D Doyle and Dolye 1990) werre ineffficient in removing polysacccharides (Fig gures 2 and 3).

3 Seveeral modifiedd DNA protocols that remove polyysacch haride have recently r been reported (Fan ng et al. 19922; Molller et al. 19992; Luro et aal. 1995; Cru uz et al. 19977; Poreebski et al. 19997). All werre unsuccessfu ul in removinng polysaccharides from f crops oof Mangifera indica, Citruus spp. and other fruuit crops. Isollating high qu uality DNA foor RFL LP analysis froom some matterials, such as a withered annd old frosted f Citruss spp. leaves, was difficult. DNA samplees weree hyaloplasm gel-like (alm most insoluble in TE buffer) (Cheeng et al. 20003); A260/280 raatios were alw ways less thaan 1.5; and a peak of 270 nm corrresponding to o the peak of a comb bination of phenol p and poolysaccharides, was usuallly scanned (Tesnieree and Vayda 1991). When n tested on 1% % ose gel, it wass observed thaat DNA samp ples with severre agaro smeaar and the DNA D concentrrated on the well, w appeareed brigh htly glow when placed undder the UV liight (Figures 2 and 3). Conductinng PCR analysis or enzymee digestion waas difficcult because polysaccharide p es inhibited en nzyme activityy.

DNA A samples showed minimuum number of o polymorphiic band ds with maxximum smearring and faiiled to creatte ampllification withh primer (Figgure 5). Fang g et al. (19922), Poreb bski et al. (19997), Schlink aand Reski, (200 02) and Sharm ma et al. (2002) reported similar rresults from th he materials of o fruit crops. Thereffore, the modiified protocol should be useed for fruit fr crops plannt like Mangifeera spp. and otther tropical annd subtrropical woodyy fruit crops too avoid the co ontamination of o DNA A samples from m high concenttration polysacccharides level.

The T data on thhe DNA contennts of sixty mango m genotypees (Tab ble 1) showedd that on an aaverage, theree had been 700-

MAJUMDER et al. – DNA extraction protocol

90% increase in the DNA contents of the studied materials over the average (1.012 µg/µL) of SDS method. Compared with the three studied DNA extraction protocols of mango, it was found that WSE method with NaCl had the highest value of average percentage (85.44%) in DNA contents of the mango genotypes. In this method, MI09 had the highest amount of DNA content (11.283 µg/µL), which was closely preceded by MI28 (10.450 µg/µL) and MI27 (10.217µg/µL), while the least amount of DNA content was recorded in MI03 (5.05 µg/µL). In case of CTAB method, the average percentage of DNA content of mango was 71.79%. MI04 had the highest amount of DNA content (5.850 µg/µL) followed by MI94 (5.516µg/µL) and MI95 (5.05 µg/µL).

The minimum amount of the DNA contents were recorded in MI88 (2.017µg/µL). On the contrary, SDS showed the average DNA content (1.012 µg/µL) in the studied mango genotypes. In this method, MI61 had the highest DNA content (1.767µg/µL) but MI82 had the lowest DNA content (0.30µg/µL).

The WSE with NaCl method removed polysaccharides efficiently before DNA precipitation. White DNA pellets

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formed (Figure 1) and were quickly soluble in TE buffer.

The DNA samples could be stored at 40C for 1.5 year.

Results of the agarose gel test and PCR or RAPD (Figures 4 and 6) analyses indicated that polysaccharides had been efficiently removed and DNA quality had been enhanced (Figure 4).

Classic CTAB (Saghai-Maroof et al. 1984) and SDS (Doyle and Dolye 1990) protocols, when combined with the NaCl and water-saturated ether treatment, produced satisfactory results. In addition, the concentration of the DNA samples were too low for RAPD analysis. Those problems were resolved in the molecular analyses of Mangifera indica and other fruit crops using the studied modified DNA extraction protocol.

Molecular marker is powerful tool over conventional fruit breeding. Breeders occasionally find interesting mutants under extreme environmental conditions or on some genetically abnormal phenotypes (Cheng et al.

2003).Nonetheless, vigorous tissue and chilling equipment were unavailable, which limited the extraction of DNA according to CTAB and SDS method.

Table 1. Variation of the DNA contents in 60 mango genotypes in three different extraction methods (SDS,CTAB, WSE method)

Genotypes

DNA concentration (µg/µL) DNA concentration (µg/µL) DNA concentration (µg/µL) Genotypes Genotypes SDS CTAB WSE SDS CTAB WSE SDS CTAB WSE

1. MI01

0.933

4.167

8.783

21. MI38

0.717

4.633

5.167

41. MI74

0.783

3.667

5.633

2. MI02

0.733

4.000

5.983

22. MI39

0.850

2.900

5.667

42. MI75

1.433

4.000

6.200

3. MI03

0.667

3.467

5.050

23. MI40

0.450

2.383

5.950

43. MI77

1.300

3.083

6.333

4. MI04

1.40

5.850

8.817

24. MI41

0.750

2.500

5.150

44. MI80

0.917

2.700

5.650

5. MI08

1.00

4.650

5.750

25. MI43

1.067

2.717

5.333

45. MI81

0.567

3.283

6.433

6. MI09

1.133

4.667

11.283

26. MI44

0.833

2.867

5.483

46. MI82

0.300

2.533

5.267

7. MI12

1.533

3.567

5.300

27. MI45

0.550

2.93

5.800

47. MI83

0.817

4.150

8.600

8. MI16

0.817

3.433

8.500

28. MI46

0.583

4.650

5.667

48. MI84

1.417

3.167

7.217

9. MI19

0.717

2.900

7.450

29. MI47

0.583

4.600

7.133

49. MI85

1.033

2.817

6.500

10. MI20

0.850

2.417

5.750

30. MI48

0.717

2.333

6.033

50. MI86

0.833

3.400

6.633

11. MI21

1.300

2.417

5.750

31. MI49

1.033

3.367

5.383

51. MI88

1.300

2.017

4.900

12. MI22

0.450

4.300

6.567

32. MI50

1.267

4.517

6.767

52. MI90

0.467

3.050

5.183

13. MI23

0.550

4.133

9.617

33. MI51

1.067

3.583

6.450

53. MI91

1.050

2.816

6.416

14. MI24

1.300

4.200

10.116

34. MI52

0.800

3.583

5.867

54. MI92

0.883

3.050

6.100

15. MI25

1.517

3.633

9.467

35. MI54

1.200

3.400

8.583

55. MI93

1.400

2.150

5.083

16. MI26

1.583

4.533

9.350

36. MI58

1.567

2.883

6.300

56. MI94

1.517

5.516

9.867

17. MI27

1.717

4.700

10.217

37. MI60

1.367

3.200

6.167

57. MI95

1.350

5.050

9.433

18. MI28

1.417

4.483

10.450

38. MI61

1.767

3.567

8.617

58. MI96

0.417

3.567

4.867

19. MI29

0.933

4.367

8.367

39. MI64

1.150

4.350

7.883

59. MI97

1.183

3.917

9.633

20. MI33

1.200

2.300

5.417

40. MI70

0.917

4.150

8.833

60. MI98

0.750

3.950

4.817

Range

0.30-1.767 2.017-5.850 5.050-11.283

Mean

1.012

3.587 (71.79%)

Note: Data in the parentheses indicate increase percentage of DNA concentration over the average of SDS method

6.951 (85.44%)

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1000 700 500 200 bp

M 1

2

3

4

8

9 12 16 19 20 21 22 23 24 25 26 27 28 29 33 38 3 9 40 41 43 44 45 46 47 48 49 50 51 52 M

Figure 5. Analysis of 34 samples of Mangifera indica, by using RAPDs with OPC-12 primer. M, 20 bp ladder. DNA bands separation were not good (where DNA samples was used, extracted by using CTAB Method).

1000 600 400 200 bp M1 1 2

3 4

8

9 12 16 19 20 21 22 23 24 25 26 27 28 29 33 38 39 40 41 43 44 45 46 47 48 M2

Figure 6. Analysis of 30 samples of Mangifera indica, by using RAPDs with OPC-12 primer. M1 100 bp ladder & M2 λ - DNA. DNA bands were properly separated (where DNA samples was used, extracted by using Water-Saturated Ether with NaCl method).

CONCLUSION

ACKNOWLEDGEMENTS

Using the modified protocol water-saturated ether with NaCl, the DNA was isolated from several tissues including matured, withered and frosted leaves, but the quality of DNA isolated from recently mature leaves was high enough to perform DNA marker analyses (Figure 7). The protocol has been performed in our laboratory since 2007.

In the past 3years more than 600 DNA samples have been extracted from different developmental stages of mango leaves. Recently, good quality DNA samples were obtained from old leaves of other tropical and sub-tropical fruit crops. Results also proved the reproducibility, reliability and practicality of this customized protocol.

This research work was financially supported by the Prime-Minister’s Advanced Studies and Research Scholarships from the Prime-Minister Office, Government of the People’s Republic of Bangladesh.

REFERENCES Cheng J, Guo WW, Deng XX. 2003. Molecular charcterization of cytoplasmic and nuclear genomes in phenotypicaly abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids. Plant Cell Rep 21: 445-451.

Cruz MDL, Ramirez F, Hernandez H. 1997. DNA Isolation and amplification from Cacti. Plant Mol Biol Rep 15: 319-325.

MAJUMDER et al. – DNA extraction protocol Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissues.

Focus 12: 13-15.

Fang G, Bammar S, Grumnet R. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biofeedback 13: 52-54.

Lodhi MA, Ye GN, Weeden NF, Reisch BI. 1994. Simple and efficient method for DNA extractions from grape vine cultivars and Vitis species. Plant Mol Biol Rep 12: 6-13.

Luro FM, Lorieux JM, Laigret Bove, Ollitrault P. 1995. Genetic mapping of an integenric Citus hybrid using molecular markers. Fruit 49: 404408.

Moller EM, Bahnweg G, Sandermann H, Geiger HH. 1992. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucl Acids Res 22: 6115-6116.

Porebski S, Bailey LG, Baum BR. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and ployphenol components. Plant Mol Biol Rep 15: 8-15.

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Prakash DP, Narayanaswamy P, Sondur SN. 2002. Analysis of molecular diversity in guava using RAPD markers. J Hort, Sci Biotech 77 (3): 287-293.

Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW. 1984.

Ribosomal DNA sepacer-length polymorphism in barley: Mendelian inheritance, chromosalmal localtionk, and population dynamic. Proc Natl Acad Sci USA 81: 8014-8019.

Schlink K, Reski R. 2002. Preparing high-quality DNA from Moss (Physcomitrella patens). Plant Mol Biol Rep 20: 423a-423f.

Sharma AD, Gill PK, Singh P. 2002. DNA isolation from dry and fresh samples of polysaccharide-rich plants. Plant Mol Biol Rep 20: 415a415f.

Tesniere C, Vayda ME. 1991. Method of the isolation of high quality RNA from grape berry tissues without contaminating tannins or carbohydrates. Plant Mol Biol Rep 9: 242-251.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 112-117 November 2011

Blood bacterial wilt disease of banana: the distribution of pathogen in infected plant, symptoms, and potentiality of diseased tissues as source of infective inoculums HADIWIYONO♥ Faculty of Agriculture, Sebelas Maret University. Jl. Ir. Sutami 36a, Surakarta 57126, Central Java, Indonesia. Tel. +62- 271-646994 Fax. +62-271646655., ♥email: hadi_hpt@yahoo.com Manuscript received: ………….. Revision accepted: ………………….

ABSTRACT Abstract. Hadiwiyono. 2011. Blood bacterial wilt disease of banana: the distribution of pathogen in infected plant, symptoms, and potentiality of diseased tissues as source of infective inoculums. Nusantara Bioscience 3: 112-117. Bacterial wilt caused by blood disease bacterium (BDB) is the most important disease of banana in Indonesia. The disease was difficult to control due to by poorly understood of ecology and epidemiology of the disease. This paper reports the distribution of pathogen infected plant, symptoms, and potentiality of diseased tissues as source of inoculums. For studying the distribution of BDB in diseased banana, a number of 14 points of plant organ tissue was sampled and the pathogen was detected by PCR using a couple of specific primer for BDB, 121F and 121R. In addition to the detection of BDB using PCR, both external and internal symptoms were observed. All the points of detection were also used as source of inoculums to search the potentiality of the tissues as source of infective inoculums. The results showed that BDB was distributed systemically in infected banana. The pathogen infection caused systemic symptom and all part of infected banana were potential as source of infective inoculums.

Key words: blood disease bacterium, banana, distribution, inoculums, PCR.

Abstrak. Hadiwiyino. 2011. Penyakit layu bakteri darah pada pisang: distribusi patogen pada tanaman yang terinfeksi, gejala, dan potensi jaringan yang sakit sebagai sumber inokulum infektif. Nusantara Bioscience 3: 112-117. Layu bakteri yang disebabkan oleh penyakit darah (BDB) adalah penyakit paling penting yang menyerang tanaman pisang di Indonesia. Penyakit ini sulit dikontrol karena ekologi dan epidemiologinya kurang dipahami. Penelitian ini melaporkan distribusi patogen pada tanaman yang terinfeksi, gejala, dan potensi jaringan yang sakit sebagai sumber inokulum. Untuk mempelajari distribusi BDB pada pisang yang sakit, sejumlah 14 titik jaringan dari berbagai organ tanaman dicuplik dan patogen dideteksi dengan PCR menggunakan sepasang primer spesifik untuk BDB, yaitu 121F dan 121R. Selain deteksi BDB menggunakan PCR, baik gejala eksternal maupun internal diamati. Semua titik deteksi juga digunakan sebagai sumber inokulum untuk mencari potensi jaringan sebagai sumber inokulum infektif. Hasil penelitian menunjukkan bahwa BDB terdistribusi sistemik pada pisang yang terinfeksi. Infeksi patogen menyebabkan gejala sistemik dan semua bagian pisang yang terinfeksi berpotensi sebagai sumber inokulum infektif.

Kata kunci: penyakit darah bakteri, pisang, distribusi, inokulum, PCR.

INTRODUCTION Banana and plantain (Musa spp.), hereafter referred to as bananas are important horticultural commodities. In the latest years, export growth of banana fruits from Indonesia is less conducive. The significant growth was occurred during 1984-1994 with growth rate in the volume and the value 57.7% and 46.7% (Setiajie 1997). After the years however, the production tend to stagnant or decline. In the period of 2001-2005 Indonesian banana production was 4.30, 4.38, 4.21, 4.20, and 4,28 million tons respectively (General Directorate of Horticulture 2006). It was seem that blood bacterial wilt disease caused by blood disease bacterium (BDB) have involved in the case of low production of bananas (Supriadi 2005).

The national loss of banana production due to blood bacterial wilt disease was estimated around 36% in 1991 (Muharam and Subijanto 1991). The damage of banana mats was extremely serious in certain districts in where ABB genomic group were planted such as Bondowoso and Lombok, the disease incidence could reach over 80 % (Mulyadi and Hernusa 2002; Supeno 2002; Supriadi 2005).

Now, the pathogen has been distributed in 90 % of provinces in Indonesia with various disease incidences from 10 thousands to millions of banana clusters (Subandiyah et al. 2006).

Blood bacterial wilt disease is remain difficult to control due to poor fundamental knowledge about the ecology and epidemiology of the disease. How long does the pathogen survive in soil? Does the pathogen associate

HADIWIYONO – Blood bacterial wilt disease of banana

with root systems of non-host plants? How widespread is the problem in the naturally occurring of Helliconia and Musa spp? It is obvious that in-depth studies on the ecology and epidemiology of blood disease bacterium is urgently required (Fegan 2005). This paper reports the distribution of pathogen, symptoms, and potentiality of diseased tissues as source of inoculums.

MATERIALS AND METHODS Sampling method Plant materials were used in this study determined with purposive sampling method using the criteria: from endemic area of BDB, early symptom of BDB, generative stage, no symptom caused by other diseases or pests. A number of 14 points of plant organ tissue was sampled from infected plants. The main parts of infected plants that were detected for the present of BDB cell were flower, brack, fruit pulp, fruit shelter, fruit stalk, bunch peduncle, middle peduncle, basal peduncle, leaf lamina, midrib, petiole, pseudodstem, corm, and root.

BDB-DNA extraction Bacterial cells of BDB were gained through the following technique. Five thin pieces of the tissue approx.

0.2x0.5x1.5 cm3 obtained from particular tissue point of diseased bananas were immersed in 5 ml sterile water in test tube and left over night for oozing. One ml of bacterial ooze was transferred into Eppendorf tube and several samples were centrifuged using microcentrifuge at 13000 rpm for 10 minutes. The supernatants were discarded and the pellets were re-suspended each with 1 ml sterile water for washing potential inhibitors of the PCR. The Supernatants were discarded again and the left pellets were used for DNA extraction. The extraction was done using “MicroLYSIS PLUS” Kit, Microzone TM. The DNA extraction Kit was containing Taq-polymerase, Anti-tagpolymerase, 2x reaction buffer (6 mM MgCl2), 400μM dNTPs, stabilizer, and blue loading dye.

Each of the clean pellets was added with 20 μl solution of Microlysis Plus. The extraction was run in Automatic Thermocycler Machine (Bio RadTM) with the program as following. Seven steps of heating were programmed for the extraction, that were step 1: 65 oC for 15 minutes, step 2: 96 oC for 2 minutes, step 3: 65 oC for 4 minutes, step 4: 96 o C for 1 minutes, step 5: 65 oC for 1 minutes, step 6: 96 oC for 30 seconds, and step 7: 20 oC for hold. After cycling, the DNA mixture was stored at -20 oC before using as a template of PCR. Before using, the DNA was centrifuged 10000 rpm for 3 minutes and clean supernatant was used as PCR template.

BDB Detection The existence of BDB in the tissue points was employed through DNA finger printing of PCR (Polymerase Chain Reaction)-based method. PCR was done using ”Mega Mix Royal” Kit, MicrozoneTM(Appendix 2) added 0.1% BSA in PCR mix. A couple of BDB specific primers 121F and 121R was used in the DNA amplification

113

(Hadiwiyono 2010).

The PCR amplification program of DNA was conducted using Automatic Thermocycler Machine (BioRadTM). Thermal cycle of PCR program was arranged as described by Fegan (Unpublished) one cycle of initial denaturizing at 96 oC for 5 minutes, followed by 30 cycles of 94 oC for 15 seconds, 59 oC for 30 seconds, and 72 oC for 30 seconds, ended with one cycle of 72 oC for 10 minutes, then hold at 11 oC. Amplified DNA fragments were visualized by electrophoresis using agarose gel 2 % (weigh/volume) in 0.5XTBE buffer for 30 minutes at 100 volt current. A volume of 1 μg/ml ethidium bromide was added in the melted agarose gel to stain the DNA, subsequently, the gel was poured in a mold to form gel wells by cooling in room temperature for ± 20 minutes.

The agarose gel was removed to be soaked in TBE running buffer in the electrophoresis tank. The PCR products at 5 μL volume was loaded into the well on the gel. The DNA fragments were visualized under UV Tranilluminator and documented by taking the photograph.

BDB Detection using plantlets-indicator A volume of one ml of washed bacterial ooze collected as described above was injected in a plantlet, Kepok Kuning having been acclimated for 3 months. If the plantlets were showing wilt symptom of blood disease, the bacterial ooze samples were considered that the tissue positively contain BDB and the tissues were potential as source of infective inoculums.

Redetection of BDB from inoculated plantlets To make sure that wilting on the plantlets were caused

by BDB, re-detection of the pathogen was done using PCR. When the PCR gives a positive result, it means that BDB is distributed in the tissue.

RESULTS AND DISCUSSION Three cultivated varieties of banana have been achieved from different location in this study, that are Kepok Arab, Kepok Kuning, and Raja Bandung sampled from Sragen, Karanganyar, and Klaten respectively (Figure 1).

For purposing detection of BDB existence in diseased plant by PCR using primers of 121F and 121R, at least 14 tissue points of individual infected plant for each cultivar were sampled. The detection using PCR showed that BDB could be detected from all of the plant tissue points. These results were supported by the established symptoms on indicator susceptible plantlets and re-detection BDB from the seedlings. It was occurred on all of cultivars, Kepok Arab, Kepok Kuning, and Raja Bandung (Figure 1, 2, 3).

This means that BDB is existent in all of the sample tissue points. Thus, these observations give evidences that BDB is systemically distributed in all parts of infected plants.

For supporting the detection results, the sign (ooze), external and internal symptom on the inoculated seedling were observed. The observations of natural infection got that the bacterial ooze was usually exuded by brack and male flower of inflorescence in the early morning and in

Â

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3 (3): 112-117,, November 2011

wet weather. Yellowing on leaves innitiated from w m leaf m margin frequeently was obbserved on eaarly symptom m and s slow wilting banana. b Someetimes howeveer, the wiltingg was

ves previouslyy. very fast without through yelloowing of leav The leaves showeed wilting andd some of leav ves were felleed hang ging drop of leeaves (Figure 4-A).

A

B

C

Figure 1. Diseaased bananas with F w early sympptom used to sttudy on BDB distribution. d Cvv. Kepok Arab (A) shows big gger canopy thaan K Kepok Kuning, Cv. Kepok Kuning K (B) show ws smaller cannopy than Kepo ok Arab, Cv. Raja R Bandung ((C) shows smaaller canopy thaan K Kepok Arab andd lighter green of o leaves than those t Kepok Arrab and Kepok Kuning K (C).

1

2

3

4

5

6

7

8

9

10

11

12

13

14 C+ C- M

A 300 bp

B 300 bp

C 300 bp

Figure 1. Amplified DNA of BDB generatedd by PCR usingg primers of 12 F 21F and 121R from 14 individdual tissue poin nts of cv. Kepook A Arab, Kepok Kuning, K and Raaja Bandung. Male M flower (1), Brack (2), Fruit F pulp (3), Fruit shelter/coat (4), Fruit stalk s (5), Buncch p peduncle (6), Middle M pedunclee(7), Basal peduuncle (8), Leaf lamina l (9), Mid drib (10), Petiolle(11), Pseudosstem(12), Corm m (13), Root (144), p positive control-pure culture off BDB (C+), neegative control-healthy pseudo ostem (C), and Ladder L (M).

HADIIWIYONO – Bllood bacterial wilt w disease of banana b

A

B

C

1115

D

Figure 2. Wiltinng symptom onn seedlings of cv.

F c Kepok Kuniing inoculated by b washed ooze of tissue poinnts of individuaal diseased plannts o cv. Kepok Arab of A 5 weeks affter inoculation. Male flower (A), ( Brack (B),, Fruit pulp (C)), Fruit shelter/ccoat (D), Fruit stalk (E), Buncch p peduncle (F), middle m pedunclle(G), Basal peeduncle(H), Pseeudostem(I), Leaf L lamina(J), Midrib(K), Peetiole(L), Corm m (M), Root (N N), C Control-positive e (C+).

C- 1

2

3

4

5

6

7

8

9

10

11

12

317 bp

13

14

15 M

300 bp

Figure 3. Ampplified DNA off BDB generateed by PCR usiing primer 121 F 1F and 121R from fr corm of sseedlings of cv. Kepok Kuninng innoculated withh washed ooze from f 14 tissue points of cv. Kepok K Arab on 5 weeks after inoculation, M Male flower (1), Brack (2), Fruuit p pulp (3), Fruit shelter/coat s (4),, Fruit stalk (5), Bunch pedunccle (6), Middle peduncle(7), Basal B peduncle((8), Leaf laminaa(9), Midrib(100), P Petiole(11), Pseeudostem(12), Corm(13), C Roott(14), positive control-pure c cullture of BDB (C C+), negative coontrol (C-), and d Ladder (M).

A

B

C

Figure 4. Specific symptoms on bacterial w F wilt banana cauused by BDB. A. A Vegetative stage s of Kepokk Kuning with hanging drop of o leaves. B. Geneerative stage off Kepok Abu with w dried inflorrescence of flow wer. C. Bunch of Kepok Kunning with dried inflorescence of o f flower extendinng to upper partss or fruits.

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3 (3): 112-117, November 2011

A

A

B

B

E E

F

II

JJ

F

C

C

D

G

H

K

L L

G

K

D

H

Figure 5. Internal/external symptoms of blood disease on organs of infected plant cv. Kepok Kuning, inflorescence with bacterial ooze (A) browning pulp at along section of the fruit and its stalk (B), browning vessel at the sliced fruit shelter (C), browning vessel at the pseudostem (D), browning vessel concentrated at the peduncle (E), browning vessel concentrated at margin of the middle peduncle (F), browning vessel concentrated at margin of the basal peduncle (G) browning vessel at the midrib (I), browning vessel at the petiole (J), browning vessel at the corm (H), yellowing leaf lamina at the margin (K), and browning diseased root (L).

Wilting of inflorescence flower on generative stage of bananas was observed frequently (Figure 4-B). The wilting inflorescence developed to upper parts of bunch including fruits (Figure 4-C). If peduncle was cut in some points from upper to lower part would be observed a gradual browning in vessel tissues which was observed most severe at the upper part. Discoloration vascular tissues represented by brown dots/points were gradually less frequent on the further lower parts of pseudostem or peduncle. Such symptom can be speculated that the infection is started from the inflorescence. Some diseased plants were in contrary, the symptom was with no or light browning at the upper parts and gradual more severe to the lower part with the most severe in the corm. The latest symptom might be

started from the mother plant the growing sucker. Globally, the browning in vessel cells usually can be occurred in the most part of plants, pulp, stalk, fruit shelter, pedundle, middle peduncle and psedustem, basal peduncle, midrib, petiole, corm, and root (Figure 5).

BDB is difficult to isolate from almost point of diseased plant tissues except from the upper peduncle and the bunch particularly from the fruits. From the fruit shelter is the most frequent to be able to isolate BDB on CPG medium whereas from lower tissue points it is very difficult due to the existence of high population of saprophytic that are suppressive the growth of slow growing BDB. Selective medium for BDB has not been developed yet. Therefore, detection of BDB using culturable-dependent approaches

HADIWIYONO – Blood bacterial wilt disease of banana

will find technique difficulties. Molecular based method through culturable-independent should be developed.

Detection of pathogenic bacteria using PCR-based method is used for BDB study. In facts, PCR-based method using BDB sequencing primers of 121F and 121R was effective and sensitive.

Indeed due to the high level of sensitivity, PCR-based detection protocol is an interesting detection tool. This method however detects dead cells, viable but not culturable, and culturable (Louws et al. 1999). For monitoring the risk of disease caused by the abundance of pathogen inoculums, BIO-PCR was devised to circumvent for this problem (Shaad et al. 1995). Samples are first plated on selective media to propagate culturable cells and subject to PCR analysis. Unfortunately, the selective media for BDB has not been available yet. For handling this problem, detection using susceptible plant indicator could be used to circumvent to the problem. In this study, susceptible seedlings cv. Kepok Kuning were used for indicator in the detecting BDB. The results showed that all of sample tissue points were existed by viable or infective cells of BDB, indicated by the establishment of symptom generated by inoculation on plantlets with washed ooze of BDB from diseased plant. These results also indicate that all of plant parts are potential as source of inoculums for disseminating or transmitting of BDB. These works reveal evidences that BDB invade systemically in diseased banana. It suggested that the bacterium is the vascular competence. The bacteria life and do their reproduction in along vascular system of the host plant. Eden-Green (1994) mentioned that BDB infection is systemic and usually spreads throughout the rhizome, affecting the young sucker, which may show wilting and act as source of inoculums. The systemic infections were not just indicated by the existence of BDB in all point of samples of diseased plants but also by visible sign, external and internal symptoms. In facts, sometimes browning vascular system is appeared, especially in advance disease symptom (Figure 4, 5). It can be speculated that systemic symptom of blood disease is caused by the existence of BDB colonizing all of points of infected plant host.

117

CONCLUSION The results showed that BDB was distributed systemically in infected banana. The pathogen infection caused systemic symptom and all part of infected banana were potential as source of infective inoculums.

REFERENCES Eden-Green SJ. 1994. Diversity of Pseudomonas solanacearum and related bacteria in South East Asia: new direction for Moko Disease.

In: Hayward AC, Hartman GL (eds) Bacterial Wilt: the disease and its causative agent, Pseudomonas solanacearum. CAB International, California.

Fegan M. 2005. Bacterial wilt diseases of banana: evolution and ecology.

In: Allen C, Prior P, Hayward AC (eds) Bacterial wilt disease and the Ralstonia solanacearum species complex. APS Press, Minnesota.

General Directorate of Horticulture. 2006. Distribution Pattern of Pests of Plant Fruits. The General Directorate of Horticulture, Indonesian Agricultural Department, Jakarta.

Hadiwiyono. 2010. Blood bacterial wilt disease: the infection and genetic characters. ᇖDissertationᇗ. University of Gadjah Mada, Yogyakarta. ᇖIndonesiaᇗ.

Louws FJ, Rademaker JLW, de Bruijn FJ. 1999. The three Ds of PCRbased genomic analysis of phytobacteria, diversity, detection, disease diagnosis. Ann Rev Phytopathol 37:81-125.

Muharom A, Subijanto. 1991. Status of banana disease in Indonesia. In: banana disease in Asia and Pacific. Proceeding of technical meeting on diseases affecting banana and plantain in Asia and the Pacific, Brisbane, 15th–18th Augustus 1991.

Schaad, NW. 2001. Initial identification of genera. In: Schaad NW, Jones JB, Chun W (eds) Laboratory guide for identification of plant pathogenic bacteria 3rd Ed. APS Press, Minnesota.

Setiajie I. 1997. Bussiness growth of fruits and vegetables through studying of export and import. J Res Develop Agric 19:135-143.

Subandiyah S, Hadiwiyono, Nur E, Wibowo A, Fegan M, Taylor P. 2006) Survival of blood disease bacterium of banana in soil. In: Proceeding of the 11th international conference on plant pathogenic bacteria, Edinburgh, 10-14 July 2006.

Supeno B. 2001. Isolation and characterization of bacterial wilt disease of banana in Lombok. In: Proceedings of 16th Congress and National Seminar of Indonesian Phyitopathological Society. Department of Pests and Diseases, Faculty of Agriculture, Bogor Agricultural Institute (IPB) and the Indonesian Phytopathological Society, Bogor.

Supriadi. 2005. Present status of blood disease in Indonesia. In: Allen C, Prior P, Hayward AC (eds) Bacterial wilt disease and the Ralstonia solanacearum species complex. APS Press, Minnesota.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 118-123 November 2011

Synthesis and study of cholosubstituted 4-aroyl pyrazolines and isoxazolines and their effects on inorganic ions in blood serum in albino rats 1

AMOL D. BHOYAR1,♥, GANESH N. VANKHADE2, PRITHVISIGH R. RAJPUT3 Department of Chemistry, P.R. Patil College of Enggineering and Technology, Kathora Road, Amravati 444607, Maharasthra, India.

Tel. +91 0721 2530342, Fax. +91 0721 230341 ♥email: amolbhoyar@rediffmail.com 2 Department of Zoology, Sant Gadge Baba Amravati University, Amravati 444602, Maharasthra, India 3 Department of Chemistry, Vidybharati Mahavidyalaya, Camp, Amravati 444601, Maharasthra, India Manuscript received: 14 March 2011. Revision accepted: 8 November 2011.

Abstract. Bhoyar AD, Vankhade GN, Rajput PR. 2011. Synthesis and study of cholosubstituted 4-aroyl pyrazolines and isoxazolines and their effects on inorganic ions in blood serum in albino rats. Nusantara Bioscience 3: 118-123. Condensation of 2-substitutied 3,5dichloroacetophenones (2a-b) obtained from the condensation of 2-hydroxy 3,5-dichloro-acetophenone (1) and benzoyl chloride were dissolved in NaOH, on treatment under Baker-Venkatraman transformation in presence of KOH with pyridine gives 1-(2-hydroxy-3,5dichlorophenyl)-3-substituted-1,3-propanediones (3a-b). Then converted into 3-aroyl-6,8-dichloroflavanones (4a-d) by using different aromatic aldehyde in ethanol containing little piperidine. The condensation of (4a-d) and phenylhydrazinehydrochloride, piperidine in DMF gives 3-(2-hydroxy3,5-dichlorophenyl)-4-substitution-1-phenyl-Δ2pyrazolines (5a-d) and condensation of (4a-d) and hydroxylaminehydrochloride gives 3-(2-hydroxy-3,5-dichlorophenyl)-4-aroyl-5-substituted isoxazolines (6a-d). The above compounds are screened for their activities and have been found to exhibit significant effects on inorganic ions in blood serum in albino rats.

Key words: flavanone, isoxazoline, pyrazoline.

Abstrak. Bhoyar AD, Vankhade GN, Rajput PR. 2011. Sintesis dan studi cholo-tersubtitusi 4-aroil pirazolina dan isoxazolina serta efeknya pada ion anorganik dalam serum darah tikus albino. Nusantara Bioscience 3: 118-123. Kondensasi 2-tersubtitusi 3,5-dikloroasetofenon (2a-b) yang diperoleh dari kondensasi 2-hidroksi 3,5-dikloro-asetofenon (1) dan benzoil klorida yang dilarutkan dalam NaOH, pada perlakuan berdasarkan transformasi Baker-Venkatraman dengan keberadaan KOH dengan piridin menghasilkan 1-(2-hidroksi-3,5diklorofenil)-3-tersubstitusi-1,3-propanedion (3a-b). Kemudian diubah menjadi 3-aroil-6,8-dikloroflavanone (4a-d) dengan aldehida aromatik yang berbeda dalam etanol yang mengandung sedikit piperidina. Kondensasi (4a-d) dan fenilhidrazina-hidroklorida, piperidina dalam DMF menghasilkan 3-(2-hidroksi 3,5-diklorofenil)-4-substitusi-1-fenil-Δ2pirazolina (5a-d) dan kondensasi (4a-d) dan hidroksilaminhidroklorida menghasilkan 3-(2-hidroksi-3,5-diklorofenil)-4-aroil-5-tersubstitusi isoxazoline (6a-d). Senyawa-senyawa di atas diuji untuk mengetahui aktivitasnya dan diketahui menunjukkan efek yang signifikan pada ion anorganik dalam serum darah pada tikus albino.

Kata kunci: flavanone, isoxazoline, pirazolina.

INTRODUCTION Pyrazole is a five membered heterocyclic azole containing two nitrogen atoms in 1,2-position and its dihydro derivative is pyrazolines (Stokes and Ridgway 1980). Last five decades, the pyrazolines ring shows spectacular presence as it has fairly accessible and shows diverse properties.

Recently, numbers of derivatives of pyrazolines are reported to have anesthetic properties (Sinha 1939; Mandal et al. 1986). Along with the traditional interest, pyrazoline is a base of number of dyes and drugs. They show bleaching, luminescent and fluorescent (Orlov et al. 1977; Krasovitskii 1994; Archana et al. 2002; Mulwad and Choudhari 2005; Li et al. 2007). properties and are reported to be useful intermediates in the synthesis of pyrazoles. The use in the development of cine-films opened a new area of applicability based on easier oxidation of 1-phenyl-3-aminopyrazolines.

Several pyrazolines and isoxazolines derivatives have been found to be posses considerable activity such as

antimicrobial (Ramlingham et al. 1977), antibacterial (Azarifar and Maseud 2002), 5-α redutase inhibitor (Amr et al. 2006), antiproliferative (Chimichi 2006), central nervous system (Brown and Shavrel 1972) and immuno suppressive stimulant (Lombardino et al. 1972), Antispermatogenic (Raji et al. 2005), hypoglycemic and antidiabetic (Adeneye et al. 2008; Ettarh et al. 2004), Hepatotoxicity, nephrotoxicitym (Shri 2003), they can also help in predictive toxicology (Rahman et al. 2001; Sahni et al. 2001; Hodgson et al. 2004; Paliwal et al. 2009), Hepatoprotective (Itoh et al. 2009), 2-pyrazoline seems to be most among the frequently studied of all pyrazolines and isoxazoline type compound. Numerous chlorinated organic compounds have various bioactivities which render them valuable active ingredient of medicine or plant protecting agents. Taking into consideration the possible beneficial effect of the presence of chlorine atom(s) in an organic compound, it appear expedient to synthesis a series of systematically chlorinated 2-pyrazolines and isoxazolines.

BHOYAR et al. – Chlorosubstituted 4-aroylpyrazolines and isoxazolines.

Toxicology is a very old concern to humans from the time of Stone Age to modern era. Now it is a separate branch of science and has its own importance. Toxicology deals with toxicity by any chemical or compound by intension or accidental exposure to living organisms.

Excess of any compound will be harmful to life and considered under toxicity studies. In the modern era, use of chemicals and compounds that will accumulate or daily exposed to humans, are harmful in many ways. Pesticides are used for welfare of human beings but by the time, they will challenge us by showing their toxicity. They can be directly exposed to us or indirectly through food chain.

Indiscriminate use of pesticides is on increase. India is one of the largest user of agricultural pesticides such as organophosphates, carbamates etc. Pesticides are toxic compounds to all living organisms however effects vary with species to species. But excessive use of these pesticides creates many problems to all of us. These days, synthetic chemical pesticides are in practice because of their active and best results. But their excessive use causes serious damage to ecosystem-terrestrial as well as aquatic and consequently the flora and fauna of surrounding. Nowadays synthetic pyrethroids have become an economically and environment friendly group of insecticides as these possess a low mammalian toxicity, rapid decomposition in soil, leave no residue in biosphere and are stable in sunlight. The persistence and continuous application of these synthetic pyrethroids may create a problem directly or indirectly in the higher tropical level of the ecosystem. Accidental exposure at the work place and their presence in the environment has aroused concern over their possible adverse effects on human health.

MATERIALS AND METHODS 2-hydroxy-3,5-dichloroacetophenone (1) on treating with different aromatic acid in the presence of pyridine and NaOH gives a compound containing aromatic group, The structures are possible for these compounds (2a, 2b). The IR spectrum of these compounds consist of a ester stretching band at 1790 cm-1, thereby suggested that there is reaction between hydroxyl group and benzoyl chloride (2a). However (2b) shows a PMR peak at δ2.60 of Ar-OCH3.

The acetophenones (2a-b) was formulated by the reaction of pyridine in KOH gives 1-(2-hydroxy-3,5dichloro-phenyl)-3-aryl-1,3-propane-dione (3a-b) which on reaction with different aldehyde gives 3-aroyl-flavanones (4a-d). These flavanones on treatment with phenylhydrazinehydrochloride in DMF medium containing small amount of piperidine gives 4-aroyl-3,5-diaryl-1-phenyl-pyrazolines (5a-d) which was confirm by its spectral analysis. In a similar fashion 3-aroyl-flavanone (4a-d) were treated with hydroxylaminehydrochloride in DMF medium containing small amount of piperidine gives 3,5-diaryl-4-aroylisoxazolines (6a-d) which was characterized by spectral analysis.All melting points were determined in open capillary tubes and are uncorrected. I.R. spectra were recorded on a Perkin Elmer Infra Red Spectro photometer 1310 using KBr disc. 1H NMR was recorded in CDCl3 on a DRX 300 spectrometer (Figure 1; Table 1).

119

The reactions were monitored on TLC on Silica gel G and the solvent system used was benzene.

2-Aroyloxyacetophenone (2a-b) 2-hydroxy-3,5-dichloroaceto-phenone(0.04mol.)and benzoyl chloride (0.05mol.) were dissolved in NaOH (10%) 30 mL, (2a), 2-hydroxy-3,5 dichloroaceto-phenone (0.04 mol) and anisic acid (0.05mol) were suspended in dry pyridine (30 mL ) with POCl3 3 mL, (2b). All the above reaction mixture was kept for overnight and then worked up by dilution and acidification with ice cold HCl (50%) to neutralize pyridine. The solid product was filtered washed with water followed by sodium-bicarbonate (10%) washing finally again with water it crystallized from ethanol to obtained 2-Aroyloxyaceto-phenones (2a-b ).

1-(2-hydroxy-3,5-dichlorophenyl)-3-aryl-1,3-propanediones (3a-b) When 2-Aroyloxyacetophenone (2a-b) (0.05 mol) was dissolved in dry pyridine 40 mL .The solution was warmed up to 600C and pulverized KOH (15 g) were added slowly with constant stirring. After 4 hours the reaction mixture was acidified by adding ice cold dil. HCl (1:1) The product thus separated was filtered washed with sodiumbicarbonate solution (10%) and finally again with water. It was then crystallized from ethanol-acetic acid mixture to get 1-(2hydroxy-3,5-dichloro-phenyl)-3-aryl-1,3-ropanedione (3ab) respectively.

3b-IR spectrum recorded in KBr (cm-1) 3030, (v),-OH; 1600, (s), >C=O;1170, (s), >C-O; 790,(s), C-Cl. PMR spectrum recorded in δ CDCl3 3.69,(s), 3H, Ar-O-CH3; 4.56,(s), 2H,-CO-CH2-CO-(Keto); 6.6, (s), 1H,-C=CH-; 6.92-8.08, (m), 6H, Ar-H; 12.75, (s), 1H, Ar-OH; 15.71, (s), 1H, -CHOH=C(enol). TLC: solvent (benzene) height: 2.7 cm, solute height: 2.3 cm; Rf value: 0.85, m.p.1120C, yield 78%.

3-Aroylflavanone (4a-d ) 1-(2-hydroxy-3,5-dichlorophenyl)-3-(4’-methoxyphenyl)1,3-propanedione 3a (0.01 mol) and benzaldehyde, anisaldehyde (0.012 mol) separately was refluxed in ethanol (25 mL) and piperidine (0.5 mL) for 15-20 min. yield 3-arylflavanone (4a-b) resp. 1-(2-hydroxy-3,5dichloro-phenyl)-3-phenyl-1,3-propanedione3b (0.01mol) and benzaldehyde, anisaldehyde (0.012 mol) separately was refluxed in ethanol (25 mL) and piperidine (0.5 mL) for 15-20 min. yield 3-aroylflavanone (4 c-d) resp. 1-(2hydroxy-3,5-dichlorophenyl-3-(2’-hydroxyphenyl)-1,3proponedione. All above reaction after refluxing, cooling the reaction mixture was acidified with dil. HCl (1:1). The product thus separated was filtered washed with sodium bicarbonate solution (10%) and finally again with water. It was then crystallized from ethanol-acetic acid mixture.

4c IR spectrum recorded in KBr (cm-1) 1637, (s), >C=O; 1562, (s), >C=O; 1213,(s), C-O-C; 825, (s), C-Cl PMR spectrum recorded in δ CDCl3 3.89, (s), 3H, ArOCH3; 5.36, (dd), 1 H, CHA-CH; 5.76 (dd), 1H, CH-CHB; 6.7-8.1, (m), 11H,-Ar-H. TLC: solvent (benzene) height: 2.0 cm; solute height: 1.7 cm; Rf value: 0.85, m.p.1780C, yield 72%.

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Cl

R2

Cl

a OH

Cl

OCO

COCH 3

Cl

b

R1

COCH 3

(1)

(2a -b)

C R4

Cl

Cl

d O

OH

R3

Cl

C O

e

R1

O

R2

C

Cl

CH2

O

O

R2

(4a-d )

R1

C

(3a-b)

g f Cl

R2

Cl O

OH

OH

C

C

R1

N

R3

N

R1

Cl

Cl N

R2

O

R3

O

Ph

R4

R4 ( 5a-d )

(6a-d)

Figure 1. Stepwise reaction of compounds from starting material to final product. a. C6H5 COCl, NaOH (10% ), b. Anisic Acid, POCl3, Pyridine, c. Pyridine, KOH, d. Benzaldehyde, Piperidine, ethanol, e. Anisaldehyde, Piperidine, ethanol, f. PhNHNH2.HCl, Piperidine DMF, g. NH2OH.HCl, Piperidine DMF

Table 1. Physical and Analytical characterisation data of newly synthesised compounds Compound 2a 2b 3a 3b 4a 4b 4c 4d 5a 5b 5c 5d 6a 6b 6c 6d

Molecular formula C15H10O3Cl2 C16H12O4Cl2 C15H10O3Cl2 C16H12O4Cl2 C22H14O3Cl2 C23H16O4Cl2 C23H16O4Cl2 C24H18O5Cl2 C28H20O2 N2Cl2 C29H22O3 N2Cl2 C29H22O3 N2Cl2 C30H24O4 N2Cl2 C22H15O3 NCl2 C23H17O4 NCl2 C23H17O4 NCl2 C24H19O5 NCl2

Molecular weight 309 339 309 339 417 447 447 477 473 503 503 533 412 442 442 472

R1 H OCH3 H OCH3 H H OCH3 OCH3 H H OCH3 OCH3 H H OCH3 OCH3

R2 H H H OCH3 H H H H H H H H H H H H

R3 H OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H OCH3

R3 H H H H H H H H H H H H

M.P. 690C 1190C 1220C 1120C 1610C 1780C 1670C 1560C 1690C 1650C 1720C 1700C 1930C 1880C 1950C 1800C

Yield (%) 72 77 82 78 87 72 75 62 85 82 68 75 86 84 71 82

Rf 0.71 0.82 0.76 0.85 0.42 0.85 0.61 0.44 0.70 0.82 0.73 0.44 0.73 0.79 0.88 0.83

Cal. (Found ) C N 58.14 (58.25) 56.41 (56.43) 58.18 (58.25) 56.48 (56.63) 61.17 (61.53) 64.55 (64.63) 64.46 (64.63) 62.99 (63.01) 68.62 (68.99) 5.35 (5.47) 67.24 (67.31) 5.32 (5.41) 67.22 (67.31) 5.32 (5.41) 65.67 (65.81) 5.01 (5.11) 64.01 (64.07) 3.31 (3.39) 62.33 (62.44) 3.07 (3.16) 62.38 (62.44) 3.10 (3.16) 60.96 (61.01) 2.81 (2.96)

BHOYAR et al. – Chlorosubstituted 4-aroylpyrazolines and isoxazolines.

4-Aroyl-Δ2-Pyrazolines (5a-d) When 3-aroylflavanone (4a-d) and phenyl-hydrazinehydrochloride (0.02mol) were refluxed in 20 mL DMF containing a few drops of piperidine for 1.5 hrs separately, after cooling the mixture was diluted with water HCl (1:1).

The product thus separated was filtered washed with sodium bicarbonate solution (10%) and finally again with water. It was then crystallized from ethanol-acetic acid mixture to yield 4-Aroyl-Δ2-pyrazolines (5a-d) respectively.

5c IR spectrum recorded in KBr cm-1 3076, (w,b),-OH; 1598, (s), > C=O; 1502, (s), >C=N;1176, (m), Ar-O-C; 837, (s), C-Cl; PMR spectrum recorded in δ CDCl3 3.89, (s), 3H, Ar-OCH3; 5.27, (dd), 1H, CHA-CH; 5.65, (dd), 1H, CH-CHB; 6.6-8.1, (m), 16 H, Ar-H; 12.08, (s), 1H, Ar-OH. TLC: solvent (benzene) height: 3.1cm; solute height: 2.6 cm; Rf value: 0.83; m.p. 1650C, yield 82%.

121

3,5-diaryl-4-aroylisoxazoline (6a-d) When 3-aroylflavanone (0.01 mol) 6a-d and hydroxylaminehydrochloride (0.02 mol) were refluxed in 20 mL DMF containing few drops of piperidine for 1.5 hrs.

Separately, after cooling the mixture was diluted with water HCl (1:1). The product thus separated was filtered washed with sodium bicarbonate solution (10%) and finally again with water. It was then crystallized from ethanolacetic acid mixture to yield 3,5-diaryl-4-aroylisoxazoline (6a-d) 6c IR spectrum recorded in KBr cm-1 3377, (w,b),-OH; 3012, (s), C-H;1691, (s),>C=O; 1599, (s), >C=N; 1382, (m), Ar-O-C; 812, (s), C-Cl PMR spectrum recorded in δCDCl3 2.35, (s),3H,Ar-OCH3; 5.21, (dd), 1H, CHA-CH; 5.63, (dd), 1H, CH-CHB; 7.26-8.14, (m), 10H, Ar-H; 9.94, (s), 1H, Ar-OH. TLC: solvent (benzene) height: 2.4 cm; solute height: 1.7 cm; Rf value: 0.79, m.p. 1880C, yield 89%

Cl

Cl OCH3

O

OH

C

Cl

O

Cl

O

Cl

CH

C

C

O

COPh

..OCH .. 3

Cl

Cl H H

OH O

COPh

C

C-

OH

N

+

OCH3

CH

+

Cl

OH

O

COPh

Cl

OH OH

H

+

OCH .. 3

CH

C

:NH2OH

Cl

Cl

-

C

C

COPh C

N ..

CH

OH COPh

-H2O Cl

+

OCH3

OH

-

C

C

N

CH

+

OCH3

OH

Cl

Cl OH

O

-H2O

C Cl N (7c)

OH

C Cl

O

OCH3

O

C N

Ph

C CH

OCH3

OH

Figure 2. A possible mechanism with NH2OH for the conversion of 3-aroylflavanone into 3, 5-diaryl-4-aroylisoxazoline

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3 (3): 118-123, November 2011

Table 2. Serum sodium, potasssium, calcium and magnesium ions changes on exposure to cythion and chlorosubstituted heterocycles in albino rats.

Inorganic ions Sodium Potassium Calcium Magnesium

1.36 + 0.17 6.08+ 0.18 6.30+ 0.20 3.5+ 0.17

1.41+ 0.18 6.95+ 0.20 8.7+ 0.29 3.8+ 0.19

Chlorosubstituted heterocycles 5d 6d 1.37+ 0.17 1.36+ 0.17 6.10+ 0.20 6.22+ 0.17 7.38+ 0.20 7.49+ 0.29 3.6+ 0.17 3.72+ 0.18

4

Sodium Potassium Calcium Magnesium

1.36+ 0.17 6.18+ 0.17 6.30+ 0.29 3.5+ 0.17

1.39+ 0.18 7.37+ 0.20 11.2+ 0.34 4.7+ 0.18

1.35+ 0.17 6.38+ 0.17 7.35+ 0.34 3.62+ 0.17

1.36+0.18 6.25+ 0.18 7.42+ 0.21 3.78+ 0.19

6

Sodium Potassium Calcium Magnesium

1.36+ 0.17 6.18+ 0.20 6.30+ 0.20 3.5+ 0.18

1.48+ 0.17 8.27+ 0.17 13.8+ 0.29 4.2+ 0.18

1.39+ 0.18 7.28+ 0.18 7.39+ 0.20 3.58+ 0.19

1.42+ 0.18 7.40+ 0.20 7.35+ 0.20 3.72+ 0.19

8

Sodium Potassium Calcium Magnesium

1.36+ 0.17 6.18+ 0.18 6.30+ 21 3.5+ 0.19

1.55+ 0.18 8.84+ 0.17 15.82+ 0.34 3.9+ 0.19

1.40+ 0.17 7.32+ 0.18 7.42+ 0.20 3.62+ 0.17

1.39+ 0.18 7.25+ 0.18 7.38+ 0.21 3.57+ 0.18

10

Sodium Potassium Calcium Magnesium

1.36+ 0.17 6.18+ 0.18 6.30+ 10.20 3.5+ 0.17

1.58+ 0.17 9.40+ 0.20 16.96+ 0.21 4.1+ 0.17

1.42+ 0.18 7.40+ 0.20 7.39+ 0.29 3.58+ 0.17

1.45+ 0.17 7.36+ 0.17 7.51+ 0.29 3.71+ 0.18

Weeks 2

Control

Induced

Effect on Inorganic Ions in blood serum in Albino rat Albino rats of either sex weighing between 80-120 gms were divided into three groups viz (A, B and C). Animals in each group maintained on specific diet . The animals of group A were fed on stock diet used as control. Animals of group B were given cythion intravenously (40 SD) body weight/day for one week. Animals from group C were given newly synthesized heterocycles.Synthesized drug doses were administered orally and pesticide cythion was injected, 0.2 to 0.3 mL/100 g body weight. Intravenous injections were given in the tail vein using 12.7 mm 24 gauge needle. The animals were restrained in a plastic holder with the tail protruding. Anesthetic ether was used as anesthetic reagents to sacrifice test animals without pain and discomfort. For inducing alteration of liver functions cythion pesticide was selected. The doses were prepared on the basis of lethal toxicity method and injected intravenously by a sterile syringe of about 12.7 mm 24 gauge.

Blood samples were collected from normal as well as insecticide treated animals and left to clot at room temperature for at least 30 minutes then centrifuged at 2000 r.p.m. to remove clot and cell debris. Equal amounts of serum from experimental and control animals were pooled in order to have sufficient material to perform all the analysis.Effects of chlorosubstituted heterocycles on induced (cythion treated) hepatotoxicity with special reference to serum inorganic ions in albino rats are tabulated in Table 2, no. from 2.

In the present study, it is evident from the Table 1 that a large decrease in the level of circulating serum inorganic ions was found in albino rats due to the cythion intoxication.

CONCLUSION When we analyzed the results obtained from newly synthesized chlorosubstituted heterocycles treated animals it was found that the decreasing trend in the levels of circulating serum inorganic ions was prevented and consequently protected the liver from cythion intoxication.

During hepatic concentration of sodium, potassium, calcium magnesium and phosphate get significantly increased. Increased in this concentration may be due to histopathological changes in kidney. Increased potassium concentration may be due to cellular necrosis which has already been reported in many tissues during hepatitis.

Dehydration during hepatics has also been reported. These cations may be present into the circulation only for excretion. Decrease SDH activity in liver and kidney might because reduction in stored energy and activity of sodium pump. The peroxidation of membranes lipid during hepatics indicate the loss of membrane integrity and membrane bound enzyme activity which in turn brought about disturbance in cellular homeostasis. Increase calcium in serum could be due to its release from bones. The level of calcium and phosphate ion in extra cellular fluid rise markedly, instead of falling. Because kidney cannot excrete rapidly enough as phosphorus being reabsorbed from bones. Since cythion cause nephrotoxicity. From table 1 it is evident that the two heterocyclic compounds viz 5d and 6d were effectively helpful in restoring the increased concentration of sodium, potassium, calcium, magnesium and phosphate to normalcy. Thus these drugs may protect liver function from cythion damage.

BHOYAR et al. – Chlorosubstituted 4-aroylpyrazolines and isoxazolines.

ACKNOWLEDGEMENT The authors expresses their sincere thanks to the Principal Vidybharati Mahavidyalaya, Amravati, India and Head Department of Zoology, Sant Gadge Baba Amravati University, Amravati, India for providing necessary laboratory facilities.

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 124-129 November 2011

Selection of parent trees for Rubber (Hevea brasiliensis) breeding based on RAPD analysis FETRINA OKTAVIA♥, MUDJI LASMININGSIH, KUSWANHADI Sembawa Research Centre, Indonesian Rubber Research Institute. Jl. Raya Palembang-Sekayu km 29, Palembang 30001, South Sumatra, Indonesia. Tel. +62-711-7439493; Fax. +62-711-7439282. ♥email: fetrina_oktavia@yahoo.com Manuscript received: 22 June 2011. Revision accepted: 1 December 2011.

Abstract. Oktavia F, Lasminingsih M, Kuswanhadi. 2011. Selection of parent trees for Rubber (Hevea brasiliensis) breeding based on RAPD analysis. Nusantara Bioscience 3: 124-129. The parent trees’ clones usually originate from the previous generation having the potential of high production with better agronomical characters. Although, phenotype characters can determine the genetic variability among accessions, they are highly sensitive to environmental factors, so it is often difficult to identify the difference between closely related clones. The genetic variability or phylogenetic relationships among rubber clones can be analysis using RAPD method, and based on the result, the parent trees can be selected. This research was aimed to analyze the genetic distance among rubber clones using RAPD method. Analysis was conducted on 45 rubber clones with 12 random primers. Pair-wise comparisons of unique and shared polymorphic amplification products were used to generate similarity coefficients. These coefficients were employed to construct a dendogram by using an Unweighted Pair-Group Method with Arithmetical Averages (UPGMA). The amplification of genomic DNA from 45 clones yielded 2408 DNA fragments, ranging in size from 250 bp to 3000 bp. The range of genetic similarity matrix was very wide (59.18%-94.23%). It indicated that most of the clones have a low level of polymorphism. The lowest genetic similarity (59,18%) was found between RRIC 110 and AVROS 352 clones, while the highest (94.23%) was between IRR 41 and IRR 42 clones. Cluster analysis showed that 45 clones of rubber were divided into two groups, the biggest group consisted of 30 clones, while the other one consisted of 15 clones with a genetic similarity value of 0,73.

Key words: rubber, RAPD, hand pollination, hevea breeding, parents trees.

Abstrak. Oktavia F, Lasminingsih M, Kuswanhadi. 2011. Pemilihan pohon induk untuk pemuliaan karet (Hevea brasiliensis) berdasarkan analisis RAPD. Nusantara Bioscience 3: 124-129. Klon-klon yang digunakan sebagai pohon induk biasanya berasal dari generasi sebelumnya yang memiliki potensi produksi tinggi dengan karakter agronomi yang lebih baik. Karakter fenotipe dapat menentukan variabilitas genetik di antara aksesi, namun sangat sensitif terhadap faktor-faktor lingkungan, sehingga sering kali sulit untuk mengidentifikasi perbedaan antar klon. Variabilitas genetik atau hubungan kekerabatan antar klon karet dapat analisis dengan menggunakan metode RAPD, dan berdasarkan hasil analisis tersebut klon-klon tetua dapat dipilih. Penelitian ini bertujuan untuk menganalisis jarak genetik antar klon karet dengan menggunakan metode RAPD. Analisis dilakukan pada 45 klon karet dengan 12 primer acak. Perbandingan pita polimorfik hasil amplifikasi digunakan untuk menghasilkan koefisien kesamaan. Koefisien ini berguna untuk menyusun dendogram dengan menggunakan Unweighted Pair-Group Method with Arithmetical Averages (UPGMA). Amplifikasi DNA genom dari 45 klon menghasilkan 2408 fragmen DNA yang berukuran 250-3000 bp. Kisaran matriks kesamaan genetik cukup luas (59,18%-94,23%). Hal ini menunjukan bahwa sebagian besar klon memiliki tingkat polimorfisme yang rendah. Kesamaan genetik terendah (59,18%) ditemukan antara klon RRIC 110 dan AVROS 352, sedangkan yang tertinggi (94,23%) antara klon IRR 41 dan IRR 42. Analisis pengelompokkan menunjukkan bahwa 45 klon karet terbagi menjadi dua kelompok, kelompok terbesar terdiri dari 30 klon, sedangkan yang lain terdiri dari 15 klon dengan nilai kesamaan genetik 0,73.

Kata kunci: karet, RAPD, persilangan buatan, pemuliaan karet, pohon induk.

INTRODUCTION Rubber tree (Hevea brasiliensis Muell. Arg.) belongs to the family of Euphorbiaceae. It is an important crop producing natural rubber which have been cultivated in South-East Asia. The plant is indigenous to the Amazon basin of South America, and has a high heterozygotic genetic base.

Recently high yielding clones have been produced as a result of selection program conducted by Rubber Research centers.

High yielding clones are generally obtained through longterm breeding programs by crossing between clones

having special characters. The goal of rubber breeding is to obtain superior clones which have a high pruduction of lateks or wood, and are resistant to diseases (IRRI, 2005).

The selected parent clones usually originate from the previous generation having a high production potential and better agronomical characters. Although, phenotype characters are helpful in determining the genetic variability among accessions, they are highly sensitive to environmental factors, so it is often very difficult to identify the difference among closely related clones. The information on genetic variability is required to select the parent in order to avoid the use of closely related clones. That Information can also

OKTAVIA et al. – Parents trees of rubber

describe correctly the level of genetic difference among clones. Crossing of the clones having high genetic distance will increase the possibility of obtaining a heterosis hybrid vigor.

Molecular markers such as isozymes (Chevallier, 1988; Chaidamsari et al. 1993, Seguin et al. 1995; Yeang et al.

1998), restriction fragment length polymorphism (RFLP) (Besse et al. 1994; Luo et al. 1995), and microsatellite (Lekawipat et al. 2003) have already been applied to investigate the polymorphism among rubber tree clones and used in varietal identification. Another technique which has been developed with detailed results is the marker of Random Amplified Polymorphic DNA (RAPD).

According to Williams et al. (1990), RAPD was one of the techniques of DNA analysis based on random amplified DNA sequences in polymerase chain reaction (PCR) by using an arbitrary primer. Among techniques for DNA polymorphism analysis, PCR-based RAPD is a relatively simple and efficient method. Here, only a small quantity of DNA is required to develop DNA fingerprints. Besides, knowledge of the targeted plant genome is not necessary and it can distinguish the closely related genotypes.

RAPD technique has already been applied in research with several aims. The RAPD has been used to determine genetic relationships for several plant species like coffee (Toruan-Mathius et al. 1998) and cocoa (Wilde et al. 1992; Toruan-Mathius et al. 1997). RAPD can also be used to identify markers related to resitance to certain diseases in coffee (Toruan-Mathius et al. 1995; Agwanda et al. 1997) and tea (Sriyadi et al. 2002). In rubber, a number of RAPD markers have been used to identify clones (Nurhaimi-Haris et al. 1998; Venkatachalam et al. 2002; Zewei et al. 2005), to identify markers related to diseases (Toruan-Mathius et al. 2002), to identify markers related to character of dwarf genom-specific (Venkatachalam et al. 2004) and to identify a sequence having partial homology with proline-specific permease gene (Venkatachalam et al. 2006).

The objective of the present research was to use RAPD markers to estimate the genetic distance among rubber clones in germplasm of Sembawa Research Station, Indonesian Rubber Research Institute. The result will be used in parents trees selection for hevea breeding program.

MATERIAL AND METHODS Planting material This trial was done on 45 cultivated clones, which consisted of elite rubber clones in Indonesia. As a source of DNA, young rubber leaves measuring about 3-5 cm long and 1.5-1.7 cm wide were used. All of the 45 accessions have been planted in hand pollination garden of Sembawa Research Station, Indonesia Rubber Research Institute.

DNA extraction and RAPD analysis DNA extractions were performed according to the procedure described by Orozco-Castillo et al. (1994) which was modified, specifically by the addition of polivinylpolipyrolidon (PVPP), in each sample at the time of grinding in liquid nitrogen to fine powder using pestle and

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mortar. The powdered was transferred to Eppendorf tube using spatula and 5 mL of DNA extraction buffer (1.4 M NaCl, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 30 mM ßmercaptoetanol) was added immediately. The mixture was homogenized by gentle shaking, and incubated at 65oC for 30 minutes. An equal volume of chloroformisoamylalcohol (24:1) was added, and then spined at 11.000 rpm for 3 minutes. The supernatant was transferred to a new Eppendorf tube. To precipitate DNA, an equal volume of isopropanol was added and the mixture was refrigerated 4oC for at least 30 minutes. The DNA was pelleted by centrifugation at 11.000 rpm for 10 minutes.

The pellet was then washed with ice cold (maaf saya kurang faham istilah ice cold) of 70% (v/v) ethanol and dried. Finally, the DNA pellet was dissolved in 1 mL TE (10 mM Tris-HCl pH 8,0; 1 mM EDTA) and stored at20oC, untill it was used as DNA template in PCR. The Quality of DNA was confirmed by agarose gel electrophoresis (0.8% agarose) with ethidium bromide in TAE buffer (40 mM Tris-acetate pH 8.1, 1 mM EDTA).

The samples were loaded into agarose gel with 0.25% bromophenol blue, 0.25% Xylene cyanol FF and 30% glycerol in water, as loading buffer. The DNA purity was determined by using a spectrophotometer based on the ratio of optical density (OD) value between 260 nm and 280 nm wave length. DNA concentration was determined, based on the value of OD at 260 nm (1 OD unit = 50 µg/mL DNA) (Sambrook et al. 1989).

In PCR analysis, arbitrary primers selection was based on its capability to produce different DNA fragments in various clones, in order to obtain polymorphic bands. Each primer consist of 10 base and contains 60-70% G and C base (Table 1). The Primer used was 20 kinds of Kit-N primers produced by Operon technologies (Alameda, USA), which had been selected randomly.

Table 1. RAPD primer nucleotide sequence Primer OPN-01 OPN-02 OPN-03 OPN-04 OPN-05 OPN-06 OPN-07 OPN-08 OPN-09 OPN-10

Primer sequences (5’ Æ 3’)

Primer

5’-CTCACGTTGG-3’ OPN-11 5’-ACCAGGGGCA-3’ OPN-12 5’-GGTACTCCCC-3’ OPN-13 5’-GACCGACCCA-3’ OPN-14 5’-ACTGAACGCC-3’ OPN-15 5’-GAGACGCACA-3’ OPN-16 5’-CAGCCCAGAG-3’ OPN-17 5’-ACCTCAGCTC-3’ OPN-18 5’-TGCCGGCTTG-3’ OPN-19 5’-ACAACTGGGG-3’ OPN-20

Primer sequences (5’ Æ 3’) 5’-TCGCCGCAAA-3’ 5’-CACAGACACC-3’ 5’-AGCGTCACTC-3’ 5’-TCGTGCGGGT-3’ 5’-CAGCGACTGT-3’ 5’-AAGCGACCTG-3’ 5’-CATTGGGGAG-3’ 5’-GGTGAGGTCA-3’ 5’-GTCCGTACTG-3’ 5’-GGTGCTCCGT-3’

DNA amplification was carried out following the method of William et al. (1990). The PCR reaction were in 25 μL volume reaction mixture containing 1.0 μL DNA template, 1.5 μL MgCl2 25 mM, 2.5 μL PCR 5x buffer, 0.5 μL dNTP mix, 0.2 μL tag DNA polymerase (5 unite), 1.0 μL primer 10 mM and demineralized water was added until the volume was 25 μL. PCR amplification by using Biometra machine was programmed for 45 cycles of

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denaturation for 2 minutes at 940C, annnealing for 1 minute at 530C, and extention for 2 minute at 720C. The last cycle was followed by incubation for 4 minute at 72oC. DNA amplification products were separated by 1.4% agarose gel in 1x TAE buffer (0.04 M Tris-acetic in 1 mM EDTA) and added 5 µL loading dye. DNA migration was conducted for 1 hour and 15 minutes at 50 volt. The gel was then stained in 0,5 µg/mL ethidium bromide, and washed with aquadest. DNA fragments were visualized by UV transiluminator and a picture of DNA fragment in the gel was taken by polaroid camera. Molecular weight of DNA were determined by the migration of DNA marker (1 Kb DNA ladder).

Data analysis The DNA fragments used in RAPD analysis were the one which could be clearly identified by determining its presence (1) or absence (0). Based on the data of DNA fragment, genetic distances were estimated by a dendogram which was constructed following the UPGMA method, and the similarity matrix among clones was analyzed by using NTSYSpc program (Rohlf 1993).

RESULT AND DISCUSSION RAPD analysis Forty primers have been used to amplify the DNA of GT 1 clone to select the best primer. The amplification could obtain 181 fragments with the range of 0-8 fragments per primer. Primers were selected according to the number of DNA fragments obtained in PCR. From 40 kinds of primers used, twelve primers (OPN-05, OPN-06, OPN-08, OPN-10, OPN-11, OPN-12 OPN-17, OPH-01, OPH-03, OPH-05, OPH-18 and OPH-19) produced the highest number of DNA fragment. These primers were then used to amplify 45 rubber clones.

DNA amplification of 45 rubber clones by 12 primers produced 2408 DNA fragments which formed 95 DNA

fragment patterns with the size of DNA fragment of 2503000 bp. The size of DNA fragments amplified depend on the DNA region surrounded by two primers (McPherson et al. 1992). In general, the fragment pattern obtained on all 45 clones rubber tree was still relatively the same (monomorphic). When a similar pattern was obtained from different clones by using a primer, it showed that primer could not be used to track genetic difference among those analyzed clones.

Among the 78 DNA fragment patterns obtained, 2 specific DNA fragments were found on certain clone i.e. fragment no. 1 which was found only on GT 1 and fragment no. 11 on PN 177 which were amplified by OPN08 (Figure 1). Beside many specific DNA fragments found only on certain clones, many fragments with certain size were also only found in a small group of clones, for example the fragment with the size of 850 bp which was amplified by OPN-10 primer could be observed on IRR 39 and IRR 44 clone only. These fragments were assumed to be related with a specific genetic character that was inherited by their parents or a specific character that is formed genetically in an individual. It could be shown in IRR 39 and IRR 44 clone which had the same characteristic in one of their parents, that is LCB 1320. We expect those specific DNA fragments can be furthermore analyzed, cloned and sequenced. It may be used as a specific marker like SCAR or CAPS. To know the relationship between a specific DNA fragments with a certain character, a more detailed molecular study is needed. This study can be carried out by taking into account the agronomical characters found in plant groups which have the same specific DNA fragment, and then doing DNA hybridization using these fragments as a probe. Another method can be applied using a more specific molecular technique such as analysis at mRNA level related to the already known agronomical characters of each clone like high production or resistance to a certain disease.

Figure 1. Amplification products generated from 45 clones of rubber by using OPN-08 primer.

Note: 1. IRR 24 8. RRIM 600 15. PN 177 22. PR 300 29. RRIC 101 36.

2. IRR 39 9. Tjir 1 16. RRIM 901 23. PB 260 30. RRIM 712 37.

3. IRR 104 10. PN 138 17. RRIM 911 24. GT1 31. IRR 42 38.

4. IRR 118 11. PB 217 18. PN 680 25. PR 303 32. IRR 41 39.

5. IRR 105 12. IRR 44 19. BPM 109 26. LCB1320 33. TM 5 40.

6. RRIM 2004 13. IRR 100 20. BPM 24 27. RRIC 100 34. TM 8 41.

7. RRIM 2020 14. BPM 107 21. BPM 1 28. RRIC 110 35. IRR 18 42.

H. benthamiana IRR 220 PB 235 IRR 204 RRIC 101 IRR 32 TM 9

43. BPPJ 3 44. BN 1 45. AV 352

OKTAVIA et al. – Parents trees of rubber

11

Table 2. Genetic similarity matrix between 45 clones of rubber based on the propotion of shared fragment IRR 24 IRR 39 IRR 104 IRR 118 IRR 105 RRIM 2004 RRIM 2020 RRIM 600 TJIR 1

1.0000 0.8852 0.8833 0.7477 0.8421 0.8000 0.8376 0.8108 0.7731 0.8644 0.8780 0.8214 0.8475 0.8293 0.8361 0.7680 0.8067 0.7863 0.8000 0.7934 0.7705 0.7826 0.7273 0.7667 0.8235 0.7627 0.6786 0.7478 0.7434 0.7414 0.7368 0.7705 0.7692 0.7788 0.7130 0.7521 0.7080 0.7193 0.6964 0.8000 0.6972 0.7636 0.8142 0.7143

1.0000 0.8448 0.8155 0.8727 0.8679 0.8850 0.8598 0.7652 0.8772 0.8739 0.8519 0.9123 0.9076 0.8644 0.8430 0.8348 0.7788 0.8099 0.8034 0.7966 0.8288 0.7521 0.7931 0.8522 0.7719 0.7222 0.7928 0.7890 0.7143 0.7091 0.7797 0.7788 0.7890 0.7748 0.7788 0.7890 0.7636 0.7222 0.7748 0.7048 0.7170 0.7890 0.7037

1.0000 0.7723 0.8333 0.8269 0.8288 0.7810 0.7434 0.8214 0.8376 0.8491 0.8750 0.8205 0.8276 0.7563 0.8142 0.7387 0.8067 0.8174 0.7759 0.7890 0.7478 0.8246 0.8319 0.8036 0.7547 0.8073 0.8037 0.7636 0.7593 0.8276 0.8108 0.7850 0.7706 0.7568 0.7290 0.7407 0.6981 0.7890 0.6796 0.7115 0.7664 0.6604

1.0000 0.8211 0.8791 0.8367 0.7826 0.7200 0.8081 0.7500 0.7742 0.8081 0.7692 0.7767 0.7170 0.7400 0.7143 0.7170 0.7255 0.7961 0.7708 0.6863 0.7723 0.7600 0.7071 0.6882 0.7500 0.7021 0.6186 0.6526 0.6990 0.7143 0.7447 0.7292 0.6735 0.7234 0.6737 0.6667 0.6250 0.6222 0.6154 0.6596 0.6882

1.0000 0.8980 0.8762 0.8283 0.7850 0.8491 0.8468 0.8600 0.8491 0.8288 0.8364 0.7788 0.7664 0.7810 0.7788 0.8073 0.7818 0.8155 0.7156 0.7963 0.8037 0.7736 0.7200 0.7961 0.7525 0.7115 0.7255 0.7636 0.7619 0.7723 0.7379 0.7429 0.7129 0.7255 0.6800 0.6990 0.6598 0.6735 0.7327 0.6800

1.0000 0.8713 0.8421 0.7767 0.8431 0.7850 0.8333 0.8627 0.8411 0.8491 0.7523 0.7767 0.7723 0.7890 0.8190 0.8113 0.8485 0.7619 0.8269 0.8155 0.7647 0.7500 0.8081 0.7423 0.7400 0.7755 0.7925 0.7921 0.8247 0.8081 0.7525 0.7835 0.7347 0.7083 0.7071 0.6667 0.6809 0.7216 0.7083

1.0000 0.8627 0.7636 0.9174 0.8421 0.8544 0.8807 0.8421 0.8850 0.7931 0.7818 0.7593 0.7586 0.7857 0.8319 0.8491 0.7500 0.8108 0.8364 0.7523 0.7379 0.8113 0.7308 0.6916 0.7238 0.7434 0.7407 0.7885 0.7736 0.7593 0.7500 0.7619 0.7184 0.7170 0.7000 0.7129 0.7500 0.7379

1.0000 0.7692 0.8544 0.7963 0.8247 0.8544 0.8148 0.8224 0.7818 0.7885 0.7647 0.7818 0.7547 0.7477 0.8200 0.7170 0.7429 0.7692 0.7184 0.7629 0.7600 0.7347 0.6733 0.6869 0.7290 0.7255 0.7755 0.7200 0.7255 0.7143 0.7071 0.6804 0.7000 0.6383 0.6947 0.7143 0.7010

1.0000 0.7928 0.7586 0.8000 0.7748 0.7586 0.7826 0.6949 0.7321 0.7636 0.7966 0.7895 0.7652 0.7593 0.7193 0.7611 0.7143 0.7207 0.7048 0.7593 0.7170 0.6789 0.6729 0.7304 0.7818 0.7358 0.7407 0.7273 0.7170 0.7103 0.6857 0.6667 0.6471 0.6602 0.6604 0.6476

1.0000 0.8696 0.8654 0.8545 0.8348 0.8947 0.8205 0.8108 0.8073 0.8205 0.7965 0.8246 0.8598 0.7434 0.8036 0.8108 0.7818 0.7115 0.8037 0.7429 0.6667 0.6981 0.7193 0.7523 0.7619 0.7477 0.7523 0.7429 0.7170 0.6923 0.7477 0.6931 0.6667 0.7238 0.6923

1.0000 0.8624 0.8522 0.8167 0.8067 0.7705 0.7759 0.7719 0.8197 0.7797 0.8235 0.8214 0.7797 0.8034 0.8276 0.8000 0.7156 0.7857 0.7636 0.7434 0.7387 0.8067 0.7544 0.7273 0.7679 0.8070 0.7455 0.7568 0.6972 0.7679 0.6792 0.7290 0.7636 0.6972

1.0000 0.8846 0.8073 0.8333 0.7568 0.7429 0.8155 0.8468 0.8411 0.8148 0.8713 0.7477 0.8113 0.8190 0.8077 0.7347 0.8317 0.7475 0.6863 0.6800 0.7407 0.7767 0.7475 0.7525 0.7184 0.7475 0.7200 0.6531 0.6733 0.6105 0.6667 0.6667 0.6122

1.0000 0.8870 0.8596 0.7863 0.7928 0.7523 0.8034 0.8319 0.8246 0.8224 0.7257 0.8036 0.8649 0.8000 0.7308 0.8037 0.7619 0.7222 0.7170 0.7895 0.7706 0.7810 0.7664 0.7339 0.7619 0.7170 0.6731 0.7290 0.6535 0.6667 0.7238 0.6731

1.0000 0.8403 0.7869 0.7586 0.7193 0.7869 0.7627 0.7731 0.7679 0.7288 0.7863 0.7931 0.7478 0.6972 0.7679 0.7455 0.7257 0.7027 0.8067 0.7544 0.7455 0.7679 0.7719 0.7455 0.7207 0.6606 0.7321 0.7170 0.6729 0.7273 0.6422

1.0000 0.8760 0.8174 0.8319 0.8099 0.8376 0.8475 0.8649 0.7521 0.8448 0.8696 0.8596 0.7778 0.8468 0.8073 0.6964 0.7455 0.7797 0.7965 0.8073 0.7928 0.7434 0.7523 0.7273 0.7037 0.7748 0.7429 0.6792 0.7339 0.7037

1.0000 0.8475 0.7759 0.7742 0.7500 0.7603 0.8070 0.7500 0.7563 0.7627 0.7863 0.7207 0.7719 0.7500 0.6435 0.6726 0.7273 0.7241 0.7500 0.7193 0.7069 0.6964 0.7080 0.6667 0.6842 0.6667 0.6239 0.7143 0.6486

1.0000 0.8182 0.8136 0.7719 0.7826 0.8148 0.7719 0.7965 0.7857 0.7748 0.7810 0.7593 0.7925 0.7156 0.7477 0.7478 0.7818 0.7736 0.7593 0.7273 0.7547 0.7477 0.7238 0.7593 0.6863 0.6796 0.7547 0.6857

1.0000 0.8448 0.8393 0.8142 0.8113 0.7679 0.8108 0.7818 0.8073 0.7184 0.7925 0.7308 0.6729 0.7048 0.7080 0.7407 0.7115 0.6981 0.6667 0.7115 0.6857 0.6602 0.6604 0.6000 0.6337 0.6538 0.6408

1.0000 0.8667 0.8430 0.8421 0.8500 0.8403 0.8305 0.8547 0.7748 0.8246 0.7679 0.7478 0.7434 0.7934 0.8103 0.7321 0.7719 0.7586 0.7500 0.7080 0.6847 0.7193 0.6296 0.6606 0.6607 0.6306

1.0000 0.8376 0.8545 0.7586 0.8348 0.8421 0.8496 0.7477 0.8364 0.7963 0.7207 0.7523 0.7863 0.8036 0.7963 0.7818 0.7500 0.7593 0.7339 0.7103 0.7273 0.6346 0.6857 0.7037 0.6542

1.0000 0.8649 0.8205 0.9138 0.8696 0.8421 0.7778 0.8108 0.7706 0.7500 0.7818 0.8136 0.7965 0.7890 0.8468 0.7965 0.7890 0.7636 0.7407 0.7387 0.6667 0.6792 0.6972 0.7222

1.0000 0.8364 0.8624 0.8704 0.8224 0.7921 0.8462 0.7843 0.7048 0.7573 0.7748 0.8302 0.8431 0.8077 0.7736 0.8039 0.7573 0.7327 0.7308 0.6939 0.7273 0.7451 0.6931

1.0000 0.8348 0.8070 0.7788 0.8037 0.8000 0.7222 0.7207 0.7523 0.7692 0.7679 0.7222 0.7455 0.7321 0.7222 0.6972 0.6729 0.6727 0.6154 0.6286 0.6667 0.6355

PR 303 LCB 1320 RRIC 100 RRIC 110 RRIC 102 RRIM 712 IRR 42 IRR 41 TM 5

1.0000 0.8496 0.8571 0.8491 0.8440 0.8224 0.7636 0.7963 0.8276 0.8288 0.8037 0.8440 0.7748 0.7477 0.7407 0.6981 0.7339 0.6796 0.6538 0.7103 0.6792

1.0000 0.9189 0.8190 0.8704 0.7925 0.7523 0.7850 0.8174 0.8000 0.7925 0.7778 0.7455 0.7736 0.7290 0.7238 0.7778 0.7255 0.6990 0.7547 0.6857

1.0000 0.8077 0.8785 0.8000 0.7407 0.7736 0.8070 0.7890 0.7429 0.7664 0.7339 0.7238 0.6981 0.6731 0.7477 0.6931 0.6471 0.7048 0.6154

TM 8

IRR 18 H.ben

IRR 220 PB 235 IRR 204 RRIC 101 IRR 32 TM 9

BPPJ 3 BN 1

AV 352

1.0000 0.8119 0.8283 0.6863 0.7200 0.7593 0.7767 0.7475 0.7525 0.6990 0.7071 0.6800 0.6735 0.6733 0.6737 0.5833 0.6667 0.5918

1.0000 0.8039 0.7048 0.7184 0.7928 0.7736 0.7451 0.7692 0.7547 0.7843 0.7573 0.7129 0.6923 0.6735 0.6465 0.6863 0.6733

1.0000 0.6990 0.7327 0.8073 0.8077 0.7800 0.8039 0.7500 0.7600 0.7327 0.6869 0.7647 0.7500 0.6598 0.7400 0.6465

1.0000 0.9423 0.8750 0.8411 0.7961 0.8190 0.8224 0.7573 0.7500 0.7451 0.8190 0.7475 0.7400 0.7961 0.7255

1.0000 0.8727 0.8381 0.8515 0.8544 0.8190 0.7723 0.7451 0.7400 0.8155 0.7423 0.7143 0.7723 0.7200

1.0000 0.8673 0.8257 0.8829 0.8850 0.8073 0.8364 0.7963 0.8108 0.7619 0.7736 0.8073 0.7222

1.0000 0.8846 0.8679 0.8333 0.8077 0.8190 0.7961 0.8113 0.8000 0.7525 0.8077 0.7184

1.0000 0.8824 0.8269 0.8000 0.7921 0.7879 0.8039 0.7708 0.7629 0.8200 0.7475

1.0000 0.8679 0.8431 0.8350 0.7921 0.8077 0.7755 0.7475 0.7843 0.7525

1.0000 0.8462 0.9143 0.8350 0.8302 0.7800 0.7921 0.8269 0.7573

1.0000 0.8911 0.8485 0.7843 0.7708 0.7629 0.8000 0.7677

1.0000 0.9000 0.7961 0.7835 0.7959 0.8317 0.8200

1.0000 0.8119 0.7789 0.7917 0.8283 0.7755

1.0000 0.8776 0.8485 0.8824 0.8119

1.0000 0.8172 0.8750 0.8000

1.0000 0.9072 1.0000 0.8333 0.8081 1.0000

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1.0000 0.9365 0.9016 0.8833 0.7664 0.8421 0.8182 0.8718 0.7928 0.7731 0.8814 0.8780 0.8393 0.8644 0.8618 0.8525 0.7680 0.7731 0.7863 0.8000 0.7934 0.8033 0.7826 0.7438 0.8167 0.8739 0.8136 0.7321 0.8000 0.7434 0.7414 0.7368 0.7705 0.7521 0.7434 0.7304 0.7521 0.7080 0.7018 0.6786 0.7826 0.7156 0.7091 0.7788 0.6786

PR 300 PB 260 GT 1

OKTAVIA et al. – Parents trees of rubber

IRR 24 IRR 39 IRR 104 IRR 118 IRR 105 RRIM 2004 RRIM 2020 RRIM 600 TJIR 1 PN 138 PB 217 IRR 44 IRR 100 BPM 107 PN 177 RRIM 901 RRIM 911 PN 680 BPM 109 BPM 24 BPM 1 PR 300 PB 260 GT 1 PR 303 LCB 1320 RRIC 100 RRIC 110 RRIC 102 RRIM 712 IRR 42 IRR 41 TM 5 TM 8 IRR 18 H.benthamiana IRR 220 PB 235 IRR 204 RRIC 101 IRR 32 TM 9 BPPJ 3 BN 1 AV 352

PN 138 PB 217 IRR 44 IRR 100 BPM 107 PN 177 RRIM 901 RRIM 911 PN 680 BPM 109 BPM 24 BPM 1

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IRR24 IRR39 IRR118 IRR44 IRR104 BPM107 PN177 RRIM600 PB217 RRIM901 RRIM2004 RRIM2020 TJIR1 BPM109 IRR100 PB260 BPM24 BPM1 PR300 PR303 LCB1320 RRIC100 RRIC102 RRIM911 PN680 IRR105 GT1 PN138 RRIC110 RRIM712 IRR42 IRR41 TM5 H.benthamiana TM8 IRR18 IRR220 IRR204 PB235 RRIC101 IRR32 TM9 BPPJ3 BN1 AV352 0.73

0.78

0.83

0.89

0.94

Coefficient Figure 2. Dendogram of 45 rubber clones based on the UPGMA method

Genetic relationship The genetic similarity matrix based on UPGMA method (Table 2) indicated that the proportion of the same DNA fragments among clones was quite high, ranging between 59.18% and 94.23%. The lowest genetic similarity (59.18%) was found between RRIC 110 and AVROS 352 clone, while the highest (94.23%) was between IRR 41 and IRR 42 clone. This showed that the genetic variability of clones analyzed by using OPN-05, OPN-06, OPN-08, OPN-10, OPN-11, OPN-12 OPN-17, OPH-01, OPH-03, OPH-05, OPH-18 dan OPH-19 primers was low. It might be caused by the limited number of DNA marker which was used to distinguish it, so that it could not differentiate the analyzed clones yet. Some publications showed that in genetic analysis to know the relationship number genetic among population need a minimum number of 200 different patterns of DNA fragments. If every primer can produce 5-9 different DNA fragments, it means that on polimorfism observation or analysis of genetic relationship among clones can use 22-40 primers to track genetic difference of these clones. While on this research we used 12 primers only and obtained a total of 95 DNA fragments, so that it still obtained a low carefulness level.

Cluster analysis of clones by using 12 primers was shown in dendogram of 45 clones (Figure 2). According to the similarity level of 0.73, 2 groups were separated, a big group consisting of 30 clones and a small one of 15 clones.

These groups could be divided further into many subgroups with different genetic distances. The dendogram showed that many clones which had the same characteristic in one of their parents and came into the same group, as IRR 41 and IRR 42 clone with LCB 1320 and F 351 clone as their parent, have a genetic similarity of 0.94. This could also be observed between IRR 24 and IRR 39 clone that have that same parent of LCB 1320 with genetic similarity 0.93, so as RRIM 2004 and RRIM 2020 clone with the same parent of PB 5/51 clone, come that into the same group with genetic similarity about 0.90. However, not all clones with the same parent come into the same group. This could be observed on PB 260 and PB 5/51 clone was not in the same group with PB 217 and RRIM 901 clone. This case was also found for IRR 24 and IRR 39 clone that came into different groups with their parent LCB 1320 clone.

Nurhaimi-Haris et al. (1998) and Toruan-Mathius et al.

(2002) reported the same condition between RRIC 100 and RRIM 600 clone which had the same clone, PB 86, as one of their parents. The analysis showed that RRIC 100 and RRIM 600 were in different groups. That could also be observed between PPN 2447 and PPN 2444 clone which originated from LCB 1320 illegitim, come into different group (Nurhaimi-Haris et al. 1998).

Some clones had high genetic similarity but really they did not have genealogy relationship such as IRR 104 and BPM 107 clone had genetic similarity of 0,91; 0,915 for

OKTAVIA et al. – Parents trees of rubber

RRIM 600 and PB 217 clone; 0,9 for PR 300 and PR 303 clone1; 0,915 for RRIC 100 and LCB 1320 clone.

Varghese et al. (1997) reported that it could happen because generally the rubber tree was a crossed pollination plant where F1 hybrid multiplied by a vegetative method and also these clones were very heterozygous. Segregation caused propotion of hybrid alleles from parents to vary..

This may be able to explain why the parents and hybrid come into different groups.

From the dendogram obtained by UPGMA method, we could know the genetic distance between 45 clones analyzed.

This genetic distance can be used as a consideration in selecting the parent clones for hand pollination. To obtain a heterosis effect, the clones crossed should have a wide genetic distance (low similarity level).

CONCLUSION The DNA polymorphism of rubber clones based on RAPD analysis could be produced using OPN-05, OPN-06, OPN-08, OPN-10, OPN-11, OPN-12 OPN-17, OPH-01, OPH-03, OPH-05, OPH-18 and OPH-19 primers. The genetic similarity among the analyzed clones was quite high i.e. between 59.18%-94.23%. The lowest genetic similarity (59,18%) was found between RRIC 110 and AVROS 352 clones, and the highest (94.23%) was found between IRR 41 and IRR 42 clones. UPGMA with cluster analysis showed that 45 clones of rubber were divided in to two groups, the first one consisted of 30 clones, while the other one consisted of 15 clones with a genetic similarity value of 0.73.

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 130-135 November 2011

Variation in oil contents, and seed and seedling characteristics of Jatropha curcas of West Nusa Tenggara selected genotypes and their first improved population BAMBANG BUDI SANTOSO♼ Faculty of Agriculture, University of Mataram, Jl. Majapahit No. 62 Mataram, 83125, West Nusa Tenggara, Indonesia. Tel. +62-0370 621435, Fax. +62-0370 640189, ♼email: bbs_jatropha@yahoo.com Manuscript received: 20 July 2011. Revision accepted: 11 September 2011.

ABSTRACT Abstract. Santoso BS. 2011. Variation in oil contents, and seed and seedling characteristics of Jatropha curcas of West Nusa Tenggara selected genotypes and their first improved population. Nusantara Bioscience 3: 130-135. This study describes variation in seed and seedling characters of Jatropha curcas Linn. of West Nusa Tenggara selected genotypes. Exploration was conducted in several areas where large population of this species grown as fences was found. Five selected genotypes were then grown in experimental fields to let mass selection to obtain the first improved population for each genotype. Seeds of wild population (P0) and those of selected trees as the first improved population (IP-1) were subjected to this study. Seed and seedling characteristics were measured. The result indicated that considerable genetic variability existed among the five J. curcas of West Nusa Tenggara selected genotypes and within each genotype population for seed and seedling characteristics. Genotypes of West Lombok, Sumbawa, and Bima performed exceedingly better than those of Central Lombok and East Lombok. Therefore, this study has suggestions for identifying potential seed sources of J. curcas and these existing genetic variability provides breeders with materials in crop improvement program.

Keywords: genetic variability, seeds, seedling, selection.

Abstrak. Santoso BS. 2011. Keragaman kandungan minyak, serta karakteristik biji dan bibit genotipe terpilih Jatropha curcas Nusa Tenggara Barat dan populasi pertamanya yang diperkaya. Nusantara Bioscience 3: 130-135. Kajian ini menjelaskan keragaman karakteristik benih dan bibit Jatropha curcas Linn. Nusa Tenggara Barat dari genotipe terpilih. Eksplorasi dilakukan di beberapa daerah dimana populasi-populasi besar jenis ini ditemukan sebagai tanaman pagar. Lima genotipe yang terpilih kemudian ditanam di lahan percobaan untuk memulai seleksi massal untuk mendapatkan populasi yang diperkaya pertama pada setiap kenotipe. Benih dari populasi liar (P0) dan pohon-pohon yang dipilih sebagai populasi yang diperkaya pertama (IP-1) menjadi subyek penelitian ini. Karakteristik benih dan bibit diukur. Hasilnya menunjukkan adanya variabilitas genetik yang cukup tinggi di antara lima genotipe terpilih J. curcas Nusa Tenggara Barat dan karakteristik benih dan bibit dari setiap populasi genotipe. Genotipe dari Lombok Barat, Sumbawa, dan Bima memiliki penampilan yang jauh lebih baik dari pada genotipe dari Lombok Tengah dan Lombok Timur. Hasil penelitian ini dapat menjadi acuan dalam mengidentifikasi potensi sumber benih J. curcas dan menunjukkan adanya variabilitas genetik yang diperlukan penangkar sebagai bahan untuk program pemuliaan tanaman.

Kata kunci: variabilitas genetik, benih, pembibitan, seleksi.

INTRODUCTION Physic nut (Jatropha curcas L.) is presently grown throughout arid and semi arid tropical and sub-tropical regions including Indonesia. In Indonesia, it is found in semi-wild condition as fences in the villages and well adapted to various kinds of critical soil conditions and commonly called jarak pagar (Santoso, 2008; Hasnam, 2006). Jatropha curcas has gained interest all over the world in comparison to other tree-borne oil seed crops because of its better adaptation to a wide range of environmental conditions, low cost of oil seed production, high oil content, small gestation period and smaller plant size that makes the seeds harvested easier (Sujatha 2006;

Sujatha et al. 2008). To reduce the dependence on crude oil and to achieve energy independence, J. curcas has been promoted to be developed as an alternative energy source.

J. curcas has attracted conciderable attention as a source of seed oil (Openshaw 2000; Jongschaap 2008; Kumar and Sharma 2008). However, growth and management of this crop have been poorly documented and little results of field experiments have been shared amongst farmersUntil now this crop has not been fully domesticated. Success in establishment and management of this crop plantation is largerly determined by factors of plant varieties used and the sources of seed within species.

Jatropha curcas has a high degree of reproductivity and naturally pollinated out crossing mating system, that

SANTOSO – Jatropha genotypes of West Nusa Tenggara

ensures large amount of heterozygosity and considerable genetic variability (Ginwal et al. 2004; Das et al. 2010; Parthiban et al. 2011; Zhang et al. 2011). No report on genetic improvement aspect of this species has been published so far in Indonesia, but restricted to few publications at the global level. Studies on genetic basis of J. curcas came mostly from India and China researchers.

However, early exploration in several locations in Indonesia found that there was variation owing to differences in location creating certain ecotypes such as colours of stem, leaf and shoot, forms of capsule, and number of seeds per capsule (Hasnam 2006). Makkar et al.

(1997) reported that from 18 provenances in West Africa, North and Central America, and Asia there was variation in seed weight, kernel weight, crude protein, and oil content.

Heller (1996) also found that, from 11 provenances of Sinegal there was variation in weight of capsule, weight of seeds per trees, and weight of 100 seeds. Therefore, those variations can be used as the basis for selection and development of high yielding genotypes.

For West Nusa Tenggara region that has a wide range of dry land and large variations in wild genotype of J. curcas, regional or local crop improvement programs will be successful only after assessing local native genetic strength and possible options toward yield improvement.

Furthermore, screening of existing populations for oil yield is needed to select the best producing genotypes. It can be used for profitable production before systematic crop improvement program can yield good cultivation and it can also serve as a source for crop improvement material.

Because seed is an important material for plant propagation, and seed containing oil is very important for economic aspect for this crop development, it is necessary to know more about seed and seedling characteristics.

Kumar and Sharma (2008) stated that, genetic variation in seed morphology and oil content of J. curcas is a great

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potential in tree improvement programs. Callahan (1999) suggested that several other characters should be taken into consideration for provenance description and breeding propose. This article describes variation in seed, oil content, and seedling characteristics of J. curcas of West Nusa Tenggara selected genotypes and their first improved population.

MATERIALS AND METHODS Plant materials Exploration of plant material was done from May until June 2006 in West Nusa Tenggara (NTB), Indonesia where large population of Jatropha curcas grown as garden fences was found. Seeds from each genotype were obtained from tree stand showing good growth and development and representative for each region.

Seeds of J. curcas were collected at least from 25 parental plants with a minimum of 10 capsules per cluster (inflorescence) were chosen from each population of eachgenotype. Those parental plants were grown as fence 100 m in length, and seeds from those plants were collected and labeled; the seeds were then prepared for seed analysis, nursery, field experiment, and storage in seed storage room. Seed sources and their climate condition are given in Table 1.

Cultivation area Study in cultivation areas was conducted at AmorAmor Village, Subdistrict Khayangan, North Lombok, West Nusa Tenggara, during 2006-2007. The area has semi-arid climate with mean annual rainfall of 600-1,000 mm, minimum temperature of 25OC, maximum temperature of 25OC, relative air moisture of 90%, and altitude of 25 m above sea level. Climatic conditions at cultivation site during experiment are given in Table 2.

A uniform pre-treatment was given to Table 1. Region of seeds’ sources and their climatic condition. the seeds prior to sowing by soaking them in warm water (50OC) for two hours, let it cool Temperature Seed sources Air and kept soaking for 24 hours. Seed were Region Altitude Rain fall (OC) (genotype, moisture sown directly in black polythene bags (subdistrict) (m) (mm) district) (%) Max. Min. containing media mixture of soil, sand and West Lombok Khayangan 50-75 600-1,000 31 25 90 manure with a ratio of 1:1:1 (by volume).

Central Lombok South Praya 30-55 900-1,300 31 24 90 Seedlings were grown into 2.5 month East Lombok Masbagik 75-100 1,000-1,500 31 25 85 oldsaplings.

Sumbawa Alas 50-75 550-1,000 32 25 85 Two and half month –old saplings of Bima Sanggar 50-100 600-750 32 24 85 each genotype were planted in the field experiment using Randomized Complete Block design at 2x2 m2 spacing. Three replicates of genotypes, each consisting of Table 2. Climate condition of experimental site during 2006-2007 24 were applied.

Climate component Rainfall (mm) Rainy month (months) Rainy day (days) Air temperature (OC) min.

Air temperature (OC) max.

Relative humidity (%)

2006

2007

965 5 56 24.7 31 90

716 5 59 25 32 91

Crop maintenance Saplings received fertilizers as follows: manure as much as 2 kg.tree-1 and urea 25 kg ha-1 (10 g.tree-1), SP36 150 kg.ha-1 (60 g.tree-1), and KCl 30 kg.ha-1 (12 g.tree-1) at the time of planting. The second urea was

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applied 2 months after planting with a dose of 25 kg.ha-1 (10 g.tree-1) (Mahmud et al. 2006). Irrigation was from rainfall only. At the second year of cultivation, the trees were fertilized with the same dose as that of the first year, except for manure fertilisation.

Mass selection to meet the first Improved Population (IP-1) Selection for improved population in this study is to change the population composition with individuals which have higher average value. Selection was based on yield characteristics such as number of capsules per inflorescence and number of capsules per tree identified during generative phase of growth. Intensity of selection was 25% of trees with higher number of capsules per inflorescence (about 1520 capsules) during the first production cycle. Seeds collected from selected tree as IP-1 were prepared for studying the seedling characteristics.

Tabel 3. Physical characters of Jatropha curcas seeds of West Nusa Tenggara genotypes.

Seed size component Length (cm) Wide (cm) Weight (g) P0 IP-1 P0 IP-1 P0*) IP-1 West Lombok 1.82±0.09 1.85±0.01 0.83±0.05 0.85±0.02 0.74 a 0.78 Central Lombok 1.81±0.08 1.83±0.02 0.85±0.06 0.86±0.03 0.64 ab 0.73 East Lombok 1.82±0.10 1.83±0.02 0.85±0.08 0.85±0.05 0.60 b 0.74 Sumbawa 1.79±0.09 1.81±0.03 0.86±0.06 0.86±0.04 0.70 a 0.78 Bima 1.78±0.24 1.80±0.05 0.80±0.06 0.83±0.01 0.68 ab 0.77 LSD 5% 0.09 ns Note for Table 3 until 6: P0: seed from wild plant population, IP-1: seed from the first improved population, *): numbers in the column with the same letter did not differ significantly at P<0.05, ns: not significant ±: value of standard deviation Genotypes

Tabel 4. Percentage of kernel weight to total seed weight and 100 seed weight of Jatropha curcas seeds of West Nusa Tenggara genotypes Genotypes West Lombok Central Lombok East Lombok Sumbawa Bima LSD 5%

Percentage of kernel weight (%) P0*) IP-1*) 67.27 ab 71.18 a 58.45 b 65.66 b 60.01 ab 68.36 ab 64.29 ab 69.08 ab 67.12 ab 69.96 ab 10.05 5.05

100 seeds weight (g) P0*) IP-1*) 69.1 a 75.7 a 66.4 ab 70.1 ab 60.6 b 65.7 ab 65.2 ab 69.5 abc 67.8 a 71.0 ab 6.45 9.33

Tabel 5. Kernel oil content of Jatropha curcas of West Nusa Tenggara genotypes

Parameters observed Genotypes Physical characters of seed and seed viabilities were measured and West Lombok seed oil content was analyzed. Seed Central Lombok East Lombok length and seed width were measured Sumbawa using a caliper. Seed weight and Bima weight of 100 seeds were measured LSD 5% using an electronic balance. Separate weights of seed coat and kernels, after the seed coat was removed, were measured to calculate kernel weight percentage.

Analysis of seed oil content was carried out using extraction the method of Folch et al. (1957) modified by Sudarmadji et al. (1997). Seed viabilities were measured by daily germination observation until 21 days in three replications, each having 100 seeds. Then, seedling growth parameters were measured for two months..

Data analyses Data were subjected to analysis of mean and standard deviation, analysis of variance and Least Significant Difference test using a Minitab-14 computer program.

RESULTS AND DISCUSSION Jatropha curcas’ seed and seedling characteristics varied among five genotypes of Jatropha from West Nusa Tenggara. Variations were also observed among regions where those genotypes were collected.

P0 *) 41.7 ab 42.3 a 38.8 b 42.9 a 41.1 ab 3.41

Kernel oil content (% b/b) IP-1 Rainy season Dry season 43.7 44.6 42.6 43.1 41.9 42.9 43.6 44.3 43.4 44.3 ns ns

Seed characters Seed length Mean performance of five J. curcas of West Nusa Tenggara selected genotypeswith respect to seed length is prsented in Table 3. Seeds collected from the five regions of West Nusa Tenggara province varied in their seed length. A higher variation was also found within each wild genotypes population (P0) than that in the first Improved Population (IP-1). Standard deviation of seed length in P0 was higher than in IP-1.

Seed width Variations among genotypes and within each wild genotype population (P0) as that observed in seed length also occured in seed width (Table 3). Variation dcreased after the first mass selection (IP-1) of wild population (P0).

Seed weight Seed weight ranged from 0.60g to 0.74 g in different genotypes within the wild population (P0). West Lombok and Sumbawa genotypes had the maximum seed weight, in

SANTOSO – Jatropha genotypes of West Nusa Tenggara

Tabel 6. Seed viabilities of Jatropha cyrcas seeds of West Nusa Tenggara genotypes

Genotypes West Lombok Central Lombok East Lombok Sumbawa Bima LSD 5%

Germination (%)

Germination rate (day)

Seed vigourity (%)

P0 84.3 79.3 82.0 81.7 80.9 ns

P0 11.40 12.97 12.49 12.67 12.40 ns

P0 89.93 82.50 89.37 89.70 86.83 ns

IP-1 88.9 86.7 85.9 87.4 88.2 ns

IP-1 10.07 11.03 11.56 10.22 11.01 ns

IP-1 90.06 85.76 90.35 90.54 89.88 ns

Dry weight of 3 weeks old sapling (g) P0*) IP-1 0.58 a 0.59 0.45 b 0.52 0.48 ab 0.53 0.55 ab 0.56 0.53 b 0.54 0.12 ns

Table 7. Seedling height, number of leaves, and collar diameter of Jatropha curcas of West Nusa Tenggara Genotypes of wild population (P0) and Improved Population- 1 (IP-1)

Genotypes

Seedling height (cm) 1 month 2 month old old

Number of leaves 1 month old

2 month old

Collar diameter (cm) 1 month 2 month old old

P0 West Lombok 15.7±1.078 22.9±0.893 4.6±0.925 8.9±0.908 1.1±1.142 1.3±0.877 Central Lombok 18.3±1.694 21.2±1.132 4.7±1.106 8.2±1.062 0.9±1.333 1.0±0.992 East Lombok 15.2±1.426 20.1±1.426 5.1±1.077 7.8±1.016 0.8±1.434 1.1±0.902 Sumbawa 19.1±1.148 22.2±0.926 5.4±0.945 9.1±0.921 1.0±1.024 1.3±0.743 Bima 17.5±0,992 21.8±0.901 3.8±0.982 7.2±0.933 1.1±1.872 1.4±0.967 IP-1 West Lombok 17.4±0.789 23.7±0.656 5.2±0.819 9.5±0.445 1.1±0.763 1.4±0.428 Central Lombok 18.9±0.992 22.4±0.793 5.6±0.925 8.9±0.545 1.0±0.784 1.1±0.457 East Lombok 17.3±0.905 21.2±0.885 5.7±0.961 8.7±0.607 0.9±0.825 1.1±0.724 Sumbawa 18.5±0.902 23.6±0.776 5.6±0.786 9.6±0.416 1.0±0.641 1.3±0.409 Bima 17.8±0.814 22.4±0.664 5.1±0.807 8.9±0.378 1.0±0.706 1.3±0.513 Note for Table 7 and 8: P0: seed from wild plant population, IP-1: seed from the first improved population, ±: value of standard deviation

Table 8. Dry weight of seedling shoot and seedling root of J. curcas of West Nusa Tenggara Genotypes of wild population (P0) and Improved Population-1 (IP-1) Genotypes P0 West Lombok Central Lombok East Lombok Sumbawa Bima IP-1 West Lombok Central Lombok East Lombok Sumbawa Bima

Seedling shoot (g) 1 month old 2 month old

Seedling root (g) 1 month old 2 month old

4.95±1.798 3.55±1.882 3.92±1.908 4.71±1.636 4.32±1.872

7.33±1.233 6.88±1.372 6.91±1.501 7.16±1.362 7.43±1.239

0.94±1.595 0.63±1.551 0.66±1.462 0.79±1.357 0.88±1.623

1.74±1.013 1.35±1.154 1.43±1.096 1.58±1.005 1.66±1.102

5.79±1.124 4.95±1.132 5.19±1.164 5.46±1.087 5.24±1.139

9.01±0.863 8.28±0.994 8.76±0.907 9.12±0.821 8.95±0.879

1.23±1.026 0.96±1.147 1.12±1.115 1.29±1.076 1.22±1.083

1.95±0.902 1.69±1.006 1.71±1.021 1.98±0.942 2.02±0.879

contrast, East Lombok had the minimum. However, there were no significant difference in seed weight of IP-1 (Table 3).

Kernel weight percentage Analysis of variance indicated that different genotype had a significant effect (P<0.05) on the percentage of kernel weight to total seed weight (Table 4). The maximum

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kernel weight was found in West Lombok, East Lombok, Sumbawa, and Bima, while the minimum was in Central Lombok. However, after mass selection, it was found that West Lombok had the maximum kernel weight, and Central Lombok possessed the minimum.

100 seed weight (seed index) The same phenomena as that found in kernel weight percentage was observed in 100 seed weight.

The maximum 100 seed weight was found in West Lombok, East Lombok, Sumbawa, and Bima, while the minimum was observed in Central Lombok. After mass selection, it was found that West Lombok had the maximum 100 seed weight with Central Lombok having the minimum (Table 4).

Kernel oil content Selected genotypes within West Nusa Tenggara province had a significant effect on kernel oil content. However, after mass selection, the five J. curcas West Nusa Tenggara selected genotypes showed no variation or no significant difference in the kernel oil content (Table 5).

Seed viabilities Seed viability component of J. curcas seed of West Nusa Tenggara genotypes are given in Table 6.

There were no significantly effects of genotypes on seed germination, germination rate, and vigourity of seed. However, seed viabilities were better after mass selection (IP-1) than the wild genotypes.

Seedling characters Seedling height Seedling height ranged from 15.2 cm to 19.1 cm for one month old seedlings and from 20.1 cm to 22.9 cm for two month old seedlings in different genotype within their wild population (P0). Within their first improved population (IP-1), seedling height also varied among different genotypes, eventhough there was a decrease in variation within each genotype. The highest (22.9 cm for P0 and 23.7 cm for IP-1) seedling height was found in West Lombok genotype and the lowest (20.1 cm for P0 and 21.2 cm for IP-1) was found in East Lombok genotype.

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Number of leaves Number of leaves varied within five genotypes and it ranged from 7.8 to 9.1 in the wild population (P0) and from 8.7 to 9.6 in the first improved population (IP-1) (Table 7).

There was improvement of homogenosity or decreased of variation due to mass selection.

Collar diameter Table 7 showed that genotype had different effect on collar diameter. It ranged from 1.0 cm to 1.4 cm within the wild population (P0), and 1.1 cm to 1.4 cm within their first improved population (IP-1). The variation within each genotype decreased after mass selection of the wild population.

Discussion This study showed that seed and seedling characteristics of J. curcas varied among different selected genotypes of West Nusa Tenggara province. Genotype had genotypic characters according to their location of seed sources. Seed sources varied in their growing habitat with respect to altitude, temperature, and rain fall. The sources used in this study had mean annual rain fall (550-1.500 mm), temperature (24-32OC), and altitude (30-100 m). Sumbawa, Bima, and West Lombok are drier than Central Lombok and East Lombok (Table 1). Therefore, variation in sources with respect to seed and seedling characteristicss are mainly due to the fact that those genotypes grow over a wide range of climatic conditions in West Nusa Tenggara.

When the genotypes were grown in experimental field at Amor-Amor, Subdistrict Kayangan, West Lombok they were influenced by local climatic condition affecting seed and seedling performance such as reduction in their variability, especially within IP-1 population). Ginwal et al.

(2004), Ginwal et al. (2005) and Kaushik et al. (2007) reported significant variations in seed morphology and seedling growth variables like seedling height, collar diameter, leaves, seed weight, and 100 seed weight in 10 accessions of J. curcas.

Since there was no different phenomenon of the influence of genotype within P0 and IP-1, it means that various climatic factors affected the vegetation collectively, but not individually. Considering this fact, genotypes may possess different climatic features that caused genotype variations. The present results are similar with the finding of Zang et al. (2011), that genetic variation among genotype or provenance may be due to geographical separation.

West Lombok’s genotypes followed by Sumbawa’s, and Bima’s were the best genotypes compared to Central Lombok and East Lombok with respect to their seed and seedling caharacteristics. There is correlation between good seed characteristic and good seedling characteristic. As Isik (1986) stated, that seed size and seed weight were two important characteristics for improving seedling productivity,hence, it was clear that seeds with greater seed weight produced seedlings with greater shoot and root growth. This may be due to greater nutrient reserves in larger seeds (Bhat and Chauhan 2002; Gonzales, 1993).

The purpose for genotype testing is to measure the value of genetic variation and to aid further selection of better adapted and highly productive seed sources or genotypes. Variation in seed and seedling characteristics of J. curcas of West Nusa Tenggara genotypes is an indicator of the possibility of selecting the best performance or the highest seed yield of the tree for further crop improvement programs. Due to the fact that no different phenomenon of genetic variability of five J. curcas of West Nusa Tenggara selected genotypes both in wild population (P0) and first improved population (IP-1), it can be said that those variability caused by genetic factor with minor effect of environment. Therefore, as Gohil and Pandya (2009) state, that if variability is largerly due to genetic cause with least environment effect, probability of isolating superior genotype is a precondition for obtaining higher yield.

The variability in seed, oil content, and seedling characteristic along with variability in early growth performance indicates that economic benefits may be obtained. Although, Parthiban et al. (2011) reported that in India, few native Jatropha species were utilized in their improvement program with limited success; the results of this study will be valuable for strategies for conservation of genetic variation, prospects of improvement and assessment of the potential of locally adapted seed sources.

As Boe (2003) mentions, that since seed is the main product of trees selection for increasing seed weight and their content (seed-oil concentration), it may become important selection criterion for new cultivar development.

CONCLUSION J. curcas improvement program will be successful only after assessing our native genetic strength and the possible option toward yield improvement. Result of the present study revealed that considerable genetic variability existed among the five J. curcas West Nusa Tenggara selected genotypes and within each genotype population for seed and seedling characteristics. Genotypes of West Lombok, Sumbawa, and Bima performed exceedingly better than that of Central Lombok and East Lombok in terms of seed and seedling characteristics. These variations could occur from genetic diversity that needs to be studied in detail for their performance on seed production potential and for further J. curcas improvement program.

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Boe A. 2003. Genetic and environmental effect on seed weight and seed yield in switchgrass. Crop Sci 43:63-67.

Callaham RZ. 1999. Provenance Research: investigation of genetic diversity associated with geography. FAO Corporate Document Repository - FAO/IUFRO Meeting on Forest Genetic, Rome.

Das S, Mirsa RC, Mahapatra AK, Gantayat BP, Pattnaik RK. 2010.

Genetic variability, character association and path analysis in Jatropha curcas. World Appl Sci J 8 (11): 1304-1308.

Ginwal HS, Rawat PS, Srivastava RL. 2004. Seed source variation in growth performance and oil yield of Jatropha curcas Linn. in Central India. Silvae Genetica 53: 186-192.

SANTOSO â&#x20AC;&#x201C; Jatropha genotypes of West Nusa Tenggara Ginwal HS, Phartyal SS, Rawat PS, Srivastava RL. 2005. Seed source variation in morphology, germination, and seedling growth of Jatropha curcas Linn. In Central Asia. Silvae Genetica 54:76-80.

Gohil RH, Pandya JB. 2009. Genetic evaluation of Jatropha (Jatropha curcas Linn) genotypes. J Agric Res 47(3): 221-228.

Gonzales JF. 1993. Effect of seed size on germination and seedling vigor of Virola koschyni Warb. Forest Ecol Manag 57: 275-281.

Hasnam. 2006. Variation in Jatropha L. Info-Tek of Physic Nut (Jatropha curcas L). Puslitbangbun, Badan Penelitian dan Pengembangan Pertanian 1 (2):3. [Indonesian] Heller J. 1996. Physic Nut, Jatropha curcas L. - Promoting the conservation and use of underutilized and neglected crop 1.

International Plant Genetic Resources Institute. Rome.

Isik K. 1986. Altitudinal variation in Pinus brutia: Seed and seedling characteristics. Silvae Genetica 35:58-67.

Jongschaap REE. 2008. A to Z of Jatropha curcas L. claims and facts on Jatropha curcas L. Plant Research International, Wageningen, The Netherlands.

Kaushik N, Kumar K, Kumar S, Roy N. 2007. Genetic variability and divergence studies in seed traits and oil content of Jatropha (Jatropha curcas L.) accession. Biomass Bioener 31: 497-502.

Kumar A, Sharma S. 2008. An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): A review. Industrial Crops Prod 28: 1-10.

Mahmud Z, Rivaie AA, Allorerung D. 2006. Technical Intruction on Jatropha curcas L. cultivation. Pusat Penelitian dan Pengembangan

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Perkebunan, Badan Penelitian dan Pengembangan Pertanian. Second Edition. Jakarta. Deptan. 35p. [Indonesian] Makkar HPS, Becker K, Sporer F, Wink M. 1997. Studies on nutritive potential and toxic constituents of different provenances of Jatropha curcas. J Agric Food Chem 45:3152-3157.

Openshaw K. 2000). A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass Bioener 19: 1-15.

Parthiban KT, Kirubashankkar R, Paramathma M, Subbulakshmi V, Thiyagarajan P, Vennila S, Sujatha M, Durairasu P. 2011. Genetic association studies among growth attributes of Jatropha hybrid genetic resources. Intl J Pl Breed Genet 5 (2): 159-167.

Santoso, BS. 2008. Characterization of morphological and agronomical aspects of physic nut (Jatropha curcas L.) ecotypes at West Nusa Tenggara. 225p. (Desertation). [Indonesian] Sudarmadji S, Haryono B, Suhardi. 1997. Analysisi Prosedure forr Food and Agriculture. Fourth Edition. Liberty, Yogyakarta. [Indonesian] Sujatha M. 2006. Genetic improvement of Jatropha curcas L. possibilities and prospects. India J Agroforest 8: 58-65.

Sujatha, M, Reddy TP, Mahasi MJ. 2008. Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L. Biotechnol Adv 26: 424-435.

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 136-144 November 2011

Litter decomposing fungi in sal (Shorea robusta) forests of central India KRISHNA KANT SONI, ABHISHEK PYASI, RAM KEERTI VERMA♥ Forest Pathology Division, Tropical Forest Research Institute, Post – Regional Forest Research Centre, Jabalpur 482021, Madhya Pradesh, India. Tel. +91-0761-2840746; Fax. +91-0761-2840484, 4044002; ♥email: rkverma28@rediffmail.com Manuscript received: 1 November 2011. Revision accepted: 25 November 2011.

Abstract. Soni KK, Pyasi A, Verma RK. 2011. Litter decomposing fungi in sal (Shorea robusta) forests of central India. Nusantara Bioscience 3: 136-144. The present study aim on isolation and identification of fungi associated with decomposition of litter of sal forest in central India. Season wise successional changes in litter mycoflora were determined for four main seasons of the year namely, MarchMay, June-August, September-November and December-February. Fungi like Aspergillus flavus, A. niger and Rhizopus stolonifer were associated with litter decomposition throughout the year, while Aspergillus fumigatus, Cladosporium cladosporioides, C. oxysporum, Curvularia indica, and C. lunata were recorded in three seasons. Some fungi including ectomycorrhiza forming occur only in the rainy season (June-August) these are Astraeus hygrometricus, Boletus fallax, Calvatia elata, Colletotrichum dematium, Corticium rolfsii, Mycena roseus, Periconia minutissima, Russula emetica, Scleroderma bovista, S. geaster, S. verrucosum, Scopulariopsis alba and four sterile fungi. Fungi like Alternaria citri, Gleocladium virens, Helicosporium phragmitis and Pithomyces cortarum were rarely recorded only in one season.

Key words: decomposition, fungi, forests, litter, sal, seasonal variation.

Abstrak. Soni KK, Pyasi A, RK Verma. 2011. Fungi pembusuk serasah pada hutan-hutan meranti merah muda (Shorea robusta) di India tengah. Nusantara Bioscience 3: 136-144. Penelitian ini bertujuan untuk mengisolasi dan mengidentifikasi fungi yang terlibat dalam dekomposisi serasah dari hutan meranti merah muda di India tengah. Sejalan dengan perubahan suksesional fungi pendegradasi serasah, maka penelitian dilakukan pada empat musim utama dalam setahun, yaitu Maret-Mei, Juni-Agustus, September-November, dan Desember-Februari. Fungi seperti Aspergillus flavus, A. niger dan Rhizopus stolonifer terlibat dalam dekomposisi serasah sepanjang tahun, sementara Aspergillus fumigatus, Cladosporium cladosporioides, C. oxysporum, Curvularia indica, dan C. lunata terlibat dalam tiga musim. Beberapa fungi termasuk fungi pembentuk ectomycorrhiza hanya ditemukan pada musim hujan (Juni-Agustus) yaitu Astraeus hygrometricus, Boletus fallax, Calvatia elata, Colletotrichum dematium, Corticium rolfsii, Mycena roseus, Periconia minutissima, Russula emetica, Scleroderma bovista, S. geaster, S. verrucosum, Scopulariopsis alba dan empat fungi steril. Fungi seperti Alternaria citri, Gleocladium virens, Helicosporium phragmitis dan Pithomyces cortarum jarang ditemukan dan hanya ditemukan dalam satu musim.

Kata kunci: dekomposisi, serasah, fungi, hutan-hutan meranti merah muda, variasi musiman.

INTRODUCTION The soil is regarded as a heterogeneous collection of minerals and organic materials. A major portion of organic matter in soil comes from plant material in the form of litter. There is considerable amount of litter fall annually in tropical dry deciduous forests. According to Burges (1958) the total litter fall in tropical forest may reach to 1.53 thousands kg/ha/yr. The leaf litter contains considerable amount of nutrients necessary for plant growth. In tropical forests most of the nutrient stock is in the form of biomass and relatively little in soil. Nutrients available in the plant litter falling in dry season are rapidly mineralized in the following monsoon and taken up by roots in the wet season, therefore the importance of forest floor as an integral part of the ecosystem has been recognized for a long time. In that way decomposing litter helps to gradually rehabilitate the soil and the soil productivity is enhanced.

The decomposition of plant litter at the soil surface is brought about by a variety of organisms including bacteria, fungi, actinomycetes, protozoa, nematodes, and insects. As a

result of microbial attack and activity the litter is subjected to chemical changes like oxidation, hydrolysis, reduction and condensation (Walksman 1952). Besides decomposition these are also involved in some other important biological process of an ecosystem (Harley 1971).

The importance of studying sal litter decomposition by above mentioned microbial agencies has been initiated long back (Lutz and Chandler 1946; Webster 1956; Hudson 1962). Absence of process of decomposition due to drought, fire, frost, insects and nutrient deficiency in soil created large scale sal mortality in central India (Khan 1953; Lal 1956; Pandey 1966; Prasad et al. 1983). It has also been emphasized that abnormally high temperature during the month of May causes sudden recession of water level. This factor adversely affects normal physiological function around the feeder root of sal forest. The phenomenon of litter layering during the month of March to May and gradual decomposition from June to December and mineralization from October to February with the well distribution of rains and suitable temperature creates balanced process of nutrient release and their uptake by sal

S SONI et al. – Liitter decomposiing fungi of sal forest f

ttrees. This pheenomenon is disturbed d by unequal u distribbution o rains. The successional of s fu fungi which caarries large am mount o enzymes, ennable to conveert complex organic of o molecuule to C 2, H2O and mineral components woould be drastiically CO d depleted from m the terrestriaal habitat of saal forest. Thiss kind o disturbancee dealt with thee succession and of a further nuutrient u uptake the deccomposition process p is a complete chainn if it is part-wise or o factor-wise dealing withh gradual minneraliz zation and furrther mycorrhizal developm ment in and arround f feeder root system s in moother sal trees or in juvvenile s seedling regenneration that was w seriouslyy disturbed. Recent R fungi o observation o the ecologgy of litter decomposing on d indicated needd for detailed study in orderr to understannd the r role of fungi in i litter decom mposition (Dw wivedi and Shhukla 1 1977; Mehrottra and Anejaa 1979; Sinhaa and Dayal 1983; S Soni and Jamaaluddin 1990; Jamaluddin et al. 1984; Maria M a Shridhar 2002; and 2 Hossainn and Othman 2005).

In the preesent study ann attempt was made to isolate, identify fungi and study the fungal successsion in the prrocess o decomposinng litter underr natural sal forest of f ecosysteem in t three states off central Indiaa. Occurrence and importannce of f fungi under vaarious stages of litter decoomposition aree also g given.

M MATERIALS S AND METH HODS Study area S The area chosen c for colllection of sall litter lies in three d different stattes of India namely Madhya M Praadesh, and Orissa. The selecteed sites inclludes: C Chhattisgarh A Amarkantak-A Achanakmar biosphere b resserve, covers both M Madhya Pradeesh and Chhaattisgarh; Am markantak (N222040' 0 E 45') and Motinala (N E81 N22°21'0" E80°54') comees in M Madhya Pradeesh, Gariyabaand (N20°38'224" E82°3'36"") and A Achanakmar ( (N22°25', E81°51') are in Chhattisgarh C w while

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i Jharssuguda (N20°440’ to 22°1’annd E82°39’ to 85°15’) falls in Orissa (Figure 1). In these sitess sal forest bellongs to seconnd and third categorry. Associateed species prresent in thesse foressts includes Buchanania B laanzan, Anogeeissus latifoliaa, Cleisstanthes collinnus and Flemingia panicula ata. This studdy was carried out duuring March 22009 to Februaary 2010. dy of litter deecomposing fu ungi Stud For F the studyy on litter decomposition by fungi thhe colleected fungi weere processed, cultured on media, m purifiedd, and identified i as per p methods, bbriefly describ bed below.

Direect observationn Leaf L litter sam mple squares w were cut into 5x5 5 mm2 smaall piecees with a steriile parallel razzor at random m from the base, midd dle and apexx. These pieeces were cleaned, staineed (Ship pton and Broowns 1962), oobserved undeer stereo-zoom m micrroscope and fuungal colonizaation was reco orded.

Prep paration of funngal culture Damp D chambeer method. S Squares of leeaf litter werre placeed in a sterilee petriplates of 9.0 cm diam meter to form a moisst chamber. Fungal flora was recorrded daily to t docu ument ecological successionn. Isolation off fungi was alsso donee on potato dextrose d agar (PDA) mediu um (Keywortth 1951 1).

Dilution D methood. Forty squuares of litter sample piecees cut as a described above a were pplaced in 60 mL m of distilleed wateer, rinsed repeatedly for ffive times theen placed in a sterillized 500 mL L conical flaskk containing 60 6 mL distilleed wateer. Flask was wrist shaken and dilution up to 10-3 waas prepared. One mL m inoculumss from each dilution werre plateed on PDA medium m and iincubated for seven days at a 270C in a BOD incubator. Eachh petridish was considered as a a un nit sample. The T frequenccy class was expressed as a sugg gested by Sakssena (1955).

MADHY YA PRADESH

2 1

4 5 3

CHHATTISGARH

ORISSA A

Figure 1. Map showing study sites in three states (Madhya Pradesh, Chhatttisgarh and Oriissa) of India. 11. Amarkantak and 2. Motinalla, F c comes in Madhyya Pradesh, 3. Gariyaband G andd 4. Achanakmaar are in Chhattisgarh, while 5. Jharsuguda falls in Orissa.

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Washed disc method. Leaf litter discs (5x5 mm2) after their treatment by dilution plate method were serially washed 10 times by successive changes of sterile water (Harley and Waid 1955), dried on sterile filter paper and plated on sterile PDA medium. Fungal colonies were identified and counted, and presence of each fungus was expressed in terms of percentage occurrence based on a total of 75 discs examined.

Direct plating method. Quantitative estimation of mycoflora associated with the collected litter samples was done as suggested by Warcup (1950) for studying soil mycoflora. Well ground pinch of litter samples were taken in petriplates and 10-15 mL of molten PDA medium was poured. The plates were allowed to solidify and incubated at 270C for 5-10 days.

Purification of fungal cultures Purification of fungal cultures was done preferably by streak plate and dilution plate method briefly described as follows.

Streak plate method. Sterilized fungal inoculating needle was touched on desired sporulating fungal colony and streaked in fresh plates of PDA medium in zigzag manner. After 48 hrs of incubation colonies were studied.

Dilution plate method. Moisten tip of fungal inoculating needle was touched over the colonies and then inoculated into 5 mL distilled water, shaken vigorously and diluted up to 10-3. One mL of each dilution was aseptically transferred into sterilized petriplate in triplicates then media was poured over the inoculum. Plates were incubated for 48-72 hrs at 270C in a BOD incubator. Then hyphal tips were transferred onto PDA medium slants by using a sterile inoculating needle.

Agar block method. A block of agar was taken on the tip of sterilized needle and gently touched to the sporulating mass of desired fungus in a petriplate. The block was gently slided over the plate containing Czepeks agar medium. The surface of the plates observed immediately under low power of stereo-zoom microscope to assure the place of a well separated spore. A piece of sterilized filter paper was adhered at the tip of inoculating needle and brought near to it. The well separated spore clung to the surface of filter paper which was then transferred to a fresh petriplate to get a pure monosporic culture (Ashara 1975).

Sporulation. Some fungi belonging to ascomycetes and deuteromycetes showing poor or no sporulation on common media were grown on specialized media and incubated at suitable temperature (250C) which stimulated the sporulation in fungi.

Calculation of occurrence and frequency of fungi Occurrences and frequencies of fungi occurring in litter were calculated and categorized by the procedures described by Saksena (1955) as per formulae given below: Number of colonies of individual species in all the quadrats studied % Occurrence =

Total number of colonies of all the species

X 100

Number of plates containing particular fungus % Frequency =

Total number of plates

X 100

Categorization of frequency classes Class I II III IV V

% Frequency 1-20 21-40 41-60 61-80 81-100

Category Rare Occasional Frequent Common Dominant

Maintenance of fungal cultures Living cultures of important fungi were maintained on PDA medium slants under low temperature in a refrigerator.

Microscopic study For microscopic study slides were prepared in lactophenol + cotton blue staining reagent and details of fungal colonization in litter was observed and recorded under stereo-zoom microscope (Leica Germany, model Wild M3Z). Micro slides were observed under advanced research microscope, Leica Germany, model Leitz DMRB/E, using 5x, 10x, 20x 40x objectives and 10x and 15x eyepieces and photomicrographs were taken. Photographs of fruit bodies of macro-fungi were taken by 12 mega pixel digital camera (Sony, model Cybershot H-50).

Identification of fungi Fungi were identified on the basis of their growth characteristics, morphological characteristics and ontogeny with the help of manuals, monographs and taxonomic papers of various authors (Gilman 1957; Grove 1967; Subramanian 1971; Ainsworth et al. 1972; Barnett and Hunter 1972; Ellis 1971, 1976; Sutton 1980; von Arx 1981; Verma et al. 2008).

RESULTS AND DISCUSSION Results A total 63 fungal species have been recorded from decomposing sal litter present on forest floor of central India. Season wise occurrences of these fungi are given below.

March-May With the start of summer season most of the fallen leaves are accumulated on the ground (Figure 2.A-B). The fallen leaves gradually dry up and are distributed throughout the stand by wind. Physically litter composed of dry, flat, partially folded, light brown leaves. It also composed of pieces of woody twigs and barks. In early litter formation freshly fallen dry leaves mixture is usually found near the tree bases. The layering occurred simultaneously as the leaf fall progressed. The sequence of colonization of leaves

SONI et al. â&#x20AC;&#x201C; Litter decomposing fungi of sal forest

indicated that mostly the oldest leaves are first to be colonized. Decomposition process begins before the plant part senescence. The organism involved is related to the type of plant part in litter and the environment. As soon as any plant part senescence saprophytic fungi began to colonize and multiply. The direct and indirect observation of litter revealed fungal population colonizing the litter at different stages of decomposition. The spread of fungal colonization was studied till the plant parts became completely fragile. The elimination and categorization of

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fungi occurring on different litter parts were made. A wide variety of fungi appeared at different stages of decay. The fungal flora changed as decomposition progressed. During this quarter the freshly fallen litter samples revealed less number of fungal species. Total 17 fungal species have been directly observed and isolated from the freshly fallen sal leaf litter. The most frequent colonizing fungi were Asterostomella shoreae, Cladosporium oxysporum, Curvularia indica and Curvularia lunata (Table 1, Figure 3).

A

B

Figure 2.A-B. Leaf litter fall in sal forest of central India

A

E

B

F

C

G

D

H

Figure 3.A-H. Litter decomposing fungi of sal forest. A. Alternaria citrina, B. Aspergillus flavus, C. Aspergillus niger, D. Cladosporium oxysporum, E. Curvularia indica, F. Curvularia lunata, G. Rhizopus stolonifer, H. Trichoderma viride

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A

B

E

C

F

G

D

H

Figure 4. A-H F H. Mycorrhizal and litter decomposing funggi of sal forestt. A. Astraeus hygrometricuss, B. Geastrum m fimbriatum, C. C G Geastrum triplexx, D. Boletus falllax, E. Russula emetica, e F. Sclerroderma bovista a, G. Scleroderm ma geaster, and H H. Scleroderma verrucosum v

JJune-August During Junne most of thhe leaf and noon leaf litter mixed m a spread thrroughout the forest and f floor annd the stratificcation p progressed.

The litter graddually changeed on the onsset of m monsoon andd weathering, started simuultaneously duue to w wind current, hot weatherr followed byy rains. The layer f formation wass observed neear tree bases,, stone bounddaries, a in depressed ground annd formed oligostratal layeer. In and t second weeek of June strong the s showeer soaked the litter a within 155 days the color of litter started and s changing to d darker.

The liitter set up in an approprriate frame onn the f forest floor. The color, textuure, and otherr chemical chaanges o occurred connsequently duue to heavy rains. The litter b becomes succuulent which were w readily colonized c by fungi. f T The decompoosing litter sheets took different shhapes d depending uppon the canoppy density annd slope of forest f f floor.

Regular rain increasedd the activity of fungal florra and t this period is very importtant for litter decompositioon as m of the soil borne fungii were very acctive. After hoolding most s sufficient moissture colonizaation by severaal centimeterss long s saprophytic f fungi were developed d annd appeared as a w whitish, light brownish andd blackish weeb of myceliuum in b between the gaps of decoomposing paads of litter. Fruit b bodies of seveeral gasteromyycetous fungi were also emeerged o forest flooor in a colonnial form whhich was geneerally on a attached to thhe feeder roott network of sal. s High moiisture i i.e.

85-95% relative r humiidity and 27--350C temperrature f favors the excellent growth of soil, mycoorrhizal and seeveral o other saprophhytic fungi. Common C ecto--mycorrhizal fungi

ata, Geastrum m noticced were Booletus fallax Calvatia ela tripleex, Russula emetica, e Sclerroderma boviista, S. geasteer and Scleroderma verrucosum (Figure 4). Marasmius M annd Myceena, were alsoo emerged on forest floor.

Over O all 32 speecies belongingg to 23 genera were w recognizeed as a colonizer of litter l decompooser during Jun ne-August. Ouut of th hese eight funggal species naamely Aspergiillus fumigatuus, G. trriplex, Lophoodermium shooreae, Marasm mius gordipees, Myceena roseus, R. R emetica, Tr Trichoderma harzianum, h annd sterille fungus-1 shown itss highest frequency fr annd categ gorized underr dominant frrequency (V) and exhibiteed highest percentagge of occurrrence. Simulttaneously fivve fung gi namely Asppergillus nigeer, C. elata, S. verrucosum m and sterile funguss-2 were incluuded in (IV) frequency annd colon nized with abundant a perccentage occurrrence. In this categ gory the natuure of fungal succession agents a are botth myco orrhizal and non n mycorrhiizal. These fu ungi are seasoon specific and theirr occurrence and colonizattion is directlly related to substratte and moisturre content of top litter layeer.

In caategory third (III), eight fuungi were recorded with thhe frequ uent colonizaation level. T These are Asp pergillus astus, Clad dosporium heerbarum, C. oxysporum, Colletotrichum m dema atium, Corticcium rolfsii, C. lunata, Phoma P exiguaa, sterille 3-4, Tricchoderma viiride and Wiesneriomyce W es javan nicus, which belong to deuuteromycete. Populations of o thesee fungi suddeenly increasedd and rapidly y colonized thhe uppeer and middlee layer of littter. In catego ory second (II) seven fungi were recorded r and categorized ass occasional.

SONI et al. â&#x20AC;&#x201C; Litter decomposing fungi of sal forest

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Table 1. Season-wise frequency and occurrence of different fungi involving in litter decomposition of sal forests in central India Name of fungi

Mar - May Freq Occur I 10.5 III 8.6 I 1.8 III 13.5 III 11.8 V 20.5 II 4.6 V 28.2 IV 18.0 IV 18.6 I 1.2 I 1.0 I 1.2 II 7.4 V 28.1 -

June - Aug Freq Occur II 11.4 III 8.2 V 25.3 IV 18.2 III 13.0 V 28.0 IV 17.6 IV 18.2 II 4.6 III 13.0 III 8.8 III 11.8 II 8.6 II 3.9 IV 7.6 IV 7.6 V 20.5 V 28.0 V 20.2 II 5.3 -

Sep- Nov Freq Occur V 24.2 V 20.5 V 22.2 II 3.9 III 11.4 II 5.3 III 12.9 II 3.8 II 3.3 II 2.6 II 2.8 II 2.4 III 12.4 II 4.6 III 2.0 II 6.7 II 2.5 II 3.6

Achlya debaryana Hump.

Alternaria alternata Fr. Keissl.

Alternaria citri Penz.

Aspergillus flavus Link.

Aspergillus fumigatus Fres.

Aspergillus niger Tiegh.

Aspergillus terreus Thom.

Aspergillus ustus Bainier Asterostomella shoreae Soni, Hosag,Pyasi & RK Verma Astraeus hygrometricus Pers.

Boletus fallax Corner Botryodiplodea theobromae Pat. Calvatia elata (Massee) Morgan Chaetomium globosum Kunze ex Fr. Cladosporium cladosporioides Link Cladosporium herbarum Pers Cladosporium oxysporum Berk Colletotrichum dematium Pers.

Colletotrichum gloeosporioides (Penz.) Sacc.

Coprinus aquatilis Peck Corticium rolfsii Curzi.

Curvularia indica Subram.

Curvularia lunata Wakker Curvularia prasadii Boedijn.

Drechslera spicifera (Bainier) Arx Fusarium concolor Reinking.

Fusarium equiseti (Corda) Sacc.

Fusarium moniliforme J. Sheld.

Fusarium semitectum Berk.

Fusarium solani Sac. Geastrum triplex Jungh.

Geastrum fimbriatum Fr. Gliocladium virens Corda Lophodermium shoreae Jamal, Dadwal & Soni Marasmius gordipes Sacc. & Paol.

Mucor circinelloides Tiegh.

Mycena roseus Pers.

Paecilomyces variotii Bainier.

Penicillium notatum Westling.

Periconia minutissima Corda Pestalotiopsis versicolor (Speg.) Steyaert Phoma exigua Desm.

Phoma macrostoma Mont.

Phoma medicaginis Malbr. & Roum.

Phoma multirostrata (P.N. Mathur, S.K. Menon & Thirum.) Dorenb. & Boerema Phoma nebulosa (Pers.) Berk.

Pithomyces cortarum Berk.

Rhizopus stolonifer Ehrenb.

III 10.6 II 10.0 Russula emetica (Schaeff.) Pers.

V 27.8 Scleroderma bovista Fr. IV 16.7 Scleroderma geaster Fr. IV 16.7 Scleroderma verrucosum (Bull.) Pers.

IV 16.7 Scopulariopsis alba Szilvinyi.

II 8.6 Sterile fungus 1 V 27.5 Sterile fungus 2 IV 18.6 Sterile fungus 3 III 10.2 Sterile fungus 4 III 11.5 Trichoderma harzianum Rifai V 28.2 Trichoderma koningii Oudem.

III 13.8 Trichoderma viride Pers.

III 10.8 Verticillium lecanii Zimm.

Wiesneriomyces javanicus Koord.

Helicosporium phragmitis HĂśhn.

Note: Freq = frequency; Occur = occurrence. The Roman numbers show the frequency classes percentage frequency and occurrence of fungi.

II II III III II III and the

Dec - Feb Freq Occur II 6.4 V IV V V II II

19.8 18.0 23.2 20.5 6.4 6.4

II II III II

3.7 2.8 13.0 5.0

II II I III II II II II III III II III

7.8 4.1 1.5 12.6 2.3 3.8 8.5 8.5 11.6 10.7 4.5 10.6

I I II II -

1.8 0.8 3.5 4.6 -

7.6 I 1.6 I 1.2 8.6 III 10.3 11.1 10.5 III 11.5 II 7.8 3.5 I 2.5 10.3 III 11.2 Arabic numbers represent

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They sometimes occur and sometimes does not depending upon the sampling time, locality, and substrate. During the month of August some specific fungi were also recorded from the dead insect body namely Scopulariopsis alba, and Verticillium lecanii from Achanakmar. Their population increased sufficiently under moist incubation chamber.

September-November During this quarter physical character of most of the litter losses their lamina and its color was also turned to dark brown. It became fragile and deposited near the base of trees and restricted by under storey crop (weed and saplings of tree species). Upper surface of litter produced well matured tiny dots of L. shoreae which is a dominant fungus of sal forest. Its range of distribution is also categorized as a common to all the selected sites. In all seventeen fungal species were identified in the litter of sal during September to November. The frequency of Aspergilli increased to dominant and categorized in fifth (V) category. These are Aspergillus flavus, A. niger and A. terreus. The frequency of phycomyceteous fungi was increased hence categorized in category (III). These are Mucor and Rhizopus and they were observed in all the samples, Penicillium notatum, Phoma nebulosa, Pithomyces cortarum and V. lecanii were grouped in the rare category (II) as they appeared in few samples.

December-February In early December both the layer of sal litter considerably changed in the physical characteristics. Most of the leaf lamina were broken into smaller pieces and turned into skeletal stage. On the basis of direct observation it was observed that this skeletal material was covered with black mycelial filamentous fungi which ran parallel as well as intermingled with the dead substrate under cool and dry condition. The sites also maintained their moisture by late winter rains and due to sub moist and cool condition the activity of coelomyceteous fungi followed by ascomyceteous fungi were recorded. The dematiaceous fungi were also recorded which colonized the final stage of sal litter decomposition. A total 32 fungal species were observed in decomposed sal litter. Zygomyceteous fungi for example, Rhizopus stolonifer was present as a rare category. The members of deuteromycetes dominated over other classes in this quarter. The frequency of Aspergilli decreased however, A. niger maintained its top order of colonization. The other two fungi, C. herbarum and L. shoreae were also exhibited dominance during the December ending. Ten members of coelomycete including species of Botryodiplodia, Coleophoma, Colletotrichum and Phoma were colonized the fragmented material of litter, pycnidia and pycnidial stomata profusely developed over the dead moist succulent part of sal litter and produced rich sporulation under cool and moist situation.

Discussion Mycoflora play an important role in the cycling of mineral nutrients by decomposing plant tissue. Their two fold action i.e. breaking down of complex organic compounds and trapping of the released elements in the

fungal bodies prevents the elements from leaching and balances the ecosystem (Witkamp1969). The decomposing sal litter possessed a great variety of fungi belonging to different taxonomic groups which have been recorded throughout the year under natural forest ecosystem. In this study the population of fungi colonizing the litter layers was studied qualitatively and quantitatively.

In sal forest leaf fall starts from the last week of February and continued till April. Initially the members of deuteromycete were the main colonizer. Alternaria alternata, A. shoreae, C. oxysporum, C. indica, C. lunata, and P. exigua were dominant colonizer on freshly fallen litter. As per their growth and pattern it appeared that these fungi were already present in senescent leaves prior to leaf fall. Dwivedi and Shukla (1977) studied fungal decomposition in relation to CO2 evolution in a tropical sal forest of Varanasi, Uttar Pradesh, India and reported that the phycomycetes are the initial colonizers, which were replaced by cellulose decomposing ascomycetes and deuteromycetes. They observed that fresh litter supported lesser number of fungi, half decomposed litter was colonized by a wide range of fungal species and the exhausted litter was invaded by only a few numbers of fungi. They also noted regular occurrence of Alternaria alternata, C. herbarum, and C. lunata, which was in the agreement with general observation as reported in tropics (Hudson 1968).

During June onwards T. harzianum, Trichoderma koningii, R. emetica, M. roseus, M. gordipes, Scleroderma verrucossum, T. viride were noticed in the fresh litter layer as well as in previous years granulated blackish humus layer. These basidiomyceteous mycorrhizal fungi extended their mycelial network in fragmented litter parts and surface grown fine feeder root system of sal. Due to sufficient moist ground the root network grew up superficially and well networked with the symbiont mycelial rhizomorphs. September was found the best month for the fungal development and decomposition process. Three species of Aspergillus i.e. A. niger, A. flavus, and A. terreus were recorded dominant. It is evident from the results that the litter after senescence is dominated by the members of deuteromycetes thus the beginning of the scheme doesnâ&#x20AC;&#x2122;t recall the general scheme as outlined by Garret (1963). The pattern of fungal succession in the litter of sal was alike in all the samples collected during all the four quarters and was similar to the finding of Hudson (1968). The member of coelomycetes was also the important component of litter decomposition of sal. Macauley and Throver (1966) established a definite succession of fungi on the leaves of Eucalyptus regnans during their decomposition. According to him coelomycetes tended to decrease with increasing decomposition. The pattern of ecological succession was followed as described by earlier workers (Ivarson and Sowden 1959; Hering 1967; Hudson 1968; Singh 1969; Jensen 1974; Dickinson 1976; Shukla 1976; Sinha and Dayal 1983; Soni and Jamaluddin 1990).

The majority of basidiomycetes appeared during August-September. The mycelium of these fungi activated decomposition process with onset of monsoon. It was due to the fact that each layer of litter became moist, succulent

SONI et al. – Litter decomposing fungi of sal forest

and porous to provide conducive environment for proper mycelial development. During September a number of such fungi appeared on the decaying litter some of which showed their fructifications in colony form while other only in mycelia form such as Coprinus aquatilis, C. rolfsii, M. gordipes, M. roseus, and several yellowish brownish and whitish mycelia forms exhibited typical character of clamp connection in their developing mycelial stage. The activity of micro-fauna was also increased especially they found dead due to growth of entomogeneous fungi Verticillium lecanii, Aspergillus sp., etc. The quantitative analysis of mycoflora showed higher frequency of member of deuteromycetes such as C. oxysporum and P. exigua.

The host specific fungi causing black mildew on senescent leaves were also present in freshly fallen leaf litter. Shukla (1976) also recorded competitive tolerance by the dominant fungal groups and tested culture filtrate of Aspergillus flavus, A. niger, A. sclerotiorum. A. terreus, and T. harzianum. Soni and Jamaluddin (1990) studied the fungal decomposition on Eucalyptus in dry deciduous forest for three successive years. They also found that members of ascomycetes, phycomycetes, and basidiomycetes were the weak colonizer whereas the deuteromycetes were the strong colonizer showing better adoptability and higher percentage distribution. According to them wide range of humidity and temperature regimes were suitable for litter decomposition. Pande (1999) compared the decomposition rate of four tree species viz, Shorea robusta, Tectona grandis, Eucalyptus and Pinus roxberghii. According to him leaf litter decomposition followed the order sal 1.67, teak 1.65, pine 1.35, and eucalyptus 1.34 (the values represent decomposition constants 'K' which were calculated by the formula x/xº= 1-ekt where xº= initial weight and x= weight remaining after the time t, Olson, 1963). In general values for higher decomposition rate were observed during rainy season and the lowest during winter. He also pointed out that rain fall, number of rainy days, soil moisture, and temperature showed positive correlation with decomposition rate.

Mirchink and Demkina (1977) studied the ecology of litter fungi. They obtained dominant presence of dark colored fungi as a proportion of total fungal species. In our study dark colored fungi recorded during later stage of sal litter decomposition. Cladosporium species is a one of the important litter colonizer that belongs to saprophytic group.

The litter of Scotts pine (Pinus sylvestris) has supported a highly characteristic mycoflora and many of the saprophytic fungi common on pine and angiosperms litters.

Results have indicated that many common soil fungi especially Trichoderma spp., member of mucorales and Penicillium spp. colonize the surface of decomposing needles (Hudson 1968). Kendrick and Burges (1962) also reported very high frequencies of these fungi on washed needles but suggested that attempts to wash the needles may have not been completely successful and that these fungi existed on the needle surface mainly as passive spore loads. One species of Penicillium and three species of Trichoderma are also recorded in the present study.

Egnnjobi (1974) studied the litter fall and mineralization in teak stand. He measured the litter fall at monthly interval

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for three years in a young stand of T. grandis dry forest zone of western Nigeria. On an average 70% of the litter fall between December and March and comprised 90% leaves. From the measurement of litter on the ground it is concluded that complete mineralization of teak litter occurred within six months. In our study we have observed that the complete mineralization of sal litter also took almost the same, time which in conformity with study of (Pande 1999). Some attempts were also made to stimulate growth of litter decomposition by fungi, for example, Lehmann and Hudson (1977) studied the fungal succession on normal and urea treated pine needles and reported that urea treatment stimulated development of C. herbarum but suppressed Lophiostroma pinastri. No such study has been conducted in tropical broad leaved forests.

CONCLUSION Sal litter on forest floor contains fungi throughout the year and most of them showed seasonal variations. The study showed that decomposition of litter continuously takes place throughout the year, however, the process intensified during the rainy season. The fresh litter generally colonizes by member of imperfect fungi including genera Alternaria, Cladosporium, Curvularia and Phoma that colonize freshly fallen litter. The majority of basidiomycetes including ecto-mycorrhizal fungi appeared during August-September and this is the best period for development of fungi and decomposition of litter. Dematiaceous fungi mostly colonize litter in later stages of decomposition.

ACKNOWLEDGEMENTS Authors are thankful to Dr. MS Negi, Director, Tropical Forest Research Institute, Jabalpur for providing necessary facilities during course of the study and ICFRE Dehradun for financial assistance under the project ID No. 136/TFRI/2009/Path 15(1).

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 3, Pp. 145-150 November 2011

Biological screening of selected traditional medicinal plants species utilized by local people of Manokwari, West Papua Province OBED LENSE♥ Faculty of Forestry, State University of Papua, Jl. Gunung Salju, Amban, Manokwari 98314, West Papua, Indonesia. Tel. +62-986-211065, Fax. +62986-211065, ♥email: obedlense@yahoo.com Manuscript received: 21 December 2010. Revision accepted: 16 November 2011.

ABSTRACT Abstract. Lense O. 2011. Biological screening of selected traditional medicinal plants species utilized by local people of Manokwari, West Papua Province. Nusantara Bioscience 3: 145-150. The aim of the research was to determine the presence of alkaloids and antimicrobial activity in extracts from selected medicinal plants from Manokwari District, West Papua, Indonesia. The method of alkaloid testing followed the standard phytochemical methods. The procedure of the Calibrated Dichotomous Sensitivity (CDS) test was used for the antimicrobial bioassays. Results of biological screening suggested that all but one of the 56 species tested contained different levels of alkaloids. Eleven species showed anti-microbial activity using bioassays of responses to two bacteria, Salmonella typhi and Klebsiella pneumoniae, and two fungi Candida albicans, and Cryptococcus neoformans; none of the plant extracts showed an antimicrobial effect against the bacteria Escherichia coli. Extract of Planconella sp. was the most active one as it showed activity against three different organisms (C. albicans, C. neoformans, and S. typhi).

Key words: biological screening, local people, Manokwari, traditional medicinal plant, West Papua.

Abstrak. Lense O. 2011. Penapisan hayati beberapa jenis tumbuhan obat tradisional terpilih yang dimanfaatkan oleh masyarakat lokal Manokwari, Provinsi Papua Barat. Nusantara Bioscience 3: 145-150. Tujuan penelitian ini adalah untuk mengetahui adanya alkaloid dan aktivitas anti-mikroba ekstrak beberapa tanaman obat terpilih dari Kabupaten Manokwari, Papua Barat, Indonesia. Metode pengujian alkaloid mengikuti metode fitokimia standar. Prosedur uji Calibrated Dichotomous Sensitivity (CDS) digunakan untuk uji hayati anti-mikroba. Hasil penapisan hayati menunjukkan bahwa ke-56 jenis yang diuji mengandung alkaloid dengan kadar yang berbeda-beda, kecuali satu jenis. Sebelas jenis menunjukkan aktivitas anti-mikroba berdasarkan respons uji hayati terhadap dua bakteri, Salmonella typhi dan Klebsiella pneumoniae, dan dua jamur Candida albicans dan Cryptococcus neoformans, tidak satupun dari ekstrak tanaman yang menunjukkan efek anti-mikroba terhadap bakteri Escherichia coli. Ekstrak Planconella sp. adalah yang paling aktif karena menunjukkan aktivitas terhadap tiga organisme yang berbeda (C. albicans, C. neoformans, dan S. typhi).

Kata kunci: penapisan biologi, masyarakat lokal, Manokwari, tumbuhan obat tradisional, Papua Barat.

INTRODUCTION Tropical rainforests with their high levels of diversity are considered to have great potential as a source of new drugs. The global trend of going “natural” or “green” has also contributed to the tropical rain forest being a target for such activities, combined with the added fear of forest depletion caused by logging, transmigration, and other developmental activities. Screening for biological activity using simple and fast bioassays is now being used to identify potentially useful plants. Phytochemical separations are routinely guided by bioassays which will ensure the isolation of bioactive agents irrespective of whether they belong to a certain class of compound or not.

The Manokwari tropical rainforest comprises a very rich and characteristic flora that covers more than 30,000 square kilometres of West Papua. Many of the plants in the

forests have been used as traditional medicines by the local people living in the area in order to treat several tropical diseases including malaria, fever, dysentery, wounds, and fungal or bacterial infections (MacKinnon 1991). However, no phytochemical analyses of medicinal plants from the Manokwari region have been conducted.

Fungi and bacteria cause important human diseases in tropical regions, especially in immunocompromised or immunodeficient patients. Despite the existence of potent antibiotic and antifungal agents, however, resistant or multi-resistant disease strains are continuously appearing, imposing the need for continuous research for and development of new drugs (Silver and Bostian 1993). In an effort to discover new compounds, many research groups have screened plant extracts to detect secondary metabolites with relevant biological activities.

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o the the pressent study waas to determinne the The aim of ppresence of alkaloids annd anti-microobial activitiees in e extracts from selected meddicinal plantss from Manokkwari D District, West Papua, Indonnesia.

M MATERIALS S AND METH HODS Collecting thee samples C Samples of o potentially useful plants were collectted in t field from February to April the A 2000 in collaborationn with t State Univversity of Pappua (UNIPA), Manokwari, West the P Papua Province, Indonesiaa. Specimens were collectted at t same timee for identificcation.. Sampples for laborratory the a analysis weree chosen from m the plants that are useed as m medicine souurces by tradditional healerrs (Martin 1995).

P Plant parts succh as leaves, fruits, flowerss, bark, stemss, and r roots were colllected for biological screenning.

Preparing and preserving the samples P Samples of o fresh plannt parts such as leaves, fruits, f f flowers, bark,, stems, and roots were broken b or cutt into s suitable sizes for transportt. Plant parts such as rootss and b bark were choopped into piieces using clippers. c All plants p w were air-driedd before beingg transported to the laboraatory, w where they were dried in an ovenn at a maxiimum t temperature off 50°C for 72 hours or moree depending on o the w water content of the samplees (Martin 19995).

Analysis the samples A s A Alkaloid screeening The methood of alkaloid testing follow wed the procedures o Culvenor and of a Fitzgeraldd (1963) and Frelich F and Marten M ( (1973).

Seven and half gram m of finely groound plant maaterial w was rapidly extracted with w 75 mL L of ammonniacal c chloroform (C CHCl3). Afteer filtration, the solution was e extracted by adding 9 mL m of sulphuuric acid. Three T m milliliters of extract e was thhen transferredd to a test tube and 9 drops of siilicotungstic acid a added (112 g silicotunngstic a acid to 100 mL m water). Thhe presence of o alkaloids in i the e extract phasee was deteccted by the formation of a p precipitate.

W Where the results were posittive, the amouunt of a alkaloid present was visuallly assessed annd ranked intoo five c classes accorrding to thee relative abbundance off the p precipitate (Coollins et al. 19990; Barr et al. 1993).

a Planchonellla Figure 1. The activvity of extracts of Litsea sp. and sp. ag gainst Candidaa albicans. The filter paper discs represent thhe plantt extracts that were w extracted uusing 50% and 90% EtOH. Thhe clear zone indicated the plant exxtract was effeective against C. C albiccans.

After A inoculattion, invertedd plates were incubated foor 18-2 24 hours at 355oC. Inhibitioon of growth of the bacteriia and fungi f by the plant p extracts was examined d by measurinng the diameter d of thhe clear zone (a microbe-ffree circle) thaat migh ht form arounnd the impregnnated filter paaper disc. If thhe disc showed clearr zones of 7 mm or moree, the microbees weree considered vulnerable tto inhibition by the plannt extraact and that thhe plant displaayed anti-miccrobial activityy.

In co ontrast, if the clear zone waas 6 mm or leess, it indicateed that the microbes were resistantt to the plant extract (Martiin 1995 5). Figure 1 shows s an exaample of agarr plate used in i anti--microbial acttivity screeninng. It shows that t the extracct of Planchonella P sp. was effeective against C. albicans, wherreas the extrracts of Litseea sp. showeed no activitty again nst C. albicanns.

RES SULTS AND DISCUSSION Anti-microbial screening A The proceddure of calibraated dichotom mous sensitivitty test ( (Bell et al. 19999) was used for the anti-m microbial bioasssays.

I the laboratoory, 2.5 g of dry In d finely groound plant maaterial w grounded into a powdeer and then divvided into sam was mples f different mixed for m with 50% and 90% ethanol, e and shhaken f 24 hours. The for T extracts were w filtered and a left to stannd for 2 hours undeer vacuum at 40 24 4 oC. Under sterile s conditioons, 5 μ of extract was μL w applied too a disc of filteer paper and placed p o an agar platte that had beeen inoculated with on w a single sppecies o bacterium (Salmonella typhi, Klebssiella pneumooniae, of a and Eschericchia coli) or fungus (C Candida albiicans, neoformanss), all of which C Cryptococcus w are huuman p pathogens.

Alka aloid screenin ng Fifty-eight F ethhanolic extraccts of variou us parts of 56 5 plantts used ass traditional medicinal plants werre investigated for thhe presence oor absence off alkaloids. All A but one o of these (555 species; 988%) contained d various levels of allkaloids (Table 1), but onlyy six appeared d to have a higgh levell of alkaloid presence p (Figuure 2).

The T results shhow a much higher percen ntage of plants givin ng a positivee alkaloid ressponse than similar s studiees elsew where. For exxample, a surrvey conducteed on endemiic species in Tasmania, Australiaa, indicated only15% o of thhe species gave a positive alkaloidd reading (Bicck et al. 19966).

In a study on alkaaloids of mediicinal plants from f Lombokk,

LENSE â&#x20AC;&#x201C; Traditional medicinal of Manokwari, West Papua

147

Table 1. Manokwari medicinal plants species giving negative and positive test results for alkaloids.

Dysentery Epilepsy, diarrhoea, queasy, fever Wound Earaches Fever, Malaria

Parts tested (results) Rhizomes (++++) Bark (++++) Leaves (++++) Stem (+++) Bark (+++++)

Wounds, gonorrhoea

Bark (++++)

Desire of having a child Cold, influenza Irritated eyes Liver diseases Malaria Chest pain Childbirth Dysentery Dysentery, irritated eyes

Leaves (++++) Leaves (+++) Leaves (++++) Bark (++++) Bark (++++) Bulb (+++) Bulb (+++) Leaves (+++) Leaves (+++) Stem (+++)

Kebar Kebar Minyambouw Wasior, Kebar Ransiki Merdey Ransiki, Anggi, Kebar

Ear pain, stomachaches, food poisoned Headaches, wounds Malaria and strong fever Fever, malaria Snake bite Fever Asthma Asthma

Moraceae

Wasior

Abscess, chest pain

Gigantochloa sp.

Poaceae

Wasior

Toothaches

Gnetum gnemon Homalantus nutans (Forst.f.)

Guillemin Horsfielda sp.

Instia palembanica Lansium domesticum Jack.

Laportea interrupta (L.) Chew.

Litocarpus brasii Litsea sp.

Loranthus sp.

Macaranga tanariius Mucuna novaguinensis Nauclea orientalis Octomeles sumatrana Miq. Palaquium sp.

Penthaphalaqium pachycarpum A.C. Smith.

Pimeliodendron amboinicum HSK Piper sp.

Pipturus repandus (Bl). Wedd.

Gnetaceae Euphorbiaceae

Merdey Ransiki,Anggi, Wasior, Kebar Merdey Merdey Wasior Kebar Kebar Manokwari, Minyambouw Merdey Ransiki, Anggi,Kebar Ransiki, Kebar Minyambouw, Merdey Ransiki, Anggi Merdey Ransiki, Anggi

New wounds Liver diseases

Leaves (++) Bark (++++) Leaves (+++) Leaves (+++) Bark (+++) Bark (++++) Bark (++++), Twigs (+++) Leaves (+++), Roots (+++) Outer bark (++++) Bark(++++) Leaves (++++)

Stomachaches Stomachaches Dysentery Malaria Muscular pain Scabies Gonorrhoea Fever (babies) Diarrhoea, malaria, fever Easy birth Fever Unspecified men sexual diseases Hinge pain

Bark (+++) Bark (++) Bark (+++) Leaves (+++) Bark (++++) Bark (++++) Leaves (++++) Leaves (++++) Leaves (+++) Shoot (++++) Bark (++++) Bark (++++) Bark (+++)

Plant species

Family

Localities

Acorus calamus L. Adenanthera microsperma Ageratum conyzoides Alpinia purpurata Alstonia scholaris R.Br.

Araceae Mimosaceae Asteraceae Zingiberaceae Apocynaceae

Artocarpus communis

Moraceae

Biophytum ptersianum Blumea saxatilis Calophyllum inophyllum L. Canarium sp..

Casuarina rumphiane Coelogyne asperata Colocasia sp.

Commelina nudiflora Cordyline fructiosa

Oxalidaceae Asteraceae Guttiferae Burseraceae Casuarinaceae Orchidaceae Araceae Commelinaceae Liliaceae

Costus speciosus (Koen) Sw.

Zingiberaceae

Ransiki, Anggi Manokwari Wasior, Minyambouw Kebar, Ransiki Ransiki, Kebar, Wasior, Manokwari Ransiki, Anggi, Kebar, Wasior, Merdey Kebar Ransiki, Anggi Ransiki Ransiki Manokwari Merdey Ransiki, Anggi Ransiki, Anggi Ransiki, Anggi, Minyambouw Merdey

Diplazium esculentum (Retz.) Sw. Disoxylon arborescens Miq. Drynaria quercifolia J.Sm Dryopteris sp.

Endospermum oluccanum Euodia sp.

Ficus sp.

Polypodiaceae Meliaceae Polypodiaceae Polypodiaceae Euphorbiaceae Rutaceae Moraceae

Ficus sp2.

Pisonia sp.

Planchonella sp.

Polygonum sp.

Polygonum sp.

Pothos scandens Pterocarpus indicus Willd.

Rhaphidophora oblongifolia Scott.

Rhaphidophora pertusa Roxb.

Myristicaceae Caesalpiniaceae Meliaceae Urticaceae Fagaceae Lauraceae Loranthaceae Euphorbiaceae Fabaceae Rubiaceae Dasticaceae Sapotaceae Clusiaceae Euporbiaceae Piperaceae Urticaceae Nyctaginaceae Sapotaceae Polygonaceae Polygonaceae Araceae Papilionaceae Araceae

Ransiki, Anggi, Kebar, Merdey Wasior, Ransiki, Anggi Ransiki, Anggi, Merdey, Manokwari Merdey Merdey Wasior, Kebar Kebar Merdey Kebar Wasior

Medical conditions

Headaches, unspecified men sexual Leaves (+++) diseases Stomachaches Leaves (+++) Fever, diarrhoea, epilepsy Bark (+++) Headaches Dysentery Scabies Dysentery Diarrhoea Dysentery New wounds

Roots (+++) Bark (++++) Root (++++) Leaves (++++) Leaves (-) Bark (++++) Leaves (++++)

Leaves (+++) Liver diseases, unspecified men sexual diseases Riccinus communis L. Euporbiaceae Ransiki Malaria, decoction before Leaves (++++) delivering a baby Schismatoglotis calyptra Roxb. Araceae Kebar Dislocated knee or arms Leaves (+++) Scindapsus hederaceaus Araceae Merdey Colds of infants Leaves (+++) Spathodea campanulata Bignoniaceae Minyambouw Tonic Bark (++++) Spathoglottis sp.

Orchidaceae Merdey Wounds Bulbs (+++) Note: The symbol in the bracket in the last column indicate the level of alkaloids presented: (-) no alkaloid, (+) very low, (++) low, (+++) medium, (++++) medium high, and (+++++) high level of alkaloids presented.

Araceae

Wasior, Merdey

1 148

3 (3): 145-150,, November 2011

Figure 2. Freqquency distribuution of the quualitative amouunt of F a alkaloids in 56 species medicinnal plants from m Manokwari District D g giving positive tests t for alkaloiids (5 is high).

223% of the medicinal m plannts tested show wed positive result r f for alkaloids (Hadi and Bremner 20001). In a siimilar a alkaloid survey from Quueensland, Auustralia, invoolving m many tropical and sub-tropiical species, 20 2 % of the sppecies t tested gave poositive result (Hadi and Brremner 2001). In a p phytochemical l survey off medicinal plants in SayapK Kinabalu Parkk, Sabah, Maalaysia, wheree 60 species were t tested for alkkaloids, onlyy eight speciies (13.3%) gave p positive resultts (Said et al. 1998).

1 Some of the species tested t for alkkaloids have been r reported to conntain alkaloids and other activve compoundss. The r rhizomes of Acorus A calam mus contain leucoantho-cy l yanins a 5,7-dihydrroxyflavanol (Cambie and ( and Brewis 1997)). The a active ingredieent in A. calamus is b-asaroone which belongs t the phenyl propanoid faamily (Baxterr et al. 1960)). The to s species of A. calamus contained the greaatest amount of ba asarone (70-966%) (Strelokee et al. 1989), including euggenol, m methyl-eugeno ol, acorin, caalamenol, calaamene, calam meone ( (Woodley 19991); cineole, linalol, pinene, resins, saafrole a tannins are also reported (Cowan 19999). and Hadi and Bremner B (2001) reported thhat the leaves, bark, a roots of Alstonia and A schoolaris and Ficcus septica coontain u unknown alkaaloids. The seeeds of these species are riich in h hallucinogenic c indole-alkaaloids (alstoveenine, venenatine, c chlorogenine, reserpine, ditamine, echitamine) and c chlorogenic a acid (a mild bladder andd urethra irrritant, r resulting in increased i sennsitivity of thhe genital reggion), w whereas the onnly alkaloids present p in thee bark and lateex are d ditamine, echiitamine, and echitenine.

Ming (1999) reported thhat Ageratum conyzoides c conntains a alkaloids, maiinly the pyrroolizidinic grouup, which suuggest t that it may be a good candidate forr pharmacoloogical s studies.

Alkalloid has beeen found in the species, with h hepatotoxic acctivity includiing 1,2-desifroopyrrolizidinic and licopsamine. Alkaloids A alsoo were found in i a hexane exxtract o A. conyzoiddes in Africa (Wiedenfeld and Roder 1991). of M Menut et al. (1993) reporrted that this species conttained h high percentagge of precoceene 1, particuularly those plants p f from Nigeria and a Cameroon which weree rich in precoocene 1 while oil extracted from 1, m Vietnamese and Fijian (S Suva) p plants containned roughly the same amounts of both

pounds. Terpeenoids, steroidds, flavonols, glucosides annd comp polyoxygenated flavones f havee been isolateed from plants from m India, Chhina, Nigeriaa and North hern Vietnam m.

Mon noterpene a-piinene and euggenol have beeen detected in i Indiaan plants, and α-farrnesene, hu umulene annd caryo ophyllene oxiide have beenn identified in n Fijian plants (Men nut et al. 19993). Hormones ageratochrromene and 77 meth hoxy-2, 2-metthylchromene (precocene-1) form 60 % of o the total t essential oils from thee flowers, leaaves, and stem ms of a Fijian variety (Aalbersbergg and Singh 19 991).

The T seeds of Lansium L domeesticum are kn nown to contaiin an am mount of an unnamed u alkalooid, 1% of an alcohol-soluble resin n (Morton 19887), and triterppenes (Bunyap praphatsara annd Saralamp 1982). Bunyapraphaatsara and Saaralamp (19822) foun nd only anti--inflammatoryy activity co onfined to thhe fracttions containinng triterpeness in seed extrracts. The nonnpolarr triterpene frraction showeed systemic activity a in a raat carraageenin-inducced model of iinflammation while the polaar fractiions reduced ear inflammattion. The find dings confirmeed the efficacy e of thee seeds of L. domesticum in i reducing eaar inflaammation (Bunnyapraphatsarra and Saralam mp 2001).

Cowan C (19999) reported tthat the seed ds of Ricinuus comm munis containned up to 3 % of the tox xalbumin ricinn.

This is one of thee most toxic suubstances kno own. They alsso contaained alkalooid ricinine,, cyanogenic glycosides, flavo onoids, steroiddal sapogeninn, garlic acid, and potassium m nitraate, and the oil is rich in riccinoleic, steariic, undecyleniic acid,, and ricinine (Grainge and Ahmed 1988)).

Moreover, M som me other geneera documenteed in this studdy havee been repoorted to conntain alkaloid ds and otheer comp pounds. The rhizomes r of A Alpinia galanga (L.) Willdd., reported to containn kaempferia,, galangin, a volatile v oil, annd galan ngol (which yields y cineole)), pinene, and eugenol (Perrry 1980 0). The extractt of stem and lleaves of Blum mea balsamiferra (L.) DC. contain alkaloids a and tannins flavonoids (Graingge and Ahmed 19888; Bhuiyan ett al. 2009). Fruits F of Pipeer guineense Schum m. & Thonn. ccontain the am mides piperine, N-iso o-butyloctadeeca-trans-2-traans-4-dienamid de, sylvatine, αα ,β-dihydropiperine and trichostaachine, and P. P nigrum haas piperrcide, dihydroopipercide, annd guineensinee (Miyakado et e al. 1989). The esssential oil from m the berries is i composed of o the terpenes: t pheellandrene, pinnene, and lim monene (Oliveer 1986 6).

Said S et al. (1998) repoorted that the t leaves of o Litho ocarpus confrragosus contaiined saponin (3+); ( the leavees and the bark of Litsea elliptibaacea contained d alkaloid (2+ +) and saponin (2+); the leavess of Ficus hemsleyana, h F F. lepiccarpa, F. rubrrocuspidata, aand F. stoloniifera containeed sapo onin (2+, 2+, 3+, and 3+ reespectively), and a Palaquium m sp. (lleaves) contaiined saponin ((3+).

Antii-microbial acctivity screen ning Of O the 56 plaant extracts ttested in an agar diffusioon assay y, 11 speciess were effecttive against the t two gram mnegaative bacteria (Klebsiella ( pnneumoniae, an nd S. typhi) annd two fungi (C. albiicans, C. neofo formans) assay yed.

Planchonella P s was the m sp. most active sp pecies, showinng activ vity against 3 different oorganisms (C C. albicans, C. C neofo formans, and S. typhi; Tablle 2 and Figu ure 3) followeed

by Adenanthera A m microsperma aand Dysoxylum m arborescens,

LENSE – Traditional medicinal of Manokwari, West Papua

both of which were effective in two bioassays (C. neoformans and Klebsiella pneumonaniaea). C. neoformans was the most susceptible of the two yeasts tested, with 7 extracts from a total of 11 extracts displaying activity against this organism. Against C. neoformans, the extracts from Ficus sp2. showed very significant inhibition (22.75 mm inhibition zone), followed by Dysoxylum arborescens (20.25 mm inhibition zone) and Laportea interrupta (17.50 mm inhibition zone). On the other hand, the extracts from Alpinia purpurata and Lithocarpus brassii showed less significant inhibition (7.5 mm inhibition zones) against C. neoformans and C. albicans respectively. None of the plant extract was effective against Escherichia coli.

The results of the laboratory-based anti-microbial activity screenings of plant species from Manokwari District suggested why the some traditional medicinal plants might

149

be effective against certain medical conditions. The bark of the stem of Planchonella sp, Adenanthera microsperma, and the leaves of Loranthus sp. are very commonly used by the native people in Manokwari District to treat dysentery, diarrhoea, and fever. The plant extracts of these species were effective against S. typhi which is one of the pathogenic microbes causing fever, diarrhoea, and headaches (Wasfy et al. 2000). The use of the bark of stems of Lithocarpus brassii in treating ringworm has also been supported by the anti-microbial screening results. The extracts of this species were confirmed effective against C. albicans which is an opportunistic organism (yeast) causing an itchy rash and occurs most often in warm, moist areas, such as under the arms, between skin folds, and in the groin (Bartie et al. 2001). Candida also causes mouth infections, particularly in babies and elderly.

Table 2. Manokwari medicinal plants species giving positive tests of Anti-microbial activity against Candida albicans (Ca), Cryptococcus neoformans (Cn), Salmonella typhi (St), Escherichia coli (Ec), Klebsiella pneumoniae (Kp) Medical conditions treated

Plant name

Part tested

Ca Acorus calamus Dysentery Rhizomes Adenanthera microsperma Epilepsy, diarrhoea, Bark nausea, and fever Alpinia purpurata Earaches Stem Colocasia sp.

Childbirth Bulbs 8.50 Disoxylon arborescens Fever, malaria Bark Ficus sp2.

Eye irritation, toothaches Leaves Instia palembanica Dysentery Bark Laportea interrupta Muscular pains Leaves Litocarpus brassii Ringworm Bark 8.13 Loranthus sp.

Fever in babies Leaves Planchonella sp.

Dysentery, diarrhoea Bark 12.25

Diameter of inhibition zones 50 % EtOH 90% EtOH Cn St Ec Kp Ca Cn St Ec 16.00 9.00 8.17 7.88

7.50 8.50

20.50 22.70 11.38 17.50

16.00 12.50 7.50

9.00 8.00 10.25

8.00

20.00 15.00 10.00 5.00

Cryptococcus neoformans Klebsiella pneumoniaea

Salmonella typhi

Figure 3. The activity of extracts of Several Manokwari medicinal plants against 5 different bioassays tested.

Planchonella sp.

Loranthus sp.

Litocarpus brasii

Laportea interrupta

Intsia palembanica

Ficus sp2.

Dysoxylum arborescens

Colocasia sp.

Alpinia purpurata

Adenanthera microsperma

0.00 Acorus calamus

Diameter of inhibition zones (mm)

25.00

Candida albicans Escherichia coli

Kp

150

3 (3): 145-150, November 2011

In addition, the anti-microbial screening indicated that the extracts of fresh leaves of the nettle Laportea interrupta and the bark of the stem of Dysoxylum arborescens were very effective against C. neoformans that can cause fatigue and fever (symptoms of pneumonia; Kopecka et al. 2000).

This finding agrees with the use of Laportea interrupta and Dysoxylum arborescens in this region to treat muscular pains for fatigue and fever, respectively (Table 2).

However there is no previous information regarding preparations of antibiotics from Laportea sp. to treat this pathogen, although Foster and Duke (1990) reported that it has shown antibacterial and central nervous system depressant activity.

CONCLUSION Initial work on Manokwari medicinal plants has resulted in fifty-six species being collected and screened for the presence of alkaloids and anti-microbial activity.

Results indicated that at least 55 species of the 56 species rainforest species analysed were shown to contain different level of alkaloids. Anti-microbial activity tests indicated that 11 species were effective against three Gram-negative (Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi) bacterial species and two fungi (Candida albicans, Cryptococcus neoformans). Planconella sp. was the most active species as it showed activity against three different organisms (C. albicans, C. neoformans, and S. typhi).

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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, Quake SR, You L. 2008. A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187. 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.

| Nu us Biosci | vol. 3 3 | no. 3 | pp. 105‐150 | Nove ember 2011| ISSN 2087‐394 48 (PRINT) | ISSSN 2087‐3956 ((ELECTRONIC) 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

Development of an efficient protoccol for genom mic DNA extraaction from m mango (Mangifera a indica) DILRUBA A ASHRAFUN N NAHAR MAJUMDER, LUTTFUL HASSA AN, MOHAMMAD ABDUR R RAHIM, MOHAMM MAD AHSANUL KABIR

105‐111

Blood bactterial wilt dissease of banaana: the distrribution of paathogen in in nfected plantt, symptomss, and potenttiality of diseaased tissues as source of infective ino oculums HADIWIYO ONO

112‐117

Synthesis aand study of cholosubstittuted 4‐aroyll pyrazolines and isoxazollines and theeir effects on inorgan nic ions in blo ood serum in n albino rats AMOL D. B BHOYAR, GA ANESH N. VA ANKHADE, PR RITHVISIGH R. RAJPUT

118‐123

Selection o of parents tre ees for Rubbe er (Hevea bra rasiliensis) bre eeding based d on RAPD an nalysis FETRINA O OKTAVIA, MU UDJI LASMIN NINGSIH, KU USWANHADI

124‐129

Variation in oil contentts, and seed aand seedlingg characteristtics of Jatroph ha curcas of W West Nusa Tenggara sselected geno otypes and th heir first imp proved popullation BAMBANG G BUDI SANTTOSO

130‐135

Litter deco omposing fun ngi in sal (Sho orea robusta)) forests of ce entral India KRISHNA K KANT SONI, ABHISHEK P PYASI, RAM K KEERTI VERM MA

136‐144

Biological sscreening of selected trad ditional medicinal plants species utilizzed by local p people of Manokwarri, West Papu ua Province OBED LENSE

145‐150

Societty for Indon nesian Biodive ersity

Sebellas Maret Univversity Surakkarta

Published three timess in one yearr PRINTED IN INDONESIIA

ISSSN 2087‐3948 (prin nt)

ISSN 2087‐3 3956 (electronic)