nus biosci | vol. 3 | no. 3 | pp. 105-150| november 2011

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

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EDITORIAL BOARD: Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia ([email protected]) Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia ([email protected]) Editorial Advisory Boards: Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia ([email protected]) Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia ([email protected]) Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India ([email protected]) Biomedical Sciences, Alfiono, Sebelas Maret University, Surakarta, Indonesia ([email protected]), Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia ([email protected]), Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia ([email protected]) Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia ([email protected]) Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia ([email protected]), Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia ([email protected]) Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt ([email protected]) Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia ([email protected]) Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia ([email protected]) Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China ([email protected]) Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia ([email protected]) Management Boards: Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta ([email protected]) Associated Editor (English Editor), Wiryono, State University of Bengkulu ([email protected]), Technical Editor, Ari Pitoyo, Sebelas Maret University Surakarta ([email protected]) Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok, Indonesia ([email protected]) PUBLISHER: Society for Indonesian Biodiversity CO-PUBLISHER: School of Graduates, Sebelas Maret University Surakarta FIRST PUBLISHED: 2009 ADDRESS: Bioscience Program, School of Graduates, Sebelas Maret University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: [email protected] ONLINE: biosains.mipa.uns.ac.id/nusbioscience

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| 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

ISSN: 2087-3948 (print) Vol. 3, No. 3, Pp. 105-111 ISSN: 2087-3956 (electronic) November 2011

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

DILRUBA ASHRAFUN NAHAR MAJUMDER1,♥, LUTFUL HASSAN2, MOHAMMAD ABDUR RAHIM3, MOHAMMAD AHSANUL KABIR4

1Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh; Tel. +8801714261388; Fax. +88029261424; ♥email: [email protected]

2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh 3Department of Horticulture, Bangladesh Agricultural University, Mymensingh, Bangladesh

4Department 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.),

3 (3): 105-111, November 2011

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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 water-saturated 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 (hexadecyltrimethyl-ammonoum 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 (polyvinyl-pyrollidone) 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

107

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 water-saturated 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 Tris-HCl pH 8.0, polyvinylpolypyrrolidone and 0.2% 2-mercaptoethanol), 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.

(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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109

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

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)

GenotypesDNA concentration (µg/µL)

GenotypesDNA concentration (µg/µL)

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%) 6.951 (85.44%)

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

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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).

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

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.

ACKNOWLEDGEMENTS

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.

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

1000

600

400

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

1000

700

500

200 bp


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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: 404-408.

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.

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: 415a-415f.

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.


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. +62-271-

646655., ♥email: [email protected]

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

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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.


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-tag-polymerase, 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 oC 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

(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.


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

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3 (3): 112-117, November 2011

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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

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HADIWIYONO – Blood bacterial wilt disease of banana

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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.

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. Dissertation . University of Gadjah Mada, Yogyakarta. Indonesia .

Louws FJ, Rademaker JLW, de Bruijn FJ. 1999. The three Ds of PCR-based 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.


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

AMOL D. BHOYAR1,♥, GANESH N. VANKHADE2, PRITHVISIGH R. RAJPUT3 1Department 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: [email protected] 2Department of Zoology, Sant Gadge Baba Amravati University, Amravati 444602, Maharasthra, India 3Department 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 3,5-dichloroacetophenones (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-3,5-dichlorophenyl)-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 hydroxylamine-hydrochloride 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-3,5-diklorofenil)-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 hidroksilamin-hidroklorida 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, nephro-toxicitym (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.

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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 organo-phosphates, 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.


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-3,5-dichloro-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 phenylhydrazine-hydrochloride 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-aroyl-isoxazolines (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).

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-propane-diones (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-(2-hydroxy-3,5-dichloro-phenyl)-3-aryl-1,3-ropanedione (3a-b) 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-3,5-dichloro-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-(2-hydroxy-3,5-dichlorophenyl-3-(2’-hydroxyphenyl)-1,3-proponedione. 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, Ar-OCH3; 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%.

3 (3): 118-123, November 2011

120

Cl

(1)

COCH 3

OH

Cl

(2a -b)

COCH3

OCO

a

b

R1

R2 Cl

Cl

C

(4a-d )

R3

Cl

Cl O

O

O C R1

R4

R2

O

C CH2

Cl

C

OH

Cl

O

R1

R2

(3a-b)

d

e

g

f

R1

Cl

Cl

OH

N N

C

O

( 5a-d ) Ph

R3

R4

R2

OOH

NO

C

Cl

R1 Cl

(6a-d)

R2

R3

R4

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 Molecular formula

Molecular weight R1 R2 R3 R3 M.P. Yield

(%) Rf Cal. (Found ) C N

2a C15H10O3Cl2 309 H H - - 690C 72 0.71 58.14 (58.25) - 2b C16H12O4Cl2 339 OCH3 H - - 1190C 77 0.82 56.41 (56.43) - 3a C15H10O3Cl2 309 H H - - 1220C 82 0.76 58.18 (58.25) - 3b C16H12O4Cl2 339 OCH3 OCH3 - - 1120C 78 0.85 56.48 (56.63) - 4a C22H14O3Cl2 417 H H H H 1610C 87 0.42 61.17 (61.53) - 4b C23H16O4Cl2 447 H H OCH3 H 1780C 72 0.85 64.55 (64.63) - 4c C23H16O4Cl2 447 OCH3 H H H 1670C 75 0.61 64.46 (64.63) - 4d C24H18O5Cl2 477 OCH3 H OCH3 H 1560C 62 0.44 62.99 (63.01) - 5a C28H20O2 N2Cl2 473 H H H H 1690C 85 0.70 68.62 (68.99) 5.35 (5.47) 5b C29H22O3 N2Cl2 503 H H OCH3 H 1650C 82 0.82 67.24 (67.31) 5.32 (5.41) 5c C29H22O3 N2Cl2 503 OCH3 H H H 1720C 68 0.73 67.22 (67.31) 5.32 (5.41) 5d C30H24O4 N2Cl2 533 OCH3 H OCH3 H 1700C 75 0.44 65.67 (65.81) 5.01 (5.11) 6a C22H15O3 NCl2 412 H H H H 1930C 86 0.73 64.01 (64.07) 3.31 (3.39) 6b C23H17O4 NCl2 442 H H OCH3 H 1880C 84 0.79 62.33 (62.44) 3.07 (3.16) 6c C23H17O4 NCl2 442 OCH3 H H H 1950C 71 0.88 62.38 (62.44) 3.10 (3.16) 6d C24H19O5 NCl2 472 OCH3 H OCH3 H 1800C 82 0.83 60.96 (61.01) 2.81 (2.96)


121

4-Aroyl-Δ2-Pyrazolines (5a-d) When 3-aroylflavanone (4a-d) and phenyl-hydrazine-

hydrochloride (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%.

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 ethanol-acetic 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%

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

OOH

NO

C

Cl

(7c)

O

C

OO

Cl

Cl

OCH3

Cl

Cl

O

OCH3....

OH

C C CH

COPh

Cl

Cl

O

OCH3..

OH

C C CH

COPh

:NH2OH

+

Cl

Cl

OH

C C

O- COPh

CH OCH3NOH

H

H +

Cl

-Cl

OH

C C

OH COPh

CH OCH3NOH

H+

-H2O

Cl

-Cl

OH

C C

COPh

CH OCH3NOH

+

Cl

OCH3

- +-

..

Ph

OCl

Cl

OH

C C

C

CH OCH3NOH

-H2O

3 (3): 118-123, November 2011

122

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

Weeks Inorganic ions Control Induced Chlorosubstituted heterocycles

5d 6d 2 Sodium 1.36 + 0.17 1.41+ 0.18 1.37+ 0.17 1.36+ 0.17

Potassium 6.08+ 0.18 6.95+ 0.20 6.10+ 0.20 6.22+ 0.17 Calcium 6.30+ 0.20 8.7+ 0.29 7.38+ 0.20 7.49+ 0.29 Magnesium 3.5+ 0.17 3.8+ 0.19 3.6+ 0.17 3.72+ 0.18

4 Sodium 1.36+ 0.17 1.39+ 0.18 1.35+ 0.17 1.36+0.18 Potassium 6.18+ 0.17 7.37+ 0.20 6.38+ 0.17 6.25+ 0.18 Calcium 6.30+ 0.29 11.2+ 0.34 7.35+ 0.34 7.42+ 0.21 Magnesium 3.5+ 0.17 4.7+ 0.18 3.62+ 0.17 3.78+ 0.19

6 Sodium 1.36+ 0.17 1.48+ 0.17 1.39+ 0.18 1.42+ 0.18 Potassium 6.18+ 0.20 8.27+ 0.17 7.28+ 0.18 7.40+ 0.20 Calcium 6.30+ 0.20 13.8+ 0.29 7.39+ 0.20 7.35+ 0.20 Magnesium 3.5+ 0.18 4.2+ 0.18 3.58+ 0.19 3.72+ 0.19

8 Sodium 1.36+ 0.17 1.55+ 0.18 1.40+ 0.17 1.39+ 0.18 Potassium 6.18+ 0.18 8.84+ 0.17 7.32+ 0.18 7.25+ 0.18 Calcium 6.30+ 21 15.82+ 0.34 7.42+ 0.20 7.38+ 0.21 Magnesium 3.5+ 0.19 3.9+ 0.19 3.62+ 0.17 3.57+ 0.18

10 Sodium 1.36+ 0.17 1.58+ 0.17 1.42+ 0.18 1.45+ 0.17 Potassium 6.18+ 0.18 9.40+ 0.20 7.40+ 0.20 7.36+ 0.17 Calcium 6.30+ 10.20 16.96+ 0.21 7.39+ 0.29 7.51+ 0.29 Magnesium 3.5+ 0.17 4.1+ 0.17 3.58+ 0.17 3.71+ 0.18

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.


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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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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: [email protected]

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

125

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 poly-morphism 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 polivinyl-polipyrolidon (PVPP), in each sample at the time of grinding in liquid nitrogen to fine powder using pestle and

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 ß-mercaptoetanol) was added immediately. The mixture was homogenized by gentle shaking, and incubated at 65oC for 30 minutes. An equal volume of chloroform-isoamylalcohol (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 at-20oC, 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 Primer sequences (5’ 3’) Primer Primer sequences

(5’ 3’)

OPN-01 OPN-02 OPN-03 OPN-04 OPN-05 OPN-06 OPN-07 OPN-08 OPN-09 OPN-10

5’-CTCACGTTGG-3’ 5’-ACCAGGGGCA-3’ 5’-GGTACTCCCC-3’ 5’-GACCGACCCA-3’5’-ACTGAACGCC-3’ 5’-GAGACGCACA-3’ 5’-CAGCCCAGAG-3’ 5’-ACCTCAGCTC-3’ 5’-TGCCGGCTTG-3’ 5’-ACAACTGGGG-3’

OPN-11 OPN-12 OPN-13 OPN-14 OPN-15 OPN-16 OPN-17 OPN-18 OPN-19 OPN-20

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 250-3000 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 OPN-08 (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 2. IRR 39 3. IRR 104 4. IRR 118 5. IRR 105 6. RRIM 2004 7. RRIM 2020

8. RRIM 600 9. Tjir 1 10. PN 138 11. PB 217 12. IRR 44 13. IRR 100 14. BPM 107

15. PN 177 16. RRIM 901 17. RRIM 911 18. PN 680 19. BPM 109 20. BPM 24 21. BPM 1

22. PR 300 23. PB 260 24. GT1 25. PR 303 26. LCB1320 27. RRIC 100 28. RRIC 110

29. RRIC 101 30. RRIM 712 31. IRR 42 32. IRR 41 33. TM 5 34. TM 8 35. IRR 18

36. H. benthamiana 37. IRR 220 38. PB 235 39. IRR 204 40. RRIC 101 41. IRR 32 42. TM 9

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


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 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.ben IRR 220 PB 235 IRR 204 RRIC 101 IRR 32 TM 9 BPPJ 3 BN 1 AV 352

IRR 24 1.0000IRR 39 0.9365 1.0000IRR 104 0.9016 0.8852 1.0000IRR 118 0.8833 0.8833 0.8448 1.0000IRR 105 0.7664 0.7477 0.8155 0.7723 1.0000RRIM 2004 0.8421 0.8421 0.8727 0.8333 0.8211 1.0000RRIM 2020 0.8182 0.8000 0.8679 0.8269 0.8791 0.8980 1.0000RRIM 600 0.8718 0.8376 0.8850 0.8288 0.8367 0.8762 0.8713 1.0000TJIR 1 0.7928 0.8108 0.8598 0.7810 0.7826 0.8283 0.8421 0.8627 1.0000PN 138 0.7731 0.7731 0.7652 0.7434 0.7200 0.7850 0.7767 0.7636 0.7692 1.0000PB 217 0.8814 0.8644 0.8772 0.8214 0.8081 0.8491 0.8431 0.9174 0.8544 0.7928 1.0000IRR 44 0.8780 0.8780 0.8739 0.8376 0.7500 0.8468 0.7850 0.8421 0.7963 0.7586 0.8696 1.0000IRR 100 0.8393 0.8214 0.8519 0.8491 0.7742 0.8600 0.8333 0.8544 0.8247 0.8000 0.8654 0.8624 1.0000BPM 107 0.8644 0.8475 0.9123 0.8750 0.8081 0.8491 0.8627 0.8807 0.8544 0.7748 0.8545 0.8522 0.8846 1.0000PN 177 0.8618 0.8293 0.9076 0.8205 0.7692 0.8288 0.8411 0.8421 0.8148 0.7586 0.8348 0.8167 0.8073 0.8870 1.0000RRIM 901 0.8525 0.8361 0.8644 0.8276 0.7767 0.8364 0.8491 0.8850 0.8224 0.7826 0.8947 0.8067 0.8333 0.8596 0.8403 1.0000RRIM 911 0.7680 0.7680 0.8430 0.7563 0.7170 0.7788 0.7523 0.7931 0.7818 0.6949 0.8205 0.7705 0.7568 0.7863 0.7869 0.8760 1.0000PN 680 0.7731 0.8067 0.8348 0.8142 0.7400 0.7664 0.7767 0.7818 0.7885 0.7321 0.8108 0.7759 0.7429 0.7928 0.7586 0.8174 0.8475 1.0000BPM 109 0.7863 0.7863 0.7788 0.7387 0.7143 0.7810 0.7723 0.7593 0.7647 0.7636 0.8073 0.7719 0.8155 0.7523 0.7193 0.8319 0.7759 0.8182 1.0000BPM 24 0.8000 0.8000 0.8099 0.8067 0.7170 0.7788 0.7890 0.7586 0.7818 0.7966 0.8205 0.8197 0.8468 0.8034 0.7869 0.8099 0.7742 0.8136 0.8448 1.0000BPM 1 0.7934 0.7934 0.8034 0.8174 0.7255 0.8073 0.8190 0.7857 0.7547 0.7895 0.7965 0.7797 0.8411 0.8319 0.7627 0.8376 0.7500 0.7719 0.8393 0.8667 1.0000PR 300 0.8033 0.7705 0.7966 0.7759 0.7961 0.7818 0.8113 0.8319 0.7477 0.7652 0.8246 0.8235 0.8148 0.8246 0.7731 0.8475 0.7603 0.7826 0.8142 0.8430 0.8376 1.0000PB 260 0.7826 0.7826 0.8288 0.7890 0.7708 0.8155 0.8485 0.8491 0.8200 0.7593 0.8598 0.8214 0.8713 0.8224 0.7679 0.8649 0.8070 0.8148 0.8113 0.8421 0.8545 0.8649 1.0000GT 1 0.7438 0.7273 0.7521 0.7478 0.6863 0.7156 0.7619 0.7500 0.7170 0.7193 0.7434 0.7797 0.7477 0.7257 0.7288 0.7521 0.7500 0.7719 0.7679 0.8500 0.7586 0.8205 0.8364 1.0000PR 303 0.8167 0.7667 0.7931 0.8246 0.7723 0.7963 0.8269 0.8108 0.7429 0.7611 0.8036 0.8034 0.8113 0.8036 0.7863 0.8448 0.7563 0.7965 0.8108 0.8403 0.8348 0.9138 0.8624 0.8348 1.0000LCB 1320 0.8739 0.8235 0.8522 0.8319 0.7600 0.8037 0.8155 0.8364 0.7692 0.7143 0.8108 0.8276 0.8190 0.8649 0.7931 0.8696 0.7627 0.7857 0.7818 0.8305 0.8421 0.8696 0.8704 0.8070 0.8496 1.0000RRIC 100 0.8136 0.7627 0.7719 0.8036 0.7071 0.7736 0.7647 0.7523 0.7184 0.7207 0.7818 0.8000 0.8077 0.8000 0.7478 0.8596 0.7863 0.7748 0.8073 0.8547 0.8496 0.8421 0.8224 0.7788 0.8571 0.9189 1.0000RRIC 110 0.7321 0.6786 0.7222 0.7547 0.6882 0.7200 0.7500 0.7379 0.7629 0.7048 0.7115 0.7156 0.7347 0.7308 0.6972 0.7778 0.7207 0.7810 0.7184 0.7748 0.7477 0.7778 0.7921 0.8037 0.8491 0.8190 0.8077 1.0000RRIC 102 0.8000 0.7478 0.7928 0.8073 0.7500 0.7961 0.8081 0.8113 0.7600 0.7593 0.8037 0.7857 0.8317 0.8037 0.7679 0.8468 0.7719 0.7593 0.7925 0.8246 0.8364 0.8108 0.8462 0.8000 0.8440 0.8704 0.8785 0.8119 1.0000RRIM 712 0.7434 0.7434 0.7890 0.8037 0.7021 0.7525 0.7423 0.7308 0.7347 0.7170 0.7429 0.7636 0.7475 0.7619 0.7455 0.8073 0.7500 0.7925 0.7308 0.7679 0.7963 0.7706 0.7843 0.7222 0.8224 0.7925 0.8000 0.8283 0.8039 1.0000IRR 42 0.7414 0.7414 0.7143 0.7636 0.6186 0.7115 0.7400 0.6916 0.6733 0.6789 0.6667 0.7434 0.6863 0.7222 0.7257 0.6964 0.6435 0.7156 0.6729 0.7478 0.7207 0.7500 0.7048 0.7207 0.7636 0.7523 0.7407 0.6863 0.7048 0.6990 1.0000IRR 41 0.7368 0.7368 0.7091 0.7593 0.6526 0.7255 0.7755 0.7238 0.6869 0.6729 0.6981 0.7387 0.6800 0.7170 0.7027 0.7455 0.6726 0.7477 0.7048 0.7434 0.7523 0.7818 0.7573 0.7523 0.7963 0.7850 0.7736 0.7200 0.7184 0.7327 0.9423 1.0000TM 5 0.7705 0.7705 0.7797 0.8276 0.6990 0.7636 0.7925 0.7434 0.7290 0.7304 0.7193 0.8067 0.7407 0.7895 0.8067 0.7797 0.7273 0.7478 0.7080 0.7934 0.7863 0.8136 0.7748 0.7692 0.8276 0.8174 0.8070 0.7593 0.7928 0.8073 0.8750 0.8727 1.0000TM 8 0.7521 0.7692 0.7788 0.8108 0.7143 0.7619 0.7921 0.7407 0.7255 0.7818 0.7523 0.7544 0.7767 0.7706 0.7544 0.7965 0.7241 0.7818 0.7407 0.8103 0.8036 0.7965 0.8302 0.7679 0.8288 0.8000 0.7890 0.7767 0.7736 0.8077 0.8411 0.8381 0.8673 1.0000IRR 18 0.7434 0.7788 0.7890 0.7850 0.7447 0.7723 0.8247 0.7885 0.7755 0.7358 0.7619 0.7273 0.7475 0.7810 0.7455 0.8073 0.7500 0.7736 0.7115 0.7321 0.7963 0.7890 0.8431 0.7222 0.8037 0.7925 0.7429 0.7475 0.7451 0.7800 0.7961 0.8515 0.8257 0.8846 1.0000H.benthamiana 0.7304 0.7130 0.7748 0.7706 0.7292 0.7379 0.8081 0.7736 0.7200 0.7407 0.7477 0.7679 0.7525 0.7664 0.7679 0.7928 0.7193 0.7593 0.6981 0.7719 0.7818 0.8468 0.8077 0.7455 0.8440 0.7778 0.7664 0.7525 0.7692 0.8039 0.8190 0.8544 0.8829 0.8679 0.8824 1.0000IRR 220 0.7521 0.7521 0.7788 0.7568 0.6735 0.7429 0.7525 0.7593 0.7255 0.7273 0.7523 0.8070 0.7184 0.7339 0.7719 0.7434 0.7069 0.7273 0.6667 0.7586 0.7500 0.7965 0.7736 0.7321 0.7748 0.7455 0.7339 0.6990 0.7547 0.7500 0.8224 0.8190 0.8850 0.8333 0.8269 0.8679 1.0000PB 235 0.7080 0.7080 0.7890 0.7290 0.7234 0.7129 0.7835 0.7500 0.7143 0.7170 0.7429 0.7455 0.7475 0.7619 0.7455 0.7523 0.6964 0.7547 0.7115 0.7500 0.7593 0.7890 0.8039 0.7222 0.7477 0.7736 0.7238 0.7071 0.7843 0.7600 0.7573 0.7723 0.8073 0.8077 0.8000 0.8431 0.8462 1.0000IRR 204 0.7018 0.7193 0.7636 0.7407 0.6737 0.7255 0.7347 0.7619 0.7071 0.7103 0.7170 0.7568 0.7200 0.7170 0.7207 0.7273 0.7080 0.7477 0.6857 0.7080 0.7339 0.7636 0.7573 0.6972 0.7407 0.7290 0.6981 0.6800 0.7573 0.7327 0.7500 0.7451 0.8364 0.8190 0.7921 0.8350 0.9143 0.8911 1.0000RRIC 101 0.6786 0.6964 0.7222 0.6981 0.6667 0.6800 0.7083 0.7184 0.6804 0.6857 0.6923 0.6972 0.6531 0.6731 0.6606 0.7037 0.6667 0.7238 0.6602 0.6847 0.7103 0.7407 0.7327 0.6729 0.6981 0.7238 0.6731 0.6735 0.7129 0.6869 0.7451 0.7400 0.7963 0.7961 0.7879 0.7921 0.8350 0.8485 0.9000 1.0000IRR 32 0.7826 0.8000 0.7748 0.7890 0.6250 0.6990 0.7071 0.7170 0.7000 0.6667 0.7477 0.7679 0.6733 0.7290 0.7321 0.7748 0.6842 0.7593 0.6604 0.7193 0.7273 0.7387 0.7308 0.6727 0.7339 0.7778 0.7477 0.6733 0.6923 0.7647 0.8190 0.8155 0.8108 0.8113 0.8039 0.8077 0.8302 0.7843 0.7961 0.8119 1.0000TM 9 0.7156 0.6972 0.7048 0.6796 0.6222 0.6598 0.6667 0.7000 0.6383 0.6471 0.6931 0.6792 0.6105 0.6535 0.7170 0.7429 0.6667 0.6863 0.6000 0.6296 0.6346 0.6667 0.6939 0.6154 0.6796 0.7255 0.6931 0.6737 0.6735 0.7500 0.7475 0.7423 0.7619 0.8000 0.7708 0.7755 0.7800 0.7708 0.7835 0.7789 0.8776 1.0000BPPJ 3 0.7091 0.7636 0.7170 0.7115 0.6154 0.6735 0.6809 0.7129 0.6947 0.6602 0.6667 0.7290 0.6667 0.6667 0.6729 0.6792 0.6239 0.6796 0.6337 0.6606 0.6857 0.6792 0.7273 0.6286 0.6538 0.6990 0.6471 0.5833 0.6465 0.6598 0.7400 0.7143 0.7736 0.7525 0.7629 0.7475 0.7921 0.7629 0.7959 0.7917 0.8485 0.8172 1.0000BN 1 0.7788 0.8142 0.7890 0.7664 0.6596 0.7327 0.7216 0.7500 0.7143 0.6604 0.7238 0.7636 0.6667 0.7238 0.7273 0.7339 0.7143 0.7547 0.6538 0.6607 0.7037 0.6972 0.7451 0.6667 0.7103 0.7547 0.7048 0.6667 0.6863 0.7400 0.7961 0.7723 0.8073 0.8077 0.8200 0.7843 0.8269 0.8000 0.8317 0.8283 0.8824 0.8750 0.9072 1.0000AV 352 0.6786 0.7143 0.7037 0.6604 0.6882 0.6800 0.7083 0.7379 0.7010 0.6476 0.6923 0.6972 0.6122 0.6731 0.6422 0.7037 0.6486 0.6857 0.6408 0.6306 0.6542 0.7222 0.6931 0.6355 0.6792 0.6857 0.6154 0.5918 0.6733 0.6465 0.7255 0.7200 0.7222 0.7184 0.7475 0.7525 0.7573 0.7677 0.8200 0.7755 0.8119 0.8000 0.8333 0.8081 1.0000

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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

Coefficient0.73 0.78 0.83 0.89 0.94

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


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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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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: [email protected]

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

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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

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.


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 Amor-

Amor 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 the seeds prior to sowing by soaking them in warm water (50OC) for two hours, let it cool and kept soaking for 24 hours. Seed were sown directly in black polythene bags containing media mixture of soil, sand and manure with a ratio of 1:1:1 (by volume). Seedlings were grown into 2.5 month oldsaplings.

Two and half month –old saplings of each genotype were planted in the field experiment using Randomized Complete Block design at 2x2 m2 spacing. Three replicates of genotypes, each consisting of 24 were applied.

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

Table 1. Region of seeds’ sources and their climatic condition.

Seed sources (genotype,

district)

Region (subdistrict)

Altitude (m)

Rain fall (mm)

Temperature(OC)

Air moisture

(%) Max. Min.West Lombok Khayangan 50-75 600-1,000 31 25 90 Central Lombok South Praya 30-55 900-1,300 31 24 90 East Lombok Masbagik 75-100 1,000-1,500 31 25 85 Sumbawa Alas 50-75 550-1,000 32 25 85 Bima Sanggar 50-100 600-750 32 24 85

Table 2. Climate condition of experimental site during 2006-2007

Climate component 2006 2007 Rainfall (mm) 965 716 Rainy month (months) 5 5 Rainy day (days) 56 59 Air temperature (OC) min. 24.7 25 Air temperature (OC) max. 31 32 Relative humidity (%) 90 91

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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 15-20 capsules) during the first production cycle. Seeds collected from selected tree as IP-1 were prepared for studying the seedling characteristics.

Parameters observed Physical characters of seed and

seed viabilities were measured and seed oil content was analyzed. Seed length and seed width were measured using a caliper. Seed weight and weight of 100 seeds were measured 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.


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.

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

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

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

Tabel 4. Percentage of kernel weight to total seed weight and 100 seed weight of Jatropha curcas seeds of West Nusa Tenggara genotypes

Genotypes Percentage of kernel weight (%) 100 seeds weight (g) P0*) IP-1*) P0*) IP-1*)

West Lombok 67.27 ab 71.18 a 69.1 a 75.7 a Central Lombok 58.45 b 65.66 b 66.4 ab 70.1 ab East Lombok 60.01 ab 68.36 ab 60.6 b 65.7 ab Sumbawa 64.29 ab 69.08 ab 65.2 ab 69.5 abc Bima 67.12 ab 69.96 ab 67.8 a 71.0 ab LSD 5% 10.05 5.05 6.45 9.33 Tabel 5. Kernel oil content of Jatropha curcas of West Nusa Tenggara genotypes

Genotypes Kernel oil content (% b/b)

P0 *) IP-1 Rainy season Dry season

West Lombok 41.7 ab 43.7 44.6 Central Lombok 42.3 a 42.6 43.1 East Lombok 38.8 b 41.9 42.9 Sumbawa 42.9 a 43.6 44.3 Bima 41.1 ab 43.4 44.3 LSD 5% 3.41 ns ns


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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

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.

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

Genotypes Germination

(%) Germination

rate (day) Seed vigourity

(%)

Dry weight of 3 weeks old sapling (g)

P0 IP-1 P0 IP-1 P0 IP-1 P0*) IP-1 West Lombok 84.3 88.9 11.40 10.07 89.93 90.06 0.58 a 0.59 Central Lombok 79.3 86.7 12.97 11.03 82.50 85.76 0.45 b 0.52 East Lombok 82.0 85.9 12.49 11.56 89.37 90.35 0.48 ab 0.53 Sumbawa 81.7 87.4 12.67 10.22 89.70 90.54 0.55 ab 0.56 Bima 80.9 88.2 12.40 11.01 86.83 89.88 0.53 b 0.54 LSD 5% ns ns ns ns ns ns 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) Number of leaves Collar diameter

(cm) 1 month

old 2 month

old 1 month

old 2 month

old 1 month

old 2 month

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 Seedling shoot (g) Seedling root (g) 1 month old 2 month old 1 month old 2 month old

P0 West Lombok 4.95±1.798 7.33±1.233 0.94±1.595 1.74±1.013 Central Lombok 3.55±1.882 6.88±1.372 0.63±1.551 1.35±1.154 East Lombok 3.92±1.908 6.91±1.501 0.66±1.462 1.43±1.096 Sumbawa 4.71±1.636 7.16±1.362 0.79±1.357 1.58±1.005 Bima 4.32±1.872 7.43±1.239 0.88±1.623 1.66±1.102 IP-1 West Lombok 5.79±1.124 9.01±0.863 1.23±1.026 1.95±0.902 Central Lombok 4.95±1.132 8.28±0.994 0.96±1.147 1.69±1.006 East Lombok 5.19±1.164 8.76±0.907 1.12±1.115 1.71±1.021 Sumbawa 5.46±1.087 9.12±0.821 1.29±1.076 1.98±0.942 Bima 5.24±1.139 8.95±0.879 1.22±1.083 2.02±0.879

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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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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.


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

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Perkebunan, Badan Penelitian dan Pengembangan Pertanian. Second Edition. Jakarta. Deptan. 35p. [Indonesian]

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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.

Zhang, Z, Guo X, Liu B, Tang L, Chen F. 2011. Genetic diversity and genetic relationship of Jatropha curcas between China and Southeast Asian revealed by amplified fragment length polymorphisms. African J Biotechnol 10 (15): 2825-2832.


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: [email protected]

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, March-May, 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

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of decomposinthree states offungi under vagiven.

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different statChhattisgarh Amarkantak-AMadhya PradeE81045') and Madhya PradeAchanakmar (

Figure 1. Map comes in Madhy

enomenon is dsuccessional funable to conved mineral co

m the terrestriae dealt with thecomposition p

or factor-wise rther mycorrhsystem in moneration that won the ecologd for detailed in litter decomtra and Anejaaluddin 19902002; Hossainesent study anand study the ng litter underf central Indiaarious stages

MATERIALS

chosen for coltes of Indiand Orissa.

Achanakmar besh and Chha

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showing study ya Pradesh, 3. G

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of litter deco

S AND METH

llection of sala namely M

The selectebiosphere resattisgarh; AmN22°21'0" E

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sites in three sGariyaband and

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unequal distribarries large amorganic molecuould be drastial forest. Thisand further nuomplete chain

h gradual minment in and ares or in juv

y disturbed. Rdecomposing r to understanwivedi and Sha and Dayal et al. 1984; M2005). s made to isssion in the prforest ecosysteand importan

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ect observationLeaf litter sames with a steridle and apexpton and Broroscope and fu

paration of funDamp chambeed in a sterilest chamber. ument ecologie on potato d1). Dilution methoas described aer, rinsed replized 500 mL

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ecomposing fuy on litter dere processed,per methods, b

n mple squares wile parallel razx. These pieowns 1962), oungal coloniza

ngal culture er method. S

e petriplates oFungal flor

cal successiondextrose agar

od. Forty squabove were peatedly for f

L conical flaskwrist shaken

mL inoculumsmedium and icubator. EachThe frequencsena (1955).

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GARH

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nd E82°39’ to s sal forest beled species pranzan, Anogeingia panicula

2009 to Februa

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Squares of lef 9.0 cm diama was recorn. Isolation of(PDA) mediu

uares of litter placed in 60 mfive times thek containing 6and dilution

s from each incubated for h petridish wacy class was

1. Amarkantak lls in Orissa.

ORISSA

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85°15’) falls ilongs to seconresent in theseissus latifoliaata. This studary 2010.

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5x5 mm2 smam from the bas

eaned, staineer stereo-zoomorded.

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sample piecemL of distilleen placed in 60 mL distilleup to 10-3 wadilution werseven days a

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3 (3): 136-144, November 2011

138

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:

Categorization of frequency classes

Class % Frequency Category I 1-20 Rare II 21-40 Occasional III 41-60 Frequent IV 61-80 Common V 81-100 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 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

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

SONI et al. – Litter decomposing fungi of sal forest

139

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

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).

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

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

A

F E H

B D

G

C

A B

1

FG J

apmwfatadfobTdftmsswbboaifo

140

Figure 4. A-HGeastrum triplex

June-August During Jun

and spread thrprogressed. Tmonsoon andwind current, formation wasand in depresthe second weand within 15darker. The liforest floor. Toccurred conbecomes succuThe decompodepending upfloor. Regular this period is most of the sosufficient moissaprophytic fwhitish, light between the bodies of seveon forest flooattached to thi.e. 85-95% rfavors the excother saproph

H. Mycorrhizal x, D. Boletus fall

ne most of throughout the fhe litter grad

d weathering, hot weather

s observed nesed ground aneek of June s

5 days the coitter set up ihe color, textu

nsequently duulent which wosing litter

pon the canoprain increasedvery import

il borne fungisture colonizafungi were dbrownish andgaps of deco

eral gasteromyor in a colonhe feeder root relative humiellent growth

hytic fungi. C

E

A

and litter declax, E. Russula e

he leaf and noforest floor andually change

started simur followed byear tree bases,nd formed olstrong showelor of litter sn an approprure, and other

ue to heavy were readily c

sheets took py density and the activity tant for litter i were very acation by severadeveloped and blackish weomposing paycetous fungi nial form wht network of sidity and 27-of soil, myco

Common ecto-

omposing fungemetica, F. Scler

on leaf litter mnd the stratificed on the onsultaneously duy rains. The , stone boundigostratal laye

er soaked the started changiriate frame onr chemical cha

rains. The colonized by f

different shnd slope of fof fungal flordecompositio

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3 (3): 136-144,

gi of sal forestroderma bovista

mixed cation set of ue to layer

daries, er. In litter

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, November 20

t. A. Astraeus a, G. Scleroderm

ced were Boex, Russula eSclerodermaena, were also

Over all 32 specolonizer of l

hese eight fungriplex, Lophoena roseus, Rle fungus-1 gorized underest percentag

gi namely Aspsterile fungusnized with agory the natuorrhizal and nific and theired to substratategory third uent colonizadosporium heatium, Corticle 3-4, Tricnicus, whiche fungi suddeer and middlen fungi were r

11

hygrometricusma geaster, and H

oletus fallax emetica, Scler

verrucosum o emerged on ecies belonginglitter decompogal species naodermium shoR. emetica, Tr

shown itsr dominant frge of occurrpergillus niges-2 were incluabundant percure of fungal non mycorrhir occurrence te and moistur(III), eight fu

ation level. Terbarum, C. cium rolfsii, choderma vibelong to deu

enly increasede layer of littrecorded and

G

C

s, B. GeastrumH. Scleroderma v

Calvatia elaroderma bovi(Figure 4). Mforest floor.

g to 23 genera woser during Junamely Aspergioreae, Marasm

Trichoderma hs highest frrequency (V) rence. Simulter, C. elata, uded in (IV) centage occursuccession a

izal. These fuand colonizatre content of ungi were rec

These are Aspoxysporum, C. lunata, P

iride and Wuteromycete. d and rapidlyter. In categocategorized as

m fimbriatum, Cverrucosum

ata, Geastrumista, S. geasteMarasmius an

were recognizene-August. Ouillus fumigatumius gordipe

harzianum, anfrequency an

and exhibitetaneously fivS. verrucosumfrequency an

rrence. In thagents are botungi are seasotion is directltop litter layeorded with th

pergillus astuColletotrichum

Phoma exiguaWiesneriomyce

Populations oy colonized thory second (Is occasional.

H

D

C.

m er nd

ed ut

us, es, nd nd ed ve m

nd is th on ly er. he s, m a, es of he I)


141

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 June - Aug Sep- Nov Dec - FebFreq Occur Freq Occur Freq Occur Freq Occur

Achlya debaryana Hump. I 10.5 II 11.4 - - - -Alternaria alternata Fr. Keissl. III 8.6 - - - - II 6.4Alternaria citri Penz. I 1.8 - - - - Aspergillus flavus Link. III 13.5 III 8.2 V 24.2 V 19.8Aspergillus fumigatus Fres. - - V 25.3 V 20.5 IV 18.0Aspergillus niger Tiegh. III 11.8 IV 18.2 V 22.2 V 23.2Aspergillus terreus Thom. - - - - - - V 20.5Aspergillus ustus Bainier - - III 13.0 - - II 6.4Asterostomella shoreae Soni, Hosag,Pyasi & RK Verma V 20.5 - - - - - -Astraeus hygrometricus Pers. - - V 28.0 - - - -Boletus fallax Corner - - IV 17.6 - - -Botryodiplodea theobromae Pat. - - - - - - II 6.4Calvatia elata (Massee) Morgan - - IV 18.2 - - Chaetomium globosum Kunze ex Fr. - - - - II 3.9 II 3.7Cladosporium cladosporioides Link II 4.6 II 4.6 - - II 2.8Cladosporium herbarum Pers - - III 13.0 - - III 13.0Cladosporium oxysporum Berk V 28.2 III 8.8 - - II 5.0Colletotrichum dematium Pers. - - III 11.8 - - Colletotrichum gloeosporioides (Penz.) Sacc. - - II 8.6 - - II 7.8Coprinus aquatilis Peck - - - - - - II 4.1Corticium rolfsii Curzi. - - - - III 11.4 - -Curvularia indica Subram. IV 18.0 - - II 5.3 I 1.5Curvularia lunata Wakker IV 18.6 - - III 12.9 III 12.6Curvularia prasadii Boedijn. - - - - II 3.8 - -Drechslera spicifera (Bainier) Arx I 1.2 - - - - - -Fusarium concolor Reinking. I 1.0 - - II 3.3 - -Fusarium equiseti (Corda) Sacc. - - - - II 2.6 - -Fusarium moniliforme J. Sheld. - - - - II 2.8 - -Fusarium semitectum Berk. - - - - II 2.4 II 2.3Fusarium solani Sac. I 1.2 II 3.9 - - II 3.8Geastrum triplex Jungh. - - IV 7.6 - - II 8.5Geastrum fimbriatum Fr. - - IV 7.6 - - II 8.5Gliocladium virens Corda - - - - - - III 11.6Lophodermium shoreae Jamal, Dadwal & Soni - - V 20.5 - - III 10.7Marasmius gordipes Sacc. & Paol. - - V 28.0 - - II 4.5Mucor circinelloides Tiegh. - - - - III 12.4 III 10.6Mycena roseus Pers. - - V 20.2 - - Paecilomyces variotii Bainier. - - - - - - I 1.8Penicillium notatum Westling. II 7.4 - - - - I 0.8Periconia minutissima Corda - - II 5.3 - - - -Pestalotiopsis versicolor (Speg.) Steyaert - - - - II 4.6 II 3.5Phoma exigua Desm. V 28.1 - - III 2.0 II 4.6Phoma macrostoma Mont. - - - - II 6.7 - -Phoma medicaginis Malbr. & Roum. - - - - II 2.5 - -Phoma multirostrata (P.N. Mathur, S.K. Menon & Thirum.) Dorenb. & Boerema

- - - - II 3.6 - -

Phoma nebulosa (Pers.) Berk. - - - - II 7.6 I 1.6Pithomyces cortarum Berk. - - - - - - I 1.2Rhizopus stolonifer Ehrenb. III 10.6 II 10.0 II 8.6 III 10.3Russula 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 III 11.1 - -Sterile fungus 4 - - III 11.5 III 10.5 - -Trichoderma harzianum Rifai - - V 28.2 - - III 11.5Trichoderma koningii Oudem. III 13.8 - - - - II 7.8Trichoderma viride Pers. - - III 10.8 - - - -Verticillium lecanii Zimm. - - - - II 3.5 I 2.5Wiesneriomyces javanicus Koord. - - - - III 10.3 - -Helicosporium phragmitis Höhn. - - - - - - III 11.2Note: Freq = frequency; Occur = occurrence. The Roman numbers show the frequency classes and the Arabic numbers represent percentage frequency and occurrence of fungi.

3 (3): 136-144, November 2011

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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 doesn’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


143

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

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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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. +62-

986-211065, ♥email: [email protected]

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 anti-microbial 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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SULTS AND

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DISCUSSIO

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LENSE – Traditional medicinal of Manokwari, West Papua 147

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

Plant species Family Localities Medical conditions Partstested (results)

Acorus calamus L. Araceae Ransiki, Anggi Dysentery Rhizomes (++++)Adenanthera microsperma Mimosaceae Manokwari Epilepsy, diarrhoea, queasy, fever Bark (++++)Ageratum conyzoides Asteraceae Wasior, Minyambouw Wound Leaves (++++)Alpinia purpurata Zingiberaceae Kebar, Ransiki Earaches Stem (+++)Alstonia scholaris R.Br. Apocynaceae Ransiki, Kebar, Wasior,

ManokwariFever, Malaria Bark (+++++)

Artocarpus communis Moraceae Ransiki, Anggi, Kebar, Wasior, Merdey

Wounds, gonorrhoea Bark (++++)

Biophytum ptersianum Oxalidaceae Kebar Desire of having a child Leaves (++++)Blumea saxatilis Asteraceae Ransiki, Anggi Cold, influenza Leaves (+++)Calophyllum inophyllum L. Guttiferae Ransiki Irritated eyes Leaves (++++)Canarium sp.. Burseraceae Ransiki Liver diseases Bark (++++)Casuarina rumphiane Casuarinaceae Manokwari Malaria Bark (++++)Coelogyne asperata Orchidaceae Merdey Chest pain Bulb (+++)Colocasia sp. Araceae Ransiki, Anggi Childbirth Bulb (+++)Commelina nudiflora Commelinaceae Ransiki, Anggi Dysentery Leaves (+++)Cordyline fructiosa Liliaceae Ransiki, Anggi,

MinyambouwDysentery, irritated eyes Leaves (+++)

Costus speciosus (Koen) Sw. Zingiberaceae Merdey Ear pain, stomachaches, food poisoned

Stem (+++)

Diplazium esculentum (Retz.) Sw. Polypodiaceae Kebar Headaches, wounds Leaves (++)Disoxylon arborescens Miq. Meliaceae Kebar Malaria and strong fever Bark (++++)Drynaria quercifolia J.Sm Polypodiaceae Minyambouw Fever, malaria Leaves (+++)Dryopteris sp. Polypodiaceae Wasior, Kebar Snake bite Leaves (+++)Endospermum oluccanum Euphorbiaceae Ransiki Fever Bark (+++)Euodia sp. Rutaceae Merdey Asthma Bark (++++)Ficus sp. Moraceae Ransiki, Anggi, Kebar Asthma Bark (++++),

Twigs (+++)Ficus sp2. Moraceae Wasior Abscess, chest pain Leaves (+++),

Roots (+++)Gigantochloa sp. Poaceae Wasior Toothaches Outer bark

(++++)Gnetum gnemon Gnetaceae Merdey New wounds Bark(++++)Homalantus nutans (Forst.f.) Guillemin

Euphorbiaceae Ransiki,Anggi, Wasior, Kebar

Liver diseases Leaves (++++)

Horsfielda sp. Myristicaceae Merdey Stomachaches Bark (+++)Instia palembanica Caesalpiniaceae Merdey Stomachaches Bark (++)Lansium domesticum Jack. Meliaceae Wasior Dysentery Bark (+++)Laportea interrupta (L.) Chew. Urticaceae Kebar Malaria Leaves (+++)Litocarpus brasii Fagaceae Kebar Muscular pain Bark (++++)Litsea sp. Lauraceae Manokwari, Minyambouw Scabies Bark (++++)Loranthus sp. Loranthaceae Merdey Gonorrhoea Leaves (++++)Macaranga tanariius Euphorbiaceae Ransiki, Anggi,Kebar Fever (babies) Leaves (++++)Mucuna novaguinensis Fabaceae Ransiki, Kebar Diarrhoea, malaria, fever Leaves (+++)Nauclea orientalis Rubiaceae Minyambouw, Merdey Easy birth Shoot (++++)Octomeles sumatrana Miq. Dasticaceae Ransiki, Anggi Fever Bark (++++)Palaquium sp. Sapotaceae Merdey Unspecified men sexual diseases Bark (++++)Penthaphalaqium pachycarpum A.C. Smith.

Clusiaceae Ransiki, Anggi

Hinge pain Bark (+++)

Pimeliodendron amboinicum HSK

Euporbiaceae Ransiki, Anggi, Kebar,Merdey

Headaches, unspecified men sexual diseases

Leaves (+++)

Piper sp. Piperaceae Wasior, Ransiki, Anggi Stomachaches Leaves (+++)Pipturus repandus (Bl). Wedd. Urticaceae Ransiki, Anggi, Merdey,

ManokwariFever, diarrhoea, epilepsy Bark (+++)

Pisonia sp. Nyctaginaceae Merdey Headaches Roots (+++)Planchonella sp. Sapotaceae Merdey Dysentery Bark (++++)Polygonum sp. Polygonaceae Wasior, Kebar Scabies Root (++++)Polygonum sp. Polygonaceae Kebar Dysentery Leaves (++++)Pothos scandens Araceae Merdey Diarrhoea Leaves (-)Pterocarpus indicus Willd. Papilionaceae Kebar Dysentery Bark (++++)Rhaphidophora oblongifolia Scott.

Araceae Wasior New wounds Leaves (++++)

Rhaphidophora pertusa Roxb. Araceae Wasior, Merdey Liver diseases, unspecified men sexual diseases

Leaves (+++)

Riccinus communis L. Euporbiaceae Ransiki Malaria, decoction before delivering a baby

Leaves (++++)

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.

3 (3): 145-150, November 2011148

Figure 2. Frequency distribution of the qualitative amount ofalkaloids in 56 species medicinal plants from Manokwari Districtgiving positive tests for alkaloids (5 is high).

23% of the medicinal plants tested showed positive resultfor alkaloids (Hadi and Bremner 2001). In a similaralkaloid survey from Queensland, Australia, involvingmany tropical and sub-tropical species, 20 % of the speciestested gave positive result (Hadi and Bremner 2001). In aphytochemical survey of medicinal plants in Sayap-Kinabalu Park, Sabah, Malaysia, where 60 species weretested for alkaloids, only eight species (13.3%) gavepositive results (Said et al. 1998).

Some of the species tested for alkaloids have beenreported to contain alkaloids and other active compounds. Therhizomes of Acorus calamus contain leucoantho-cyaninsand 5,7-dihydroxyflavanol (Cambie and Brewis 1997). Theactive ingredient in A. calamus is b-asarone which belongsto the phenyl propanoid family (Baxter et al. 1960). Thespecies of A. calamus contained the greatest amount of b-asarone (70-96%) (Streloke et al. 1989), including eugenol,methyl-eugenol, acorin, calamenol, calamene, calameone(Woodley 1991); cineole, linalol, pinene, resins, safroleand tannins are also reported (Cowan 1999).

Hadi and Bremner (2001) reported that the leaves, bark,and roots of Alstonia scholaris and Ficus septica containunknown alkaloids. The seeds of these species are rich inhallucinogenic indole-alkaloids (alstovenine, venenatine,chlorogenine, reserpine, ditamine, echitamine) andchlorogenic acid (a mild bladder and urethra irritant,resulting in increased sensitivity of the genital region),whereas the only alkaloids present in the bark and latex areditamine, echitamine, and echitenine.

Ming (1999) reported that Ageratum conyzoides containsalkaloids, mainly the pyrrolizidinic group, which suggestthat it may be a good candidate for pharmacologicalstudies. Alkaloid has been found in the species, withhepatotoxic activity including 1,2-desifropyrrolizidinic andlicopsamine. Alkaloids also were found in a hexane extractof A. conyzoides in Africa (Wiedenfeld and Roder 1991).Menut et al. (1993) reported that this species containedhigh percentage of precocene 1, particularly those plantsfrom Nigeria and Cameroon which were rich in precocene1, while oil extracted from Vietnamese and Fijian (Suva)plants contained roughly the same amounts of both

compounds. Terpenoids, steroids, flavonols, glucosides andpolyoxygenated flavones have been isolated from plantsfrom India, China, Nigeria and Northern Vietnam.Monoterpene a-pinene and eugenol have been detected inIndian plants, and α-farnesene, humulene andcaryophyllene oxide have been identified in Fijian plants(Menut et al. 1993). Hormones ageratochromene and 7-methoxy-2, 2-methylchromene (precocene-1) form 60 % ofthe total essential oils from the flowers, leaves, and stemsof a Fijian variety (Aalbersberg and Singh 1991).

The seeds of Lansium domesticum are known to containan amount of an unnamed alkaloid, 1% of an alcohol-solubleresin (Morton 1987), and triterpenes (Bunyapraphatsara andSaralamp 1982). Bunyapraphatsara and Saralamp (1982)found only anti-inflammatory activity confined to thefractions containing triterpenes in seed extracts. The non-polar triterpene fraction showed systemic activity in a ratcarrageenin-induced model of inflammation while the polarfractions reduced ear inflammation. The findings confirmedthe efficacy of the seeds of L. domesticum in reducing earinflammation (Bunyapraphatsara and Saralamp 2001).

Cowan (1999) reported that the seeds of Ricinuscommunis contained up to 3 % of the toxalbumin ricin.This is one of the most toxic substances known. They alsocontained alkaloid ricinine, cyanogenic glycosides,flavonoids, steroidal sapogenin, garlic acid, and potassiumnitrate, and the oil is rich in ricinoleic, stearic, undecylenicacid, and ricinine (Grainge and Ahmed 1988).

Moreover, some other genera documented in this studyhave been reported to contain alkaloids and othercompounds. The rhizomes of Alpinia galanga (L.) Willd.,reported to contain kaempferia, galangin, a volatile oil, andgalangol (which yields cineole), pinene, and eugenol (Perry1980). The extract of stem and leaves of Blumea balsamifera(L.) DC. contain alkaloids and tannins flavonoids (Graingeand Ahmed 1988; Bhuiyan et al. 2009). Fruits of Piperguineense Schum. & Thonn. contain the amides piperine,N-iso-butyloctadeca-trans-2-trans-4-dienamide, sylvatine, α-,β-dihydropiperine and trichostachine, and P. nigrum haspipercide, dihydropipercide, and guineensine (Miyakado etal. 1989). The essential oil from the berries is composed ofthe terpenes: phellandrene, pinene, and limonene (Oliver1986).

Said et al. (1998) reported that the leaves ofLithocarpus confragosus contained saponin (3+); the leavesand the bark of Litsea elliptibacea contained alkaloid (2+)and saponin (2+); the leaves of Ficus hemsleyana, F.lepicarpa, F. rubrocuspidata, and F. stolonifera containedsaponin (2+, 2+, 3+, and 3+ respectively), and Palaquiumsp. (leaves) contained saponin (3+).

Anti-microbial activity screeningOf the 56 plant extracts tested in an agar diffusion

assay, 11 species were effective against the two gram-negative bacteria (Klebsiella pneumoniae, and S. typhi) andtwo fungi (C. albicans, C. neoformans) assayed.

Planchonella sp. was the most active species, showingactivity against 3 different organisms (C. albicans, C.neoformans, and S. typhi; Table 2 and Figure 3) followedby Adenanthera microsperma and Dysoxylum arborescens,

Alkaloid level (precipitate

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LENSE – Traditional medicinal of Manokwari, West Papua 149

both of which were effective in two bioassays (C. neoformansand Klebsiella pneumonaniaea). C. neoformans was themost susceptible of the two yeasts tested, with 7 extractsfrom a total of 11 extracts displaying activity against thisorganism. Against C. neoformans, the extracts from Ficussp2. showed very significant inhibition (22.75 mminhibition zone), followed by Dysoxylum arborescens(20.25 mm inhibition zone) and Laportea interrupta (17.50mm inhibition zone). On the other hand, the extracts fromAlpinia purpurata and Lithocarpus brassii showed lesssignificant inhibition (7.5 mm inhibition zones) against C.neoformans and C. albicans respectively. None of the plantextract was effective against Escherichia coli.

The results of the laboratory-based anti-microbial activityscreenings of plant species from Manokwari Districtsuggested why the some traditional medicinal plants might

be effective against certain medical conditions. The bark ofthe stem of Planchonella sp, Adenanthera microsperma,and the leaves of Loranthus sp. are very commonly used bythe native people in Manokwari District to treat dysentery,diarrhoea, and fever. The plant extracts of these specieswere effective against S. typhi which is one of thepathogenic microbes causing fever, diarrhoea, andheadaches (Wasfy et al. 2000). The use of the bark of stemsof Lithocarpus brassii in treating ringworm has also beensupported by the anti-microbial screening results. Theextracts 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, moistareas, such as under the arms, between skin folds, and inthe groin (Bartie et al. 2001). Candida also causes mouthinfections, 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)

Plant name Medical conditionstreated Part tested

Diameter of inhibition zones50 % EtOH 90% EtOH

Ca Cn St Ec Kp Ca Cn St Ec KpAcorus calamus Dysentery Rhizomes 16.00Adenanthera microsperma Epilepsy, diarrhoea,

nausea, and feverBark 9.00 8.17

Alpinia purpurata Earaches Stem 7.88 7.50Colocasia sp. Childbirth Bulbs 8.50 8.50Disoxylon arborescens Fever, malaria Bark 20.50 16.00Ficus sp2. Eye irritation, toothaches Leaves 22.70Instia palembanica Dysentery Bark 11.38 12.50Laportea interrupta Muscular pains Leaves 17.50Litocarpus brassii Ringworm Bark 8.13 7.50Loranthus sp. Fever in babies Leaves 9.00 8.00Planchonella sp. Dysentery, diarrhoea Bark 12.25 8.00 10.25

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

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3 (3): 145-150, November 2011

150

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).

REFFERENCES

Aalbersberg WGL, Singh Y. 1991. Essential oil of Fijian Ageratum Conyzoides. Flav and Frag J 6: 117-120.

Ayensu ES. 1981. Medicinal plants of the West Indies. Reference Publications, Inc., Algonac, MI.

Barr A, Chapman J, Smith N, Wightman G. 1993. Traditional aboriginal medicines in the Northern Territory of Australia. Conservation Commission of Northern Territory of Australia, Darwin.

Bartie KL, Williams DW, Wilson MJ, Potts JC, Lewis MAO. 2001. PCR Fingerprinting of Candida albicans associated with chronic hyperplastic candidosis and other oral conditions. J of Clin Microbiol 39(11): 4066-4075.

Baxter RM, Dandiya PC, Kandal SL. 1960. Separation of the hypnotic potentiating principles from the essential oil of Acorus calamus L. of Indian origin by liquid-gas chromatography. Nature 185: 466-467.

Bell DT, King LA, Plummer JA. 1999. Ecophysiological effects of light quality and nitrate on seed germination in species from Western Australia. Australian J of Ecol 24: 2-10.

Bell SM, Gatus BJ, Pham JN. 1999. Antibiotic susceptibility testing by the CDS method. A concise laboratory manual 1999. Arthur Productions Pty., Ltd. Sydney, Australia.

Bhuiyan Ni, Chowdhury Ju, And Begum J. 2009. Chemical Components In Volatile Oil From Blumea Balsamifera (L.) Dc. Bangladesh J Bot 38 (1) : 107-109.

Bick IRC, Bremer JB, Paano AMC, and Preston NW. 1996. A survey of Tasmanian Plants for Alkaloids. University of Wollongong, Australia.

Bunyapraphatsara N, Saralamp P. 1982. Thai crude drugs : their preparations and pecifications. J Pharmacol Sci 9(4):83-87.

Cambie, R.C. and Brewis, A..A. 1997. Anti-fertility plants of the Pacific. CSIRO, Collingwood.

Collins DJ, Culvenor CCJ, Lambertson JA, Loder JW, Price JR. 1990. A chemical and pharmacological survey of plants in the Australian Region. CSIRO, Melbourne.

Cowan MM. 1999. Plant products as antimicrobial agents. Clin Microbiol Rev 12 (4): 564-582.

Culvenor CCJ, Fitzgerald JS. 1963. A field method for alkaloid screening of plants. J Pharmacol Sci 52:303-306.

Foster S, Duke J. 1990. Medicinal plants. Houghton Miffin Company, Boston.

Frelich JR, Marthen GC. 1973. Quick test for reed canarygrass alkaloid concentration. J Crop Sci 13: 548-551.

Grainge M, Ahmed S. 1988. Handbook of plants with pest-control properties. Wiley and Sons, New York.

Hadi S, Bremner JB. 2001. Initial studies on alkaloids from Lombok Medicinal Plants. Mol 6: 117-129.

Kopecka M, Yamaguchi M, Gabriel M, Takeo K, Svobodaa A. 2000. Morphological transitions during the cell devision cycle of Cryptococcus neoformans as revealed by transmisson electron microscopy of ultrathin sections and freezer-subtitution. Scrip Med (BRNO) 73 (6): 369-380.

MacKinnon K. 1991. Economic value of biodiversity; Conservation Indonesia. News of the WWW Indo Prog 7(3): 4 - 6.

Martin GJ. 1995. Ethnobotany: A people and plants conservation manual. Chapman and Hall, London.

Menut C, Sharma S, Luthra C. 1993. Aromatic plants of tropical central Africa, Part X—Chemical composition of essential oils of Ageratum houstonianum Mill. and Ageratum conyzoides L. from Cameroon. Flav and Frag J 8(1):1-4.

Ming LC. 1999. Ageratum conyzoides: A tropical source of medicinal and agricultural products. p. 469-473. In: Janick J (eds.), Perspectives on new crops and new uses. ASHS Press, Alexandria, VA.

Miyakado M, Nakayama I, Ohno N. 1989. Insecticidal unsaturated isobutylamides from natural products to agrochemical leads. In: Arnason JT, Philogene BJR, Morand P (eds) Insecticide of Plant Origin: ACS symposium series 387. American Chemical Society, Washington.

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Wasfy MO, Oyofo BA, David JC, Ismail TF, El-Gendy AM, Mohran ZS, Sultan Y, Peruski LF. 2000. Isolation and antibiotic susceptibility of Salmonella. J Health Pop Nut 18(1): 33-38.

Wiedenfeld H, Roder E. 1991. Pyrrozidine alkaloids form Ageratum conyzoides. Plan Med 57:578-579.

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Institute, Papua New Guinea.

A-1

Authors Index

Abd Rabou AFN 82Abd-ElGawad AAE 36Abdur-Rahim M 105Ade R 68Al-Hammady MAM 73Ammar MSA 36, 73Aznar E 64Bhat MY 92Bhoyar AD 118Bonde S 59Fitriawan F 28Hadiwiyono 112Hassan L 105Hassanein M 36Jabbari S 23Kabir MA 105Kuswanhadi 124Lasminingsih M 124Lense O 145Madkour HA 36Majumder DAN 105Moayeri H 23Navgran SZ 15Nurliana S 98Nuryandani E 1

Obuid-Allah AH 73Oktavia F 124Pala SA 92Poernomo 64Pourbabaei H 15Prayitno A 64Prayitno E 1Pyasi A 136Rahmani R 23Rai M 68Rajput PR 118Sajidan 7Santoso BB 130Setyawan AD 44Sohrabi V 23Soni KK 136Sugiyarto 7Sunarto 28Sutarno 28Taat-Putra S 64Trimanto 7Vankhade GN 118Verma RK 136Wani AH 92Wiryono 98

A-2

Subject Index

Anodonta woodiana 28, 29, 30, 31, 32, 33,34, 35

antibacterial 49, 50, 56, 59, 60, 62,63, 96, 118, 150

appropriate 1aspect 15, 17, 18, 19, 21, 22banana 105, 112, 113, 114, 115,

116, 117biflavonoid 44, 45, 46, 47, 48, 49,

51, 52, 53, 54, 55, 56,57, 58

biological screening 145bleaching 73, 74, 75, 76, 78, 79,

80, 81, 118blood disease bacterium 112, 113, 117botanical knowledge 98, 103cadmium 28, 30, 31, 32, 33, 34, 35callus induction 68carnivores 82, 83, 84, 86, 89, 91,Colocasia esculenta 6, 7, 8, 10, 11, 12, 13, 14concern for biodiversity 98coral 37, 40, 42, 43, 73, 74,

75, 76, 79, 80, 81decomposition 30, 119, 136, 139, 141,

142, 143, 144developing countries 64, 65, 66diameter and height classes 23, 24, 26, 27distribution 6, 16, 21, 22, 30, 38, 52,

57, 58, 59, 61, 62, 66,80, 81, 83, 87, 90, 91,112, 114, 117, 136, 142,143, 144

DNA extraction 1, 2, 4, 5, 6, 65, 105,106, 109, 111, 113, 125

edible 92, 94, 96, 97Egypt 36, 37, 40, 41, 42, 43,

73, 74, 76, 79, 81, 82,84, 85, 86, 87, 88, 89,90, 91

elevation 15, 16, 17, 20, 21, 22eutrophication 73, 80,flavanone 47, 118, 119, 121floristic 15, 16, 17, 22Foeniculum vulgare 59, 60, 61, 62, 63forestry students 98, 99, 100, 102forests 15, 16, 17, 22, 24, 26,

27, 43, 87, 92, 93, 96,97, 98, 136, 141, 143,144, 145

fruit crops 105, 106, 108, 109, 110fungi 47, 50, 52, 60, 73, 77,

79, 80, 92, 94, 95, 96,97, 111, 136, 137, 138,139, 140, 141, 142, 143,

144, 145, 146, 148, 150Gaza 82, 83, 84, 85, 86, 87,

88, 89, 90, 91genetic variability 72, 124, 128, 129, 130,

131, 134, 135gills 28, 29, 30, 31, 32, 33,

34, 35, 94, 96Gloriosa superba 68, 69, 70, 71, 72hand pollination 124, 125, 129hevea breeding 124, 125HPV 64, 65, 66, 67indices diversity 23inoculums 112, 113, 117, 137, 138,isoxazoline 118, 119, 121, 123isozyme 7, 8, 9, 11, 12, 13, 14,

129Jatropha curcas 1, 2, 3, 4, 5, 6, 101, 130,

131, 132, 133, 134, 135Kashmir Himalayas 92, 94, 97kidneys 28, 29, 31, 32, 34, 35kinetin 68, 71, 72leaf 1, 3, 4, 5, 6LM20 59, 63local people 36, 82, 84, 86, 87, 89,

90, 145macrofungi 92, 93, 95, 96, 97mammals 52, 82, 83, 84, 85, 89,

90, 91management programs 36, 37Manokwari 145, 146, 147, 149, 150Marsa Alam 36, 37, 40, 43medicinal 6, 44, 45, 46, 47, 53, 54,

55, 56, 57, 58, 60, 63,68, 69, 72, 92, 96, 97,145, 146, 147, 148, 149,150

morphology 7, 8, 13, 14, 94, 100,131, 134, 135, 1

multiple shoots 68, 69, 70, 71Muwardi Hospital 64, 65natural products 44, 45, 46, 51, 52, 53,

54, 150oil pollution 73, 75, 79, 80OSCC 64, 65, 67Palestine 82, 85, 86, 87, 88, 89, 91parents trees 124, 125pathogenesis 64PCR 5, 6, 65, 67, 105, 106,

107, 108, 109, 112, 113,114, 115, 117, 125, 126,150

plant diversity 8, 13, 15,polysaccharides 2, 97, 105, 106, 107,

109, 110

A-3

pyrazoline 118, 119, 121, 123RAPD 6, 105, 106, 107, 108,

109, 110, 111, 124, 125,126, 129,

Red Sea 36, 37, 40, 43, 73, 74,75, 76, 79, 80, 81

rubber 14, 100, 124, 125, 126,127, 128, 129

sal 136, 137, 138, 139, 140,141, 142, 143, 144

seasonal variation 90, 136, 143sedimentation 32, 73, 75seedling 16, 113, 115, 117, 130,

131, 132, 133, 134, 137,seeds 2, 53, 54, 55, 68, 69,

130, 131, 132, 133, 134,148

Selaginella 44, 45, 46, 47, 49, 50,52, 53, 54, 55, 56, 57, 58

selection 36, 37, 41, 42, 43, 74,99, 124, 125, 130, 131,132, 133, 134

selection criteria 36, 41, 42, 43sensitivity significance 36, 37, 38, 39, 40, 41, 42silver nanoparticles 59, 60, 61, 62, 63structure diversity 23, 26taro 5, 7, 8, 9, 11, 12, 13, 14,tourist use 36, 40, 41, 42, 43traditional medicinal plant 56, 145, 149,trehalose 44, 45, 51, 52, 53, 54,

55, 56, 57, 58tunnel trade 82, 85, 86, 87, 89, 90,water-saturated ether 105, 106, 107, 109, 110West Papua 145western Iran 15, 16wildlife hunting 82, 90,zoological gardens 82, 83, 84, 85, 86, 87,

89, 90, 91

A-4

List of Peer Reviewer

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

Alka Grover Division of Crop Improvement, Central Potato Research Institute, Shimla 171001,Himachal Pradesh, India

Chuan-Chao Dai Jiangsu Key Laboratory for Bioresource Technology, Life Science College of NanjingNormal University, Nanjing 210097, China.

Evi Mintowati Faculty of Mathematics and Natural Sciences, Lambung Mangkurat University,Banjarmasin, South Kalimantan, Indonesia

FX Susilo Faculty of Agriculture, Lampung University, Bandar Lampung 35145, Indonesia

Gillian Dean Haughn Laboratory, Department of Botany, University of British Columbia, Canada

Hassan Sher Centre of Botany and Biodiversity Conservation, University of Swat, Pakistan

Irma Dewiyanti Department of Marine Science, Coordinatorate of Marine and Fisheries, Syiah KualaUniversity, Darussalam, Banda Aceh 23111, Indonesia.

John R.S. Tabuti College of Agricultural and Environmental Sciences, Makerere University, Kampala,Uganda.

José Veríssimo Fernandes Departamento de Microbiologia e Parasitologia, Centro de Biociências, UniversidadeFederal do Rio Grande do Norte (UFRN), Natal, RN, Brasil

Mahendra Kumar Rai Department of Biotechnology, SGB Amravati University, Amravati 444602,Maharashtra, India.

Muhammad Ahsin Rifai Faculty of Fisheries, Lambung Mangkurat University, Banjarmasin, South Kalimantan,Indonesia

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

Rainer W. Bussmann Curator of Economic Botany, Missouri Botanical Garden, St. Louis, MO 63166-0299,USA

Sancia E.T. van der Meij Department of Marine Zoology, Netherlands Centre for Biodiversity Naturalis, NL-2300RA Leiden, The Netherlands

Sanjog Thul Plant Biotechnology Division, Central Institute of Medicinal and Aromatic Plants,Lucknow, India

Sankar Harish Rice Research Station, Tamil Nadu Agricultural University, Ambasamudram,Tirunelveli, Tamil Nadu, India

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

Supachitra Chadchawan Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330,Thailand

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

Sutomo Bali Botanic Garden, Candikuning, Baturiti, Tabanan 82191, Bali, Indonesia

Victoria Reyes-Garcıa Institució Catalana de Recerca i Estudis Avançats y Institut de Ciencia i TecnologiaAmbientals, Universitat Autonoma de Barcelona, Bellatera 08193, Barcelona, Spain

Yelda Ozden-Tokatli Plant Tissue Culture Laboratory, Department of Biology,Faculty of Science, GebzeInstitute of Technology, Istanbul 41400, Gebze, Kocaeli, Turkey

Yulianti Research Institute for Forest Tree Seed Technology, Forest Research and DevelopmentAgency, Ministry of Forestry, Bogor 16001, West Java, Indonesia.

A-5

Table of Contents

Vol. 3, No. 1, Pp. 1-58, March 2011

Optimization of DNA extraction of physic nut (Jatropha curcas) by selecting the appropriate leafEDI PRAYITNO, EINSTIVINA NURYANDANI

1-6

Characterisation of taro (Colocasia esculenta) based on morphological and isozymic patterns markersTRIMANTO, SAJIDAN, SUGIYARTO

7-14

Study on floristic and plant species diversity of the Lebanon oak site (Quercus libani) in Chenareh,Marivan, Kordestan Province, western IranHASSAN POURBABAEI, SHIVA ZANDI NAVGRAN

15-22

Evaluation structural diversity of Carpinus betulus stand in Golestan Province, North of IranVAHAB SOHRABI, RAMIN RAHMANI, SHAHROKH JABBARI, HADI MOAYERI

23-27

Microanatomy alteration of gills and kidneys in freshwater mussel (Anodonta woodiana) due tocadmium exposureFUAD FITRIAWAN, SUTARNO, SUNARTO

28-35

Site suitability to tourist use or management programs South Marsa Alam, Red Sea, EgyptMOHAMMED SHOKRY AHMED AMMAR, MOHAMMED HASSANEIN, HASHEM ABBASMADKOUR, AMRO ABD-ELHAMID ABD-ELGAWAD

36-43

Review: Natural products from Genus Selaginella (Selaginellaceae)AHMAD DWI SETYAWAN

44-58

Vol. 3, No. 2, Pp. 59-103, July 2011

A biogenic approach for green synthesis of silver nanoparticles using extract of Foeniculum vulgare and itsactivity against Staphylococcus aureus and Escherichia coliSHITAL BONDE

59-63

Oral squamous cell carcinoma patients which human papilloma virus infection: a case control study inMuwardi Hospital Surakarta, Central Java, IndonesiaADI PRAYITNO, ELYANA AZNAR, POERNOMO, SUHARTONO TAAT PUTRA

64-67

Multiple shoot formation in Gloriosa superba: A rare and endangered Indian medicinal plantRAVINDRA ADE, MAHENDRA RAI

68-72

Corals differential susceptibilities to bleaching along the Red Sea Coast, EgyptMOHAMMED SHOKRY AHMED AMMAR, AHMED H. OBUID-ALLAH, MONTASER A. M. AL-HAMMADY

73-81

The Palestinian mammalian fauna acquired by the Zoological Gardens in the Gaza StripABDEL FATTAH N. ABD RABOU

82-91

Six hitherto unreported Basidiomycetic macrofungi from Kashmir HimalayasSHAUKET AHMED PALA, ABDUL HAMID WANI, MOHMAD YAQUB BHAT

92-97

The knowledge of Bengkulu University’s forestry students of tree diversity in their campusWIRYONO, STEFFANIE NURLIANA

98-103

A-6

Vol. 3, No. 3, Pp. 105-150, November 2011

Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica)DILRUBA ASHRAFUN NAHAR MAJUMDER, LUTFUL HASSAN, MOHAMMAD ABDUR RAHIM,MOHAMMAD AHSANUL KABIR

105-111

Blood bacterial wilt disease of banana: the distribution of pathogen in infected plant, symptoms, andpotentiality of diseased tissues as source of infective inoculumsHADIWIYONO

112-117

Synthesis and study of cholosubstituted 4-aroyl pyrazolines and isoxazolines and their effects on inorganicions in blood serum in albino ratsAMOL D. BHOYAR, GANESH N. VANKHADE, PRITHVISIGH R. RAJPUT

118-123

Selection of parents trees for Rubber (Hevea brasiliensis) breeding based on RAPD analysisFETRINA OKTAVIA, MUDJI LASMININGSIH, KUSWANHADI

124-129

Variation in oil contents, and seed and seedling characteristics of Jatropha curcas of West Nusa Tenggaraselected genotypes and their first improved populationBAMBANG BUDI SANTOSO

130-135

Litter decomposing fungi in sal (Shorea robusta) forests of central IndiaKRISHNA KANT SONI, ABHISHEK PYASI, RAM KEERTI VERMA

136-144

Biological screening of selected traditional medicinal plants species utilized by local people of Manokwari,West Papua ProvinceOBED LENSE

145-150

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