Diversity of Nitrogen Fixing Bacteria Based on nijH Gene in Rice Fields

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ADVANCES IN ENVIRONMENTAL BIOLOGY

Monthly ISSN: 1995-0756

AM-EURASIAN NETWORK SCI INFORMATION-AENSI, AL HUSSEIN BIN TALAL UNIV,

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1. Zoological Record

2. AMERICAN-EURASIAN JOURNAL OF AGRICULTURAL

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SCIENCES

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INT DIGITAL ORGANIZATION SCIENTIFIC INFORMATION-IDOSI, PO BOX 40468,

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AEB Volume 8, Number 14: Special7, 2014

ＭｾＭＭＭＭＭＭＭＭＭＭＭＭＭ

Preliminary Study of Laotian Black Crested Gibbon Activity Budget in Ban Toup, Nam Kan National Protected Area, Lao PDR

SingphoneLuangleuxay, Kham Youanechuexian and Pongthep Suwanwaree

1-6

The Status of Laotian Black Crested Gibbon Nomascus concolor lu in Nam Kan National Protect ed Area, Lao POR

Kham Youanechuexian, Phaivanh Phiapalath and Pongthep Suwanwaree

7-13

Relation of Ganoderma Ergosterol Content to Basal Stem Rot Disease Severity Index

Chong, K.P .• Eldaa, P.A. and Jedol Dayou

14-19

Increasing the Energy Output from Living-plants Fuel Celts with Natural Photosynt hesis.

Choo Ying Ying and Jedol Dayou

20-23

Kittiphop Promdee, Omrumpha Soubsawwong, Chintana Sanvong, Tharapong Vitidsant

24-29

---Some Interpretations on FTIR Results for the Detection of Ganoderma Boninense

in Oil Palm Tissue

Amnyitte Alexander, Jedol Dayou, Coswald Stephen Sipaut, Chong Khim Phfn and Lee Ping Chin

30-32

Fed-batch Production of Valuable Biosurfactant, Rhamnolipid, from Waste Cooking Oil

by

Indigenously Isolate Pseudomonas aeruginosa USM-AR2

Zainatul ·Asyiqin Samsu, Zulail<ha Yusof, Mohd Syafiq Awang, Nurasshifa Md Noh, Ahmad Ramli Mohd Yahya

33-38

Effects of Varied Amount of Carbon Dioxide and Pressure on Asphaltene Stability

Josefina Barnachea Janier, Mohamad Afzal B. Jalil, Radzuan B. Razali, Afza Bt. Shafie, Samsul Ariffin B. Abdul Karim

39-43

Room Temperature Synthesis of Water-Soluble Starch-Stabilized CdSe Quantum Dots for Latent Fingerprints Detection

Tanutkuh Palakawong Na Ayudhaya, Panida Viwattana, Kheamrutai Thamaphat, Khemika Lomthaisong

44-49

Community Succession of Methanotrophic Bacteria Based on PMOA GENE In Rice Fields

Hendri Sutanto, 'Iman Rusmana, Nisa Rachmania Mubarik

50-56

Blomolecular React ion and Heat Controlled In the Reactor for Synthesis of Charcoal and Blo-011 Derived from Mixed Grass

Kittiphop Promdee, Chintana Sanvong, Somruedee Satitkune, Tharapong Vitidsant

57-62

Diversity of Nitrogen Fixing Bacteria Based on nilH Gene in Rice Fields

,

I

63-69

---·---·---Improving Power Output Prediction from Ocean Salinity and Temperature Energy Converter using Viscosity Model

Fuei Pien Chee, Shu Kim Lee, Jedol Dayou, Ejria Saleh and H.L.H. Chong

70-77

_

...

---·---Initial Investigation on Using Copperas By-Product to Remove Colour from Domestic Wastewater by Coagulation and Flocculation

Hamidi Abdul Aziz and Wan lzatul Saadiah Wan Kamar

78-82

---·---·---·---Removal of COO and Colour from Landfill Leachate using Ferric Chloride by Coagulation.flocculation Treatment

Hamidi Abd Aziz and Sifi Fatihah Ramli

83-90

Selected Heavy Metals and Polycyclic Aromatic Hydrocarbon in Commercial Fishes Caught From UMT Enclosed Lagoon, Terengganu, Malaysia

Ong M.C., Yong J.C. , Khoo

X.

Y., Tan Y.F. and Joseph B

91-98

Effect of Rainfall on the Transmission Model of Conjunctivitis

Jantraporn Suksawat and Surapol Naowarat

99-104

.

---·---·---·---·---·---·---Microbiological Risk Assessment of Fresh Water Aquaculture Fish: From Farm to Table

Ibrahim, A.B., Mohd Khan, A., Norrakiah, A.S.

105-1 11

School's Indoor Air Quality and Respiratory Health Implications among Children

Chua Poh Choo, Juliana Jalaludin, Zailina Hashim

112-121

Efficiency in Beverage Manufacturing Firm in Thailand

Jarun Bootdachi and Sirirak Khaochaimaha

122-130

In-vivo Blood Glucose Control

by

Phenolic Compound from

Vernonia amygdalina,

Andrographls

panlculata,

and

Plthecellob/um jlrlnga

in Streptozotoxin-lnduced Diabetic Rats

Asna Mohd Zain and Amirul Abu

12' Frequency

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• Dr. Nuray MISIR Karadeniz Technical University, Faculty of Forestry, 61080, Trabzon,Turkey.

• Dr. Bragadeeswaran CAS in Marine Bilogy, Annamalai University, Parangipettai 608 502 Cuddalore, Tamil Nadu, India

• Dr. Sevil TOROGLU Biology Department, Faculty of Arts and Sciences, University of KSU, 46100 Avsar Campus, Kahramanmaras, Turkey.

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• Assist. Prof. Dr. NOket SIVRI Istanbul University, Faculty of Engineering, Dept. of Environmental Engineering, Avcilar Kampus 34320. Istanbul TURKIYE .

• Or. Kamelia Mahmoud Osman Ahmed Department Microbiology, Faculty Veterinary Medicine, University Cairo, Egypt.

• Dr. Slavomlr Ceri'lansk Comenius University in Bratislava, Faculty of Natural Sciences, Department of Ecosozology and Physiotactics, Mlynska dolina 1, 842 15 Bratislava, Slovakia.

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• Dr. ａｭ｡ｾ･ｳｨ＠ Chandra Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi - 284003 India.

• Or. KALIMUTHU KANOASAMY Department of Botany, Government Arts College. Coimbatore - 641 018. India.

• Or. Abdul Latief

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• Prof. Magdy Tawfik Khalil Zoology Dept, Fae. Science, Ain Shams Univ., Cairo, Egypt. Aquatic Ecology, Biodiversity, Management & Conservation

• Dr. Halil Erhan EROGLU Department of Biology, 66200 YOZGAT, Bozok University, Faculty of Science and Arts, Turkey.

• Prof. Shikui DONG chool ofE11vironmental Sciences, eijing Normal University, No. 19, Xingjiekou Waidajie, aidian District, Beijing, 100875,

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Dr. Naveed Ahmed Khan Senior Lecturer in Microbiology, School of Biological & Chemical Sciences, Birkbeck College, University of London, Malet Street, London WC1 E 7HX, England, U.K.

• DR. DHRUVA KUMAR JHA Department of Botany, Gauhati University, Guwahati-781 014, Assam, India.

• Dr. lgnacy Kitowski Department of Nature Conservation, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, PL-20-033 Lublin, Poland

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The instructions for authors include informabon about preparing a manuset1pt for submlSSion to the Journal of interest. er 1ter1a for pubticabon and the onbne submission process

Ori\Jinal research pap8'S, review articles, technical reports and short communications in all aspects of Agricuhure. Biological. lnformat10n. Health & ｌｾ･＠ Sciences, Zoology, Humanity, Social and Applied Sciences etc .. can be submitted on the understanding that the 'Mll'k is not prev10usly published or undef C011S1deration f()( pubficatvn elsewhere.

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Short Communica1i on: It must not exceed 6-12 manuscnpt pages (wlh figures) and must conlam lhe Abstracl, Key words. Abbrev1alions. the core of the pap8' Acknowledgements. References. Tables. and Legends lo figures II should report a C<JOl)leled 'Mll'k but not preliminaty findings.

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Acknov.tedgement References

Abs1rKt

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.ebstrad of ｾ＠ w:irds should be provided summenmg tr iel lntroduc:bon, methods llSed. results and concluslon of the stud/. No Sib headngs should be given in tins secbon.

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References :

A Examp!esc'crtahon :i teYI

The use of an'l!utha's name (wllhout 1mtials} followed by a dale ex year of pubhcallon is used lor references I01Jnd 111 !ext

Peiry (2003} early proved thal This is 111 agreement ｾｬｨ＠ the resulls obtained by severnl authors {Brown 1999, Kramer, 2004, Smllh. 2008)

Zhang and C!teng (2001) repcxted that This was later round to be incor•cct (KurtlJf and Ahmed. 2000)

When there •e more than two authors. only the irsl author's name should be menboned loUo"'9d by et :.r as seeo below Prince et al (1990} staled that S1rnlar results 'M!re reported recenriy (Smilh et al. 2003)

In the event that an author crted has had two or mcxe W01ks published during the same yeai !he relerence. bolh 1n the '.ext and in lhe reference hst. should be ldenhhed by a tower case lel!er hke 'a and 'b after the date lo disllngu1sh the wOiks

Fortnstance(Slephen, 2001a,b}

Journal Artrcies Ouyang, D J Bartholic and J Selegeao. 2005 Assessing Sedtmenl Loading from Agricultural O'oplands 1n !he Great Lakes Basin J<unal ol American Science. 1(2) 14-21

A Book. Durbin, R . S R Edcty A Krogh and G Mltchison. 1999 81ologicaJ Sequence Analysis Probabi1sbc Models ol Prolerns and Nuc:lelc Acids Canillqje lJfwersdy Press

A Report MakarelMcz, J.C., T. Lev.is and P. Be11ram, 1995 Epttimnetic phy1oplanklon and zooplanktonbiomass and species composition in Lake Michigan. 1983-1992 U.S. EPA Great Lakes National Program, Chicago, IL. EPA 90&-R.es-009

Conference Proceedngs: Slock, A, 2004. S19nal Transduction in Bacteria. In the Proceedings of the 2004 Maik.ey Scholars Conference. pp 80-89,

A Thesis· Strunk, J.L .. 1991. The ex1raction of mercury iom sediment and the geochemical partilioning of mercu.ry in secimenls from Lake Superia, M. S thesis, Michigan Slate Univ., East Lansing, Ml

Abbreviations. Unrts Etc.,

Authors should follow internationally agreed rules especially those adlpted by the IUPAC·IUB Commissi:ln on Biochemical Nomenclature (CBN), The journal will essentially follow the rules defined in the IUPAC Manual of symbols and terminology for physico-chemical quanblies

and ｵｮｾｳ＠ (Bul1erworths, London). 1970.

Copyright and Permlnlons

By submilbng a manuscript to the editor or publisher yoo are deemed to have granted permtSSi:ln to publish ttie manuscripl and distribute 11 electronically or in any otner form to different databases and abstracling seNices including lharies. ｵｮｩｶ･ｲｮｾｩ･ｳ＠ and any>Mlere else Authorship

For papers to be published in this Journal, each authct should have participated sufficiently in the v.or1< to take pubic responsibi1ty for the content (ThlS statement is taken from the authQ'ship policy adlpted by the International Commitee of Medical Journal Ｆｊｾｯｲｳ＠ and published in the Uniform Requirements for Manuscrl>ls Submitted to Biomedical Jo001als, 1994 and N Engl J Med 336:300-315, 1997.)

Corrections to published articles. If necessary, cmectilns or sign ｾｩ｣｡ｮｴ＠ errors in published articles wil be published in a later issue of the Journal Within one months after publication, authors are requested to bring any errors to the attention of lhe managing editor.

Proofs ¥.ill be sent as an Aaobal PDF (Portable Documenl FQ'ffiat) file. Acrobat Reader will be required in order lo read lhe PDF. This sottware can be downloaded from the foRowing 'M!bsite·

http.!fwww ｡ｾＯｲｸｯ､ｵ｣ｴｳＯ｡｣ｲｯ｢｡ｕｲ･｡､ｳＬＱＱｭＲ＠ blml

Th1s\\1U enable Ille file to be opened, read on screen and pnnted cul 1n order for any correcbons to be added Reprints

pon final pubhcabon, an tSSue will be made ｡ｶ｡ｾ｡｢ｬ･ｯｮｬｩｮ･ Ｎ＠no hard copies ol the reprints or 1ournal copy WIN be supphed To facilitate edltor1al work. please keep the edrtonal office informed of any changes in your acttess, e-mail adctess and

Advances ia Eaviroamcntal BioloJO-, ｾＨＱＴ Ｉ＠ Special 2014, Pages: 63-69

AE'NSJ Journal$ .

Advances

in

Environmental Bioiogy

Diversity of Nitrogen Fixing Bacteria Based on

nijH

Gene in Rice Fields

l Ran di Hadianta, 21man Rusmana, 2_{Nisa Rachmania Mubarik}

1_{Gruciua/i: Schwl. Bugur .,grimlt11ra/ Uniw:rsily. Darmugu Cumpuv, Bugur, West .!<Nu, lnJunesia 166/JIJ}

1D 1purtrm:nl of Bi<>lugy. F<1t11lty u{Mu1ltt!l11u1icy um! Nuturul Scirm r, Bugw Ag11t-ulturul UmvcrsllV. Dunnusu Cumpu>, Bugor, We.11 Juvu,

Indonesia 166.'SU

ARTICLE INFO Ardd' history: Reuned 15 Jwtt 1014 Recm-ed m rensl!ii form

8 Julv W/4

Aatpted 14 Stptember 2014

ａＮｶ｡ｩｬ＼Ｑ｢ｬｾ＠ on/int 27 September :llu

Key,.VJrtls:

B1vfe11i/izcr. DGGJ:;. mfH gen.•, ｮｩＱｾｧ｣ｮＬｴｬｸｩＱＱｧ＠ hat1eri11

ABSTRACT

Bjological nnrogen fixation is ao important ｰｲｯ｣ｾｳ＠ in changing Nl gas in the lllmQlphe-c into ammcn1um "llilich pcrfonncd by nitrogen fixing baaeriL The molecular analysis of nifil g.:ne is coonnooJy ｵｾ＠ aod pro'·'d more a.ccurate for

ddccung die dm:n1ty of niirogcn fixing bacteria coowncd in !he nee ficld soil. The

di,·mi1y of n11rogen-fixing bacteria based on n11ll !!..:nc can be determined by molo:.."1lbr approM;hcs, LC. DGGE (Dcnallll'alll OnM.hcnt Gel El..'CU'Ophorcsis). This

ｲｾｨ＠ were cooducted to study the d1\'ersny of n1l(Ogen fuiog bacteria in rice fields soil and also to mea<nlre the plant gro'Mll. The plant gn,.,,.1h parameters oi rice shuwed the trcat111ct11 ｷｨｩｾ｢＠ soaked by biofcrtilizcr were better than comm! tn.'af.lllCUt aod

treallUent w1ucb ｾｰｲ｣｡､＠ by biofcnilaer. However, tho; divc"1ty of nitrogco fixing

l>aqcria was '11<)re vary tn control treabUcut bused on l)CJGE ｲ･［［ｵｨｾ Ｎ＠ There were lhre.: of eight b3.o.ds which always appears in .:very month. The ,.;qucn1:1ng n.-sult$ sbow.:d all se<lucnccs wcrc ｣ｷｾＱｦｩ･､＠ u uncultured bacterium clone nitrogcna<e i.ron prOtein (niflI)

gcoc, patttal .:ds.

() 2014 1\1':1'.SI Publisher All rk>h1 s resened To Cite Thi$ Artiri.-: Rllld1 Had1anta. Iman Rusmana. Nisa !Uchnwna Mubarik. Diversity of Nnrogcn Ft'llD" Bactcna S.,,,.,d on rutH Gene in Rice F1el<b .. fdv. F.nl'irntl. JJWJ., 8(14), 113-119. WU

INTRODUCTION

Nitrogen is a macro clement required by plants but the ｣ｯｭｰｯｾｩ ｴｩ ｯｮ＠ of78% ni trogen in the atmosphcr.: cannot

be used directly by ｰｬ｡ｮｾ Ｎ＠ Plants can only abso1b soluble nitrogen in the soil by using root. Supplying the nitrogen in the soil can be done by fertili2a1ion or naturally with the help of m1croorganim1S. B1ological 11.itrogeu fixation i!> an unpotulllt prm:ess in nature which performed by nHrogen-fix.ing bactena in changing free nitrogen gases in the: atnio,phere into 11r1moruum Ammonmn 1• nr 1mr "'..l!' ｲｊｴｲｯｧｾﾷｴ＠ source: in th:: c:cosysrc:ru_ l.iit.'"C'gen

fixing bactena 1s able to hvc freely (free living) and can also hvc in symbiosis with plant<>. Riological nitrogen fixation is limited to prokaryotes while there is no report for eukaryotes as uiirogen fixer

The ability of rulrogcn fixing bacter1c1 to convert nitrogen ia the ｡ｵｮｯｾｰｨ･ｲ｣＠ 1nw ammoruum due 10 11;

nitrogenasc cnqme. Nicrogenase enzyme consists ol ｬ｜ｾｯ＠ subunits that dinitrogcnase and dmitrogenase reducrase. Dinitrogenase enzyme is encoded by 11i}D and nrjK gene, while dmitrogeoasc: reductase enzyme: encoded by nijH

gene. The diversity of nitrogen-fixing b:icteria can be identified by molecular ｵ ｰｰ ｲｯ｡｣ｾｳＮ＠ One of the m<llecular te<'hniques that can be u:.cd is DGGE (Denimu ing Gradienl Gel Electrophoresis).

DGGE can d istinguish species based on differen ces in the GC composition of tbi: analy.(Cd DNA ｾ｣ｱ ｵ･ｮ｣｣ｳ＠

[ 10]. This is caused hy the uc;e of denaturant gradienl gel uc;cd. T he diYer<;i1y of nitrogen-fixing hacteri:1 sn1died

by nijH gene. The use of 11(/l I gene in DGGE is commonly used and proven to be more accurate for the detection of diversity nitrogen fixing bacteria contained in the soil [21,-1,5.11 ].

Accordmg 10 report<; by Pingak [l::!], the use of bio-tertih7ers in rice fields cao ｭ｣ｲ｣Ｓｾ｣＠ nee protlucti\ ity. Rice producliv1ry can be supported by a various factors, such as nitrogen. Nitrogen elemem itself can be acquired from ferti li;ration or nitrogen fixing bacteria. Research related to the relalionsh1p between the ｵｾ･＠ o l bio-lertihzcr and nee proJuctJ"ity associated wid1 the di,·ersiry of nitrogen fixing bacteria in nee fieldc; hac; not hc.:n rcpo11eJ in Sukabumi. This re!>carch aimed to study the diversity of nitrogcn-tixmg bactena in rice fields ｨｾ＠ OGG[

technique.

Corresponding Author: 1111.in R.usmana, Bogor Agricultural University, Depanment ofBiolog,y, Faculty of Mathematics

64

Jman Rosmana ｾ Ｇ＠ 111, 2014

Adn MH ia EaviroamHIU Biology, 8(14) Special 2014, Pagl!' b3-69

MATERIAL AND M ETllODS

This study was conducted in June 2013 - May 2014. Field research was performed in rice fields at Cidahu village, Cicurug, Sukabumi, West Java. The analysis was performed at the Laboratory of Microbiology and Laboratory oflntegrated, Department of Biology, Bogor Agricultural ｕｮｩｶ･ｾｩｴｹＮ＠ ·1 he treaunent used in this study were control, soaked and spread. The control treatment using 300 kg/ha of NPK fertilizer. Soaked and spread treatment using 200 kg/ha of NPK fertilizer and biofertilizcrs. Rice clump in the soaked treatment was soaked with biofertilizer while in the spread treatment, biofertilizer was spread directly into rice fields.

Soil sampling.

Rice field soil in the control, soaked and spread treatment were taken by using a 10 ml syringe. 2 repJjcatioos were used per treatment. Control treatment has an area of 150 m2 while soak and spread plots ha\·e an area of 600 m2. Soil sampling is done every 30 days during the rice planting namely 30 DAP (days after plantiug), 60 DAP and 90 DJ\P. A total of± 1 kg of soil in each treaonent was taken in the beginning and end of the growing season then scot and analyzed a1 lhe Soil Research institute, Bogor to determine the physical and chemical characteristics of the soil.

Plalll Growth Meas11rement:

Plant growth measurement was carried out by measuring the plant height and number of tillers. There were I 0 clumps that had been used in this study. Measurement of wet weight and dry weight of the clump, roots and seeds are also carried Ollt in the end of the planting.

DNA £ttmrno11.

DNA extraction was done by using the Power Soil DNA Isolation Kit (Mob10 laboratories, Carlsbad, CA. USA). Extrncaon was done according to the procedures of the company. The quality of DNA then checked using Nano Drop 2000 (Thennu Scientific, Wilmmgton, DE, USA).

Po(1 muase Cham Reaction

DNA was amplified by USIJlg PolF I PolR primer sets f 13 ]. 40 CIC sequences were attached to ｴｨｾ＠ end of the forward pnmer. PCR was performed by using KAPA Hot Stan Read}mix (KAPA Biosystems. Wilmington, MA. USA). Each PCR reaction contained 12.5 µL KAPA Hotstart Readymix, 1.25 µL forward primer (0.5 uM), 1.25 µL reverse primer (0.5 uM), 3 µL template (-100 ng) and 7 µL nuclease free water. PCR was performed using T-Gral,lient Thcrfnocycler (Biomelra GmbH. Goeuingcn. Gerrnanr). The PCR thermal cycling coodilions were as follo"v-;: m111al denaturation at 95 °C for I min; 30 cycles of dena1ura1ion at 95 °C for I 5 s. annca1mg at 55 °C for 15 sand elongation at 72 C for 15 s; and a final elongation step at72 °C for 5 mm. Products were r'Jll at 1.5 % agarose gd for checked correct ｾｩＷ･Ｌ＠ and stored at -20 °C until analyzed on OGGF

DGGE Analysi.f

OGGE wa-: ｾｲｦｯｲｮｭｬ＠ using the D Code Univer<>al Muuition .Detecuon Sy-;um lBio-Rad. llen:ules, CA. USA). 'the DGGf geb were prepared and nm under the following conditions 2S uT of the PCR p1oduct was loaded omo I mm thick IS"'o (w/\') polyacrylam1de gels (acrylamide-b1sacrylam1de (37.5.1)] in I TAE buffer (40mM Tris, 20mM acetic acid, aml lmM EDTA) using urc:a as the denaturing agent with a denaturing gradient from 35% 10 60% (100% denaturant corre::.ponded to 7M urea and 40% (v/v) deionized fonnamide). Electrophoresis was perfonncd at 60 °C and IJO V for 6 h. After clectrophores1s. the gel was stained for 60 min in SYBR Safe {l11vi1Togcn- \itolecular ｐｲｯ｢･ｾＮ＠ Carlsbad, CA, USA) and visualized by G:BOX (Syngene, Frederick, MD. USA). Band-: appearing on the gels were excised and translerred to micro tubes containing 50 ul ddH20. l'hc bands were incuba1ed at 4 °C' ovcnught Band analy7ed using Phorctix ID (Touil Lab) LO estimate total band that appeared.

Se'luencing tmd Phyloge11111ic 7i·ce Co11s1ruc1io11:

Supernatant from each band was checked usmg Nano Drop 2000 to dc1cnmnc the quanuty of DNA from excised band. A 10 µL (-SO ng) supematalll ｷＱＱｾ＠ re-amplified under the PCR conduions described aoove using non GC pnmcr. The PCR productS were sent to I stBASE Malaysia. Sequencing rc-;ults will be analy7ed using BLAST program \\ith NIRI database and then the phylogenclic tree created using MEGA 5.2 [J 7].

RF.SULT A ｾｄ＠ OISCUSS101'1

Snil Charac1eris1ics:

65 Iman Rusm:ina et al, 2014

AcfnnttS ill Enviro•111tntal Biology, 8(14) Special 2014, Pages· "'·69

48% clay. Soil in I.he control treatment, soak and spread was tbe same pH range, ic pH 5. Carbon content in the

control treatment, soak and spread is 1.97%, 1.81 %, and 1.91 %, respectively. Nitrogen content of the control treatment, soak and spread is 0: 17%, 0: 17% aoJ 0: 14%, respecLivdy. CIN ratio of each trcaullent

was

12, 11 aod 14, respec1ivcly. Based on 1he criteria of the USDA [19], 1he soil in the control treatment classified as Silty Clay, while soaked treatment classified as Clay and spread treatment classified as Clay. Based on the criteria of

BA.LIT ANAH [2], those soil'! was classified as acidic soil (pH 4.5 - S.5) with carbon and nitrogen was low while

the CIN ratio of the 1hree soil was classified as moderate. According AGRlSNET [I), rice plants can grow well in Clay soil, Silty Clay and Silly Clay Loam. The opumum pH condition which required to grow rice well ranged

5-7. So. the 'iOil characteristics in this study fairly well for rice planting.

Rice growth:

Height measurement results indicated that rice plants at soaked treatment showed muimum height than

other treatments. Control treatment occupied the lowest position among others. The number of tillers in soaked treatment also showed compared to the other two treatments. Spread treatment place at second place followed by control treatment (Figure l ). Soaked treatment had the highest wet weight and dry weight compared to the other treatments in all parametm measured (Figure 2). These results were supported with reportS [12] that the $Qaked treatment resulted in the best growth response than the other two treatments.

He Wit and Number of Tille11 100

Ill

so

6,)

-J _{26 26}

lO

ｾ ｬｬ ｒ＠

0

ｈ･ｾｴｬ＠ Tilltr

[image:18.618.182.350.266.368.2]

l'I CCIM'O' . \Q.\.f'(J ｑ ｃ｜ｰｾＮ､＠

Fig. 1: I lc:1ght and number of tillers tn the control. soaked and spread treatment.

WelWelgbl

180

160 H9.S

140

111

ｾﾷ ＲＰ＠

ｾ＠ JOO _74.75

·r

80

15t?S

ii i

3: 60

38

I

It)

20

ｾ＠

u ...

B (u.irul Ｇ｣ＮＺｩＮＧｊＧＧｪＡ ｓｰｩＭ ｾ＠

(a)

DryWf'ight

lllO

3

ISO

l: .'!!!

..

1ro

ｬｾ Ｎ ｾ ｾ＠

ｾ ﾷ ｩｩｩ＠

3: so _{11 'lb l 'l.6.S6}

0

=-• Fnl

(ump Root G,a,.,

P•rt of Plant

Ｂ ｣ＮＮ｣ｾ Ｑ ＱＰ＠ • \1 •4"•f el ｾｲ＾Ｇｾ､､＠ (b)

66 1nun Rusmana Dal, 2014

Ad\l oett in ｅｾｕＭｯｭｦｬｬｬｬｬ＠ Bioloc. 8(14) Spec1al 2014, Page'!: 63-69

DNA Extraction:

DNA was extracted from soil showed various results ranged arouod 24 - 28 ng/uL Tbc purity of the DNA obtained from the extraclioo is quite good wilh an average A260/A280 parameter ranges 1.9 (Table 1).

Re\.'Ommcnded value of A260/A280 ratio is ranged 1.8 - 2.0 (14). Good quality of DNA template was important for downstream process hke PCR.

DNA Amplificatio11:

Nine samples were successfully amplified by PolF-GC/PolR primer with fragment size obtained as expected, around 360 bp (Fig 3). PolF I PolR primer was designed to amplify nijH genes possessed by nitrogen fixing bacteria and has been shown to successfully ampltfy 19 imponant species of oitrogen-fixmg bacteria such as

Azospirillum. Agrobacterium, Burkholderia, Pseudomonas, Rhizobium, ｓｴｲ･ｰｴｯｭｹ｣ｾＮ＠ and Xamhomonas (13].

Table I : T DNA be No I 2 3 4

s

6 7 8 9

p _{S I DN bol}

cooccntrallnn us111sr: ower 01 A atJon L

Treatment DNA Concentration lnot .. 1 )

Control 3-0 DAP 26

Soaked. 30 DAP 24.4

Soread, J() DA P 24.5

Control, 60 DAP 28

Soaked, 60 OAP 24.4

S1>rcild 60 DAP 27.9

Control 90 DAP 27.4

<;oakcd. 90 OAP 26.6

Sorcad. 90 D . .\P 27.2

M a b c d e f g : h

3000 bp

1000 bp

500 bp

A260/A280 1.96 I 97 1.9 1.94 l.95 l.9R 199 1.'ll l.9S

360 bp

Fig. 3: The ni/H gene amphlica1ion resul1 wi1h PolF-GCfPolR rinmer on 1.5% agarose gel. \Veth from left 10 nght 100 bp marker (a) control 30 OAP. (b) ｾｯ｡ｫ･､＠ 30 OAP, (c) spread 30 DA.P, (d) •'Mtrol ｾｏ＠ DAl>, tc) ｾ｡ｫ･､＠ 60 OAP. (f) spread 60 OAP, (g) control 90 OAP, (h) soaked 90 DAP, (i) spread 90 OAP. DGG£ Profile:

DGGE results showed all analyzed s.imple were separated into several separate bands. f.ach separate bands represent one of us own species. The results showed there were three bands that always appeared in lht: treatment every month. Control treatmenl showed the highest number of ｢｡ｮ､ｾ＠ with different vanattons of each month. Snaked t:reaunem tended 10 be stable every month with 4 bands. Spread trearmcnt showed incrcasms number of bands from 4 bands in firs1 month into 6 bands in the 1hird month (Figure 4).

67

7

ＭＮｾ＠ ' - -..__ ...__

! b c

.:

_,...,.

ActuocH ill Eniro1mtntal Biology, 8(14) Special 2014, Pagct· 63-69

!

b

c d e f

-

--

-

--2 3

I ₄

5

I

-

....

-6 ₇

8

---g h

-

-

-

I

6 8

Fig. 4: DGGE profile analysis of nijH gene, Left: Photo ofG: BOX and Righi: Interpretation of Pboretix lD software. The number showed the bands which cut to be rcamplified. Wells from left to right: (a) control 30 OAP, (b) soaked 30 OAP, (c) spread 30 DAP, (d) control 60 OAP, (e) soaked 60 DAP , (f) spread 60

OAP, (g) control 90 OAP, (h) soaked 90 DAP. (i) spread 90 DAP.

Seque11ci11g a11d Phylogenetic Tree·

BLAST results showed that all sequence!> obtained were uncultured bactena ､ｩｮｩｴｯｾ｣ｮ｡ｳ･＠ (111jH) gene from various bactena (Table '.!). Thi:. mtfa:atcJ tJ1at the sequences obtained bdung to unculrurable bacteria. It ｮｬｾｯ＠ showed that the sequence found was relatively new a$ a percentage identity of the results of the ｢ｬｾｴ＠ sequence databases under 95% of the - 360bp sequences of bases. Band I. 6. and 8 were showed on DGGt gel on each treatment every month. The three of bands were suspected to dominant bacteria in nee field due to appear m each tn:atment 1n every month The ｲ･ｳｵｬｌｾ＠ showed that hand I hatl ｣ｬｯｳ･ｮ･ｾｾ＠ to U11c11/t11red bai:terwm c:/011e

MDE_amb_35jl d111i1rogenase reductase (nijH) gene. partial eds for 89%. Baud 6 bud closeness to Uncultured

bactel'i11m clone clo.4-42 11itroge11ase iron protein (nifli) gene, partial eds for 88% and band 8 had closeness to

[image:20.626.35.534.51.672.2]

Uncultured bacterium clone Sipa-L24 mtrogcnase iro11 protein (mjl I) gene partial ｣､ｾ＠ for 92%.

Table 2: Results of Bl AS'l seQuencc of1he 11iJI I 11cne

Blmd ｄ･ｳｾｲｩｰｮｯｮ＠ Query E Value Ba...e/H;i,c llknmy Acc:.i.100

Cover i'OumbC!'

I Un.:ulruml ba..1cnum done 100% Se-114 JOR W! 89'!. Kf'84Mll9.l

ｾｉｄｅ｟｡ｭ｢｟ＳＵｦＲ＠ ､ｭｮｲｯｾ･ｮ｡ｳ･＠ re<luc1asc

(.(11/ll) 11.ene, oMial ｾ､ｾ＠

2 Uncultured ｢ｾ･ｲｩｵｭ＠ clone Sipa-10 97°'t 5e-1)4 JO'JJJI 93•. JX'.!6!4'.17.l

lllllO!ICna<c 1100 J>fOICln tn1/H) gene,

Ｍｾ｣､Ｇ＠

'

Uncuh111ed bal:tcrium cl"ne Sir"-'" '19'% So-149 Ｑｾ｜Ｇ＠ '1 .f .. !)<•. ＮｲｸＧＮＡｴＱｾｈ＼＠ I ni1rogcn:hc 11on prou:rn tnifH} gene.

n;uuaJ c,t,

4 l}ocuhwcd baclcnum clooc Sipa-14 97"/o 3e-t26 JO' Hl 92•. JX26X413J ｮｩｬｲｯｬＡ･ｴＱＳｾ＠ iron 1uutcm (11i/H) gene.

nartial eds

5 Unculrurtd bancrium clone ｊｓｒｾＭＺＡ＠ ＹｾＢＭｾ＠ 9c-107 29.: 111) :Sil"• HM750439.I

-JUllll'Ogcna.se l'NucUl.5e (111/l l) !!el'le pnrual eds

6 Uncultured bae1enum doll<! do.\-42 ＹＷｾＧＮ＠ 2e-102 2SS ｾＱｩ｜＠ ｒｾＢ＠ ｊｘＲＶｾＲＷＲ＠ I nicrogcnnsc iron prolcm tnr/H) gene.

nanial eds

7 Un.;ul1urctl ti.cicrium CIC111e ac;2 ) 97°• 3c- I Iii !9) 1' 7 ｾＮ＠ JX07%20 I

､ｮＱｩｴｲｯｧＮＺｩｩ｡ｾ＠ •TOO pf\\ICIR (nijH) g.:nc ,

oarual ｣､ｾ＠

ｾ＠ Uocuhurcd bdcleriuni d<lnc S1pa-L24 JOO% ＵＬｾ Ｑｷ＠ 33 I 1(,1) 92°. Kf032J72 I

ni1rogc11a<e iron pru1ci11 111ijH) gene. oarual ｾｊＬ＠

From the phylogeny tree (fig. 5) 11 can be seen that the bands I, o ..1.nd ):( hatl closeness 10 Pse11do111011as

WITZl!l'I, Anaeromywbacter sp ｈ｡ｬｯｲｨｯ､ｵ Ｎｾ ｰｩｲ｡＠ haloµhila with compan:il 1estricted to - 360 bp range only

Pseudomcmas stutzeri is a bacteria which has broad habitat and relati,·el) resistant to changes in envcronmental

[image:20.626.69.495.388.643.2]

68 Iman Rusmana D al, 2014

Aduoct:s ia Ea viroameotal Biology, 8(14) Special 2014, Page<;· 63-69

predominantly in the soil [7). IT. ha/op/11/a was classified into gamma prmeobactcria, purple sulfphur bactena, Gram-negati\'e and photorotroph. H. halophila is belong to potential nitrogen fixing bacteria and djspersed quite widely. Some research and studies have shown that H. halophila bas

11!/H

gene and capable of fixing nitrogen [18] [4] (161. Anaeromyxobacter sp. is belong lO the detta proteobacteria bacteria, rod-shaped, Gram-negath·e,

capable of fanning spores, and natural habitat in soil (15]. Anaeromyxobacter sp. also capable of fixing atmosphenc nitrogen and from several studies has been shown that it has nifH gene (8) l16] (20].

·-·

Urctlfured beel•num clone cloA-42 ｉｖｾ＠ llUn protOln (,,.n '' (lef)tt f"tJtfUJI t:"9-JX268212 1

•""""'

... _ , , ... llG23...._,_ ... r-o--•-.JX079d».

Uncu1!<""'1 _ , _

c-

ｾ＠ M-•Jron

"'°'""'

ｾｬｬｦｦｦＩ＠ gMi. f*1Rt1..i. l(FIJ32t12.1

ａｾＩＧＩｏ｢｣ｴｷ＠ $f>.. CP{)()()llJA I

• b-.d5

.,,

,...,,,,,..., 11J. FJ&e7'n I ｐｳｾ＠ $p F.Ja1,T• 1

U'llCiJltUl'ed b-..J.,i..vn ｾ＠ S•.fO ｮｩｴｾＢＧﾰＢ＠ Pf°'..,. (n tH} ｾ＠ l*fW cm JXU&f31 t

ｌｫｬ｣ｴｬｬｾ＠ b1JK.l•n"'n c.((;VlO ｓｾＺｊｦＺ＠ ｮ､ｲｾｾﾷ＠ H<N• OfOI•:'' (1Hftt) gon. "''"'Ill ct# JX2684-.1S T

UlCt.itul8d bll(.lerun c/onit ｓｾＮＧｕ＠ ｍｾ･＠ NOl'l Ｌｏｲｯｦｾ＠ (nt/HJ l.k"MI l*'r-' Cd$ Jl!"'4'M.1111

-- 1' A b - 2

A b-14

Ｎａｺｾ＠ .. "-' hydrty:Jlulus ｅｆＶＲｃ ｾ＠ r

ｾＭＭＭＭＭＭＭＭＭＭ - - - - -- J.ltf,._..«OCC,... ｾａｖＱ＿ＱＱＳｃｯ＠ r

Fig. 5: Phylogenetic tree of8 nijH gene :.equences obtained from DGGI" anaJ)'sis Phylogeny tree ｷ｡ｾ＠ constrvcted with the Neighbour Joining method wich IOOOX boo1srrap Yaluc.

Eight bands were successfully sequenced didn '1 have closeness wi1h merano1roph bacteria which W&S ｵｾ｣､＠ as b1ofertililer and added to the rice fields. Some metanotroph bactena ilia.I added as a biofenih:zcr has been tested an<l analyzed the ability of nitrogen fixation

f3].

However, this study found no sequence which simjJar 10 metanotroph bacleria. It was because the different primer used in ｴｨｩｾ＠ research instead to recognize the mcthanotroph bactena PolF PolR primer designed to amplify the nitrogen fixing bacteria m w1dcl:,. coverage while the primers used by Bintarti [3) \V:l.S a special primer designed b} Dedysh [6] for m1.:tanotroph bactcna !h:ll had nhility of nnrngen fixation. The target amplicuns from cwo pnmcr alsG J1ffor.:nt. f'olF PolR 1 .• im.:r h .. J

a target of360 bp. \\hi le primers designed Dedysh had a target of 453 bp (6).

Co11ciusio11: '

The dfrcrsity of nitrogen-fixing bacteria obtained from rice fields Cicurug village, Sukabumi tend to vary. Control treatment had the most dhersit} in comparison \\1th the soaked and ,,,read trea1men1. Sequencing results showed that all th-: bands were belong into uncultured bacteria dinitogcnase (n(fJI) gene.

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