Bul. Agron.

(31) (1) 21 - 25 (2003)

SCAR (Sequence Characterized Amplified Region) Analysis for Blast Resistant Evaluation on 12
Genotypes of Rice

~ o b i r " ,Harmi ~ n d r i a n ~ t a "Mukelar

, ~mir"

ABSTRACT

Resistance evaluation to blast disease (Pvricularia & on I2 paddy genotypes was carried out in the grew
house by using spray inoculated method with race 033 and 041 of P. grisea , and SCAR (Sequence Characterized
Amplified Region) marker by using Pib primer pairs. The results revealed that among I2 paddy genotypes were
classifed into six resistance groups. The first group comprised two genotypes (Jatiluhzir and Asahan) having three
resistance genes. The second group comprised hvo genotypes (- malampuzhaensis and 0. punctata) having two
resistance genes against race 033 and 041. The third group had one resistance gene against race 033, comprised one
genotype (Way Rarem). Thefourth group comprised one genotype (Danau Tempe) having two resistance genes againsr
041 race and Pib. The fifth group comprised three genotypes (Kalimutu, Maninjau and Laut Tawar) having two
resistance genes against race 033 and Pib. The sixth group comprised hva genotypes (Kencana Bali and Cirata) haviiz~r
no resistance gene to blast race 033 and 041, and Pib. These results indicated that Pib gene did not confer resistance
to race 033 and 041 of Pvricularia &. Resistance to race 033 and 041 might be controlled by different resistant
gene

Key words :SCAR, Blast resistant, Rice

INTRODUCTION characterized Pib gene, one of the genes conferring

Rice blast, caused by the fungal pathogen
I'yricularia grisea, is the most serious disease for
upland. However, recently it has been reported that the
pathogen also infest irrigated rice (Amir et a/., 2000).
The fungus attacks leaves during early growth stages,
develops lesions that are followed by premature leaf
senescence of infected tissues, especially in case of
heavy infections. After heading, the pathogen infects
the panicles or the neck, giving high lost of yield. The
use of resistant cultivars is the most effective means on
controlling the diseases, however, the useful life span of
many cultivars is only few years, due to breakdown of
the resistance in the face of high pathogen variability of
the fungus (Kiyosawa, 1982).
The genes,confen.ing resistance to rice blast has
been studied extensively. So far at least 30 resistance
loci have been identified in rice (Inukai et al., 1994).
and several loci have recently been mapped by using
Restriction Fragments Length Polymorphism (RFLP)
markers (Yu et a/., 1996; Nakamura et a/., 1997).
Wang et a/. (1999) was successfully isolated and
resistance to rice blast disease, by using map-based
cloning strategy. The availability of information
regarding the complete sequence of Pib gene leads to
the possibility of developing specific primers to mark
the Pi-b gene. These markers are classified as Sequence
Characterized Amplification Region (SCAR) markers.
which offer advantage on accuracy over RAPD markers.
since the primer consist of more than 20 bases, and
simplicity over RFLP markers. Detection of SCAR
markers does not need laborious steps of blotting.
hybridization and detection (Sobir, 2000).
Resistance to blast diseases in rice is conferred by
R-genes that named as Pi genes (Ou, 1985). The PI
genes act as major gene, which recognize specific rice
blast race, following gene-for-gene hypothesis (Ebron et
al., 2002). To date 25 Pi genes have been identified
already (Fukuta et a/., 2002). located in several loci on
rice genome (Wang et a1.,1999). To date, based on
reactions to 7 differential varieties, in Indonesia
have been identified 27 races of P. grisea (Amir el 01..
2000), but was not available information, wheather
resistance to each of these races controlled by specific

I ) Department o f Agronomy, Bagar Agricoltural University

JI. Meranti Kampns IPB Darmuga. TelpiFax (0251)629353
2 ) Food Crop Research Institute. M u m , Bogor

SCAR (Scqrro7ce Char-ncte~iiedAnzplrfiedRegion)Analysis for .......... 21

~~~ ~~. .. . ~ !
I
i
Bul. Agron. (31) (1) 21 - 25 (2003)
Pi gene or not. The dominant Pi-b gene is confers high
resistance to most Japanese blast race, but in Indonesia,
is not well identified yet, particularly in what fungus
race that the gene to be confers and what varieties that
carrying the gene.
MATERIALS AND METHODS
Eleven rice genotypes, consisting of two wild
species of (1) Oiyza malampuzhaensis, (2) O v z a
punctata and nine cultivated varieties of (3) Jatiluhur,
(4) Cirata, (5) Way Rarem, (6) Laut Tawar, (7)
Maninjau, (8) Danau Tempe, (9) Kalimutu, (10) Asahan
(as control of resistant genotype) and (1 1) Kencana Bali
(as control of susceptible genotype) were examined.
Two races of fungus, race 033 and 041, were used
in this experiment, since both of them widely found in
paddy field in Indonesia (Amir et a[., 2000). Inoculation
materials were developed from fresh isolated conidia
from the leaf, which infected by P. grisea race 033 and
041. They were cultured in PDA (Potato Dextrose
Agar) media for 5 days, subsequently transferred to
OMA (Oat Meal Agar) media and cultured for 10 days.
Blast Infection Assays
All evaluated genotypes were planted in a culture
box containing clay soil 6 days after gemination in
greenhouse. Inoculation was conducted to the rice leaf
18 days after planting, by using compressor connected
glass atomizer; each box was sprayed with 50 ml fungus
spore, containing 3 x 1 0 ~sporell. After inoculation the
plants were placed in humid room for 2x24 hours, and
then transferred into greenhouse for observation.
Observation of diseases infection intensity was
couducted 5 and 9 days after inoculation based on IRRl
criteria, and obtained data were analyzed by the
following equation (IRRI, 1996).
Z=X&
NV
Where: Z = infection intensity
ni= plant number-i
vi= score of plant number-i
N= number of observed plant
V= maximum score base on IRRl criteria
SCAR Analysis
DNA sample of the 12 evaluated genotypes were
extracted from 1 g young leaves of 6 days rice seedling
by using CTAB extraction method (Doyle and Doyle.
1987) with slight modification (Sobir, 2000). Quantity
and quality of extracted DNA was examined by
electrophoresis method.
SCAR analysis was conducted by amplification
DNA samples of 12 genotypes of rice by using pair of
20 mer Pi6 primer designed from mRNA sequence of
Pib gene (Wang eta/., 1999). The primers sequence are
5'-AGGGAAAAAT GGAAATGTGC-3' (sense) and
5'-AG TAACCTTCTGTGCCCAA-3' (anti-sense).
Polymerase Chain Reaction (PCR) was performed in 25
ml reaction containing of 2.5pLof 10X buffer, 1SpL of
25 mM MgCI2, 1pL of 2.5 mM dNTPs, 1pL of 10 pM
of each primers, IpL of 100 ng DNA template and 1
unit of Taq DNA polymerase enzyme. Amplification
was carried out by using Perkin Elmer 9700 PSR
machine under following conditions Pre-PCR at 94 C
for 5 minutes, followed by 30 cycles of 9 4 ' ~for 30
seconds, 5 8 ' ~for 30 seconds and 7 2 ' ~for I minute.
PCR was stopped at 72'C for 7 minutes. PCR products
were resolved in 1.O% of agarose gel.
RESULT
Observation was conducted on the susceptible
type spot, as indicated by gray color on the center of the
spot, since this spot is the source of conidia for
secondary infection. Based on spot observation at 5
days after inoculation (dai) and 9 dai, infection intensity
data were presented in Table 1. Infection intensity score
below 25 indicates that the genotype is resistant and
higher than 25 indicates that the genotype is susceptible.
Based on the infection intensity score criteria, it was
found that Danau Tempe, Cirata and Kencana Bali
genotypes were susceptible to Pyricularia grisea race
033. and Laut Tawar, Maninjau, Kaliniutu, Cirata and
Kencana Bali genotypes w e r e ~ ~ u s c e ~ t ito
b l Pyricularla
e
grisea race 04 1.
Sobir, Harmi Andrianyta, Mukelar Amir
- ~~ ~ ~.~
~ ~... ~- .. .. . . .. ..
Bul. Alrnn. (31) (1) 21 - 25 (2003)

Table I . Infection intensity on 5 days after inoculation (dai) and 9 dai of the fungus race of 033 and 041 on I I ricc
genotypes.
Race 033 Race 04 1
Genotype
5 dai 9 dai 5 dai 9 dai
Orvza ~nalarnpuzl~aenms 2.5 0.0 1.6 06
iIrvzo punclala
Jatiluhur
Asahan
Laur Tdwar
Maninjau
Kalimutu
Way Rare111
Danau Ten~pe
Cirata
K e ~ ~ c a nBall
a
dai (day after inoculation)

Esarnination of Pib existence in the genome oT
evaluated genotypes was detected with single band of
730 base pairs of amplification product Pib SCAR
primer. The Pib SCAR marker analysis revealed that
Jatiluluw. Maninjau, Kalimntu, Way Rarem, Danau
Tenipe and Cirata genotypes carried Pib gene (Fibarc I),
Correlation analysis showed that the existence nT Pih
gene is not corresponding to the resistauce responses of
the evaluated genotypes, neither to race 033 or race 04 1.

Figure I. SCAR niarker analysis by using Pib primer pair. Lane no 1 to no I I represent Orvza n~alm~p~~zhnen~vi
Ocvza pundoto, Jatiluhnr, Asahan, Laut Tawar. Maninjau. I<alimutu. Way Rarem. Danau Tempe. Cirata.
and Kencana Bali, respectively. M is DNA size markers (%-DNAlkIi1?d-I11digest).

Based on resistance responses to race 033 a i d racc gem. Third group consisted oCl(alimutu, Maninjau and
041. and existence of Pib gene in the genome. ~ h c Laut Tawar. which were resistant to racc 033 and
evaluated genotypes can be classified into six groups. canied Pib gene. Fourth group consisted of Danau
First group consisted of Jatiluhur and Asahan, wluch Tempe, wlucli rvcre resistant to race 041 and canying
wcre resistant to race 033 and race 041, and canied Pih Pib gene. Fifth group consisted of Way Rarem. w11icl1
gene. Second group consisted of Otyo was resistant to race 033 only. Sixth group consisted of
~ ~ ~ a l a ~ t ~ p u zand ~ i . pui~cfoto, which wcrc
h o e 0,:vza Cirata and Kencana Bali, which were susceptible to racc
resistmt to race 033 and race 041, but did not cany Pib 033 and 041. did not c a n , Pib gene (Table 2).
Bul. Agron. (31) (1) 26 -30 (2003)

F l u o r e s e n Klorofil Benih: Parameter Baru d a l a m P e n e n t n a n M u t u B e n i h

Seed CIzloroplzyl Fluorescence: A New Parameter in Quality Seed Testing

Mohamad Rahmad ~ u h a r t a n t o "

ABSTRACT

It has been shown that chlorophyll content of seeds was negatively correlated with germinability towards the end
of maturation. Physiological maturity was achieved when the chlorophflfluorwcence reached a n7inimunz. The
presence of chlorophyll in seeds and its relation with the progress of seed maturation has gained renewed interest affer
the development of Laser Induced Fluorescence (LIF). This equipment is able to nzeasure and analyze chlorophyll
fluorescence in the seed instantaneously and non-destructively The use of LIF makes it possible to perform
physiological and biochemical assays affer chlorophyfl fluorescence measurement in the same seeds. Based on the
results from some experiments as well as the literature, the role of chlorophyll in developing seeds is presented The
overall conclusion is that chlorophyll is required during seed development, but undesirable during maturation. We
hypothesize that the presence of chlorophyll during seed maturation is undesirable since it is associated with lower
quality, particularly lower seed longevity. Chlorophyll may also be aprimary source offree radicals. Seed chlorophyN
fluorescence was affected by endogenous abscisic acid, gibberellins andphytochrome. Light, temperature and relative
humidity may also influence the chlorophyll fluorescence of seeds.

Key words: Chlorophyl, Fluorescence, Parameter, Seed testing

PENDAHULUAN yang masih hijau memiliki daya berkecambah yang

Mutu benih merupakan sebuah konsep yang
kompleks yang mencakup sejumlah faktor yang masing-
masing mewakili prinsip-prinsip fisiologi, misalnya daya
berkecambah, viabilitas, vigor dan daya simpan. Hal ini
menimbulkan kesulitan memperoleh penciri (marker)
fisik, biokimia maupun molekular yang mampu
menduga mutu benih. Lebih dari 15 tahun ini sejumlah
proses biologi telah berhasil diidentifikasi yans
kesemuanya itu berhubungan erat dengan mutu benih,
seperti proses-proses replikasi DNA, perkembangan sel,
degradasi endosperm, aktivitas enzim-enzim hidrolitik
dan potential air dalam hubungannnya dengan
perkembangan dan perkecambahan benih. Dalam
tulisan ini akan diulas kemungkinan penggunaan
fluresen klorofil benih sebagai salah satu penciri fisik
dan biokimia dalam penentuan mutu benih.
Secara alamiah dalam proses pemasakan atau
penuaan tanaman adalah terjadinya degradasi klorofil
yang dengan mudah dapat dilihat pada daun dan buah.
Sebenarnya proses yang sama juga terjadi pada benih.
Penelitian Kwong (1991) pada benih geranium
(Pelargoniun? x hortorun2) menunjukkan bahwa benih
rendah, namun kemampuan berkecambah benih-benih
tersebut meningkat bila dikecambahkan dalam media
yang mengandung nutrisi. Klorofil dalam benih sudah
banyak diteliti pada benih rapeseed (Brassica oleracea)
dalam kaitannya dengan produksi minyak. Minyak yang
berasal dari benih yang mengandung klorofil tinggi akan
rendah mutunya. Hingga kini, kaitan antara klorofil
dalam benih dengan mutu benih (daya bekecambah.
vigor, daya simpan dan lain-lain) belum banyak diteliti.
Jalink (1996) menemukan alat pemilah benih
berdasarkan fluoresen dari klorofil. Alat ( L I P Laser
Induced Fluorescence) ini mampu mendeteksi fluoresen
dari klorofil dengan sensitifitas yang tinggi. Penernuan
ini membangkitkan keinginan untuk menggali inforinasi
tentang peranan klorofil dalam benih, karena
keunggulan utama alat ini adalah selain sangat sensitif
juga dalam proses pengukurannya tidak merusak benih.
Setelah dianalisis benih dapat digunakan untuk kegiatan
penelitian atau pengujian fisiologis dan biokimia
lainnya. Berbeda dengan alat pemilahan benih
berdasarkan wama (color separator) lainnya yang hanya
mampu memilah benih bila dalam lot benih tersebut
memiliki perbedaan yang jelas dan menyolok (biasanya

" Jarusan Budidaya Penanian. Fakultas Pertanian IPB

JI. Mcranti Kampus IPB Darmaga, Bogor. TclpIFax. (0251) 629353
1'-nlall: m.r.suhartanto@ipbac.id

Mohamad Rahmad Suhartanto

.
Bul. Agron. (31) (1) 26-30 (2003)

bisa dibedakan dengan mata), alat pengukur dan pemilah
benih berdasarkan fluoresen klorofil ini mampu memilah
benih yang memiliki perbedaan warna (hijauklorofil)
yang sangat kecil yang tidak mampu diamati dengan
lnata telanjang, seperti pada benih tomat, cabe, kubis,
wortel dan lain-lain. Benih-benih ini sangat sulit
dipisahkan karena pada periode pemasakan memiliki
ukuran, bentuk dan berat yang relatif sama. Karena
relatif merupakan parameter baru, fluoresen dari klorofil
benih diharapkan dapat bersinergi dengan parameter
fisiologis lainnya untuk mengungkap masalah mutu
benih
Fluoresen Klorofil sebagai Pencir~Mutu Bmih: Kasus
Benih Tomaf
Kandungan klorofil pada benih tomat berkorelasi
negatif dengan daya berkecambahnya (Gambar I).
Masak fisiologis yang dicerminkan oleh daya
berkecambah mencapai maksimum pada saat kandungan
klorofil mencapai minimum. Mutu benih sangat
ditentukan oleh tingkat kemasakan benih tersebut.
sehingga dapat dikatakan juga bahwa kandungan
klorofil benih juga menentukan mutu benih tersebut.

21 27 33 39 45 51 57 63 69 75

Hari setelah berbunga (hari)

Gambar 1. Maksimum daya berkecambah dan berat kering benih terjadi saat fluoresen dari klorofil benih mencapai
minimum (Suhartanto, 2002).

Dengan menggunakan LIF tingkat kemasakan (basah) maupun kering (Gambar 2). Jalink el al. (1998)
benih tersebut dengan mudab dapat ditentukan, karena juga mendapatkan bahwa mutu benih kubis meningkat
fluoresen benih tomat berkorelasi secara eksponensial saat fluoresen dari klorofil dalam benihnya menurun.
dengan kandungan klorofilnya, baik pada benih segar

600 2600 4600 6600 600 2600 4600 6600

Fresh seed CF (mV) Dry seed CF (PA)

Gambar 2. Kandungan klorofil benih kering dan basah berkorelasi secara eksponensial dengan fluoresen klorofilnya
selama periode pembentukan benih (21-75 hari setelah berbunga; Suhartanto, 2002).
Bnl. Agron. (31) (1) 26 - 30 (2003)
Beberapa Faktor yang Akmpengaruhi Kandungan
Klorofil Benih
Sejumlah faktor-faktor abiotik yang mem-
pengaruhi degradasi klorofil dalam benih ialah
temperatur, cahaya dan kelembaban. Wards et a/.
(1992) melaporkan bahwa kandungan klorofil benih
rapeseed (Brassica oleraceae) menurun pada saat
masak, dan laju penurunan tersebut lebih rendah bila
suhu lingkungan rendah. Johson-Flanagan el al. (1994)
menunjukkan bahwa benih canola (Brassica napus)
yang ditempatkan pada kelembaban 97% menghasilkan
penurunan laju pigments (temasuk klorofil) sampai
25%. Penelitian pada benih rapeseed menunjukkan
penurunan kandungan klorofil pada benih seiring
dengan penurunan kadar air benih tersebut (Johnson-
Flanagan dan McLachlan, 1990a, b).
Mekanisme cahaya mempengaruhi kandungan
klorofil masih belum jelas, karena diketahui bahwa
cahaya dapat menghambat atau mempercepat proses
degradasi klorofil (Biswal dan Biswal, 1984). Pada
daun padi, degradasi klorofil dihambat oleh penyinaran
yang kontinyu dengan intensitas rendah (0.5 pmol
photon.m-2.detik"), namun dengan intensitas lebih dari
10 pmol photon.m~2.detik~'proses penghambatan
tersebut berkurang atau laju degradasi klorofil
meningkat (Okada et al., 1992). Suhartanto (2002)
melaporkan bahwa proses degradasi klorofil pada benih
tomat masih terjadi meskipun benih sudah dikeringkan.
Benih yang disimpan dalam ruang simpan dengan
cahaya merah menurun kandungan klorofilnya, namun
bila disimpan di ruang gelap kandungan klorofilnya
relatif tetap. Hal yang menarik ialah daya simpan benih
tomat dalam ruangan dengan cahaya merah lebih baik
dibanding di ruang gelap. Diduga klorofil dari benih
dapat menjadi sumber radikal bebas yang dapat
me~npercepatpenurunan viabilitas benih.
Benih tomat hasil pertanaman musim semi
memiliki kandungan klorofil yang lebih rendah
dibanding hasil musim gugur (Suhartanto, 2002).
Intensitas cahaya dan panjang hari diduga
mempengaruhi perbedaan kandungan klorofil benih-
benih tersebut. Suhartanto (2002) juga melaporkan
bahwa degradasi klorofil benih yang berasal dari buah
berukuran kecil terjadi lebih cepat dibanding buah
berukuran besar. Secara umum telah diketahui bahwa
cahaya mengendalikan perkembangan kloroplas.
Perubahan kualitas cahaya mengakibatkan perubahan
keseimbangan ekspresi gen kloroplas pada fotosistem I
dan I1 (Pfannschmidt et al., 1999). Pada daun
Arabrdops~s thaliana, intensitas cahaya tinggi akan
mengurangi jumlah thylakoid granal per kloroplas,
proporsi klorofil b terhadap klorofil a, dan akumulasi
dari polipeptida utama LHC (Weston et al., 2000).
Asam absisat (ABA) dan giberelin (GAS) sangat
berperan dalam perkembangan benih (Bewley dan
Black, 1994). ABA dan GA endogen mempengaruhi
kandungan klorofil benih. Benih tomat yang defisien
GA memiliki kandungan klorofil yang lebih tinggi
dibanding tetuanya (wild type), sedangkan benih yang
defisien ABA memiliki kandungan klorofil paling
rendah (Suhartanto, 2002). Hal ini kemungkinan dapat
disebabkan oleh adanya hubungan proses biosintesis
ABA, GAS dan klorofil. Defisiensi GAS setelah
terjadinya hambatan dalam tahap spesifik dalam
biosintesis GA akan mengakibatkan peningkatan
pigmentasi, baik klorofil maupun kamtenoid. Lebih
lanjut Maluf et al. (1997) menunjukkan bahwa pada
mutan benih jagung yang defisien ABA juga akan
mengalami defisiensi klorofil dan karotenoid. Mereka
juga menunjukkan bahwa mutan ini memiliki ekspresi
geranil-geranil pirofosfat sintase yang rendah. Enzim ini
bertanggung jawab dalam proses sintesis geranil-geranil
pirofosfat, yang merupakan precursor dari ABA,
karotenoid dan klorofil. Lebih lanjut dilaporkan bahwa
benih mutan yang memiliki kandungan fitokrom rendah
(phytochrome defsient mutant) akan memiliki
kandungan klorofil yang rendah pula dan benih dari
mutan ini memilki dormansi yang tinggi.
P e r m Klorofl dalam Benih
Sedikit sekali informasi tentang peran dan fungsi
klorofil dalam benih. Sugimoto el al. (1987)
rnennnjukkan bahwa benib kedelai yang sedang tunibuh
memiliki aktivitas fotosintesis. Hilangnya kemampuan
untuk berfotosintesis diduga disebabkan oleh
menurunnya intensitas cahaya yang dapat mencapai
kloroplas benih akibat terjadinya akumulasi zat-zat
cadangan makanan selama periode pemasakan benih.
Suhartanto (2002) juga membuktikan bahwa benih
tomat memiliki aktivitas fotosintesis in vitro sampai
dengan 40 hari setelah berbunga. Lebih lanjut juga
ditunjukkan bahwa bila buah tomat ditumbuhkan dalam
kondisi gelap akan menghasilkan benih dengan kualitas
rendah dibandingkan bila buah tersebut tumbuh dalam
kondisi penyinaran cahaya alami. Benih dari buah yang
tumbuh di tempat gelap akan memiliki masa dorrnansi
yang lebih lama. Fungsi klorofil dalam benih diduga
sangat berhubungan dengan proses evolusi seperti yang
ditunjukkan oleh Suhartanto (2002) bahwa benih-benih
tomat yang berasal dari turunan varietas liar memiliki
kandungan klorofil dan aktivitas fotosintesis yang lebih
tinggi dibanding varietas yang telah dibudidayakan. Hal
ini merupakan indikasi bahwa kandungan klorofil dan
fungsinya mengalami penurunan selama proses
domestikasi. Li et al. (2000) berhipotesis bahwa selama
proses evolusi, protein kompleks pemanen cahaya (LHC
atau light-harvesting complex protein) dengan fungsi
sebagai phototoprotective muncul terlebih dahulu
dibanding dengan fungsinya sebagai pemanen cahaya
(fotosintesis).
Bul. Agron. (31) (1) 31 - 36 (2003)

fertilizer, it indicates that there was some amount of
nitrogen transferred from legume to oat.
The of value of S "N units uptake was calculated using
equation :
6 "N units uptake= 16 "N m i l receiver - 6 '*N unit control ( C l ) j ( I )
itotal N receiver -total N (C1)l
whereas S "N unit (receiver or control) was :
S "N unit = S ' 5 x~ total N
In the experiment 2, the transfer of N was
estimated using a method known as 'Donor root
enrichment' (Giller et al., 1991). The proportion of N in
the receiver plant derived from donor plant root (% Ndft
root) was calculated under assumption that N from
donor plant deposited in the rhizosphere and taken up by
the receiver plant had the same 'IN enrichment during
the labelling period as the donor root at the time of
harvest.
atom % "N excess ,p,.
% Ndfl root = x 100 (3)
atom % 'IN excessdonorroot
The amount of N transferred (mg plant -') is calculated
as :
N transfemed = % Ndft root x total N receiver (4)
% N transfer = N transferred x 100 (5)
(total N donor + N transferred)
RESULTS AND DISCUSSION
The data of experiment 1 was found to have
greatly variance within replications, therefore only the
mean values were showed in Table 1.
The oat growing together with faba beans
showed the lowest 6. "N value, but it presumably as a
result of competition, since total N uptake was lower
than that from oat growing alone surrounded by faba
(2) (treatment m and s). If the intercropped non legume had
less "N as well as more total N than the sole crop non
legume it would indicate that the 'IN had been diluted
from the legume (Martin et a[., 1991). The S "N uptake
of oat growing together with cv. Minica was much lower
than 6 "N value of labelled N fertilizer. The lower
value of 6 1 5 uptake
~ could be caused by uptake N from
mineralization (Giller and Wilson, 1991). During the
first weeks of this experiment, faba beans were
transplanted twice because the microclimate inside the
glasshouse was not favorable. This situation could lead
to mineralization of decayed-seed.

Table I. Means value of shoot dry matter, N uptake, N content and 6 "N uptake of oats in experiment I.
Treatment Dry Matter Total N uptake 6 "N. N content S I5N uptake
(w) (%J rl"")
C1 (control -'IN) 170 1.01 68.208 0.59
C2 (control "'N) 220 1.51 4366.095 0.65
OM 170 2.68 2278.638 1.12 9040.974
0s 210 1.42 3939.530 0.65 19630.479
Om 370 1.91 5585.948 0.72 15817.665
0s 200 1.57 4348.230 0.7 1 16133.405
Note : OM = o a t growing together with faba bean cv.Minica
OS = oat growing together with faba bean cv. Scirocco
Om = oat growing surrounded by faba bean cv. Minica
0 s = o a t growing surrounded by faba bean cv. Scirocco

In experiment 2 faba bean was 16 days older than
oat. Although the oat grew poorly, oat in the 'Receiver'
pot did not get any additional N fertilizer. The source
for N for oat, solely, came from soil and N, which
deposited by faha bean. The half root of faba bean
growing in 'Donor' pot was not included for calculating
dry lnatter weight, total N uptake and "N enrichment.
Since this half root was directly contacted to N fertilizer,
it would add a considerable amount of N and 'IN to the
whole plant basis.
There were no different in plant dry weight and
total N uptake of oat growing with ISN-fertilised faba
beans or unlabelled-fertilized faba beans, although the
root of oat growing together with unlabelled-fertilized
Minica contained lower N compared to oats root
growing together with '5~-fertilized Minica. Oat
growing with 15~-fertilized Scirocco showed higher "N
enrichment compared to oat growing with unlabelled-
fertilized Scirocco, whereas in oat growing with IsN-
fertilized Minica only the root had higher I5N
enrichment (Table 2).
Bul. Agron. (31) (1) 31 - 36 (2003)
Table 4. Estimated N-transfer') from faba bean cv Minica and Scirocco to oat
Treatments
N transfer2'
(%I
Oat growing 0.68
With Minica
Oat growing 0.58
With Scirocco
HSD(0,OS) ns
Note : I) =calculation for plant excluded the half root in pot 'donor'
2) = N transferred as percentage of faba bean-N
3) = N transferred as percentage of oat-N
ns = no significant.
Despite the low N nutrition condition created in
'Receiver' pot to enhance faba bean-N-fixation, there
seemed no immediate benefit o f N to oat. At the time of
harvest, Minica contained in average I .98% N and
Scirocco 1.99% N. This N uptake of faba beans was
roughly three times of total N in oat but only less than
0.68% of faba bean's N was transferred to oat. Giller et
a/. (1991) and Jensen (1996) found that the amounts of
N transferred from bean to companion plant in
intercropping might improve under a severe limitation
on the growth of the beans such as an insect attack or
shading.
We found no correlation between the amount of N,
which transferred to oat, neither to the amount of N
taken up by faba beans, nor the amount of N left in the
~nediaof 'Receiver' pot after harvest.
Jensen (1996) explained that the donor root
eurichment method may give the most reliable
estimation o f N transfer in continuous split root labelling
because the "N enrichment of donor root is probably
similar than the ' 5 enrichment
~ o f N deposited. But, we
inust also consider that at 90 days after sowing, faba
bean was at the end of pod filling stage. The "N
enrichment of faba beans roots might be different with
the "N enrichment of N deposited and taken up by oat
since deconiposition and senescence of nodules and root
had been occurred.
We concluded that transfer of N from faba bean to
associated plant does occur, although the amount of N
transferred was very small (less than 0.68% of faba
bean's N). The result of experiment also showed that
there was no difference in the amount of N transfer
between the two tested cultivars, Minica and Scirocco.
ACKNOWLEDGEMENTS
We would like to thank R. Langell for helping in N
analyses, Gaby and Mrs Hofmann for helping during
preparation. The work was funded by Prof. Werner
Donor root enrichment method
N transferred (mg ~ d froot3)
t
N planf I)
1.280 2.51
0.927 1.62
ns 11s
Schulze-Stiftung im Stifterverband fur die Deutsche
Wissenschaft.
REFERENCES
Bulson, H. A. J., Snaydon, R.W., C. E. Stowes. 1996.
Effects of plant density on intercropped wheat and
field beans in an organic farming system.
Agricultural Botany Department, University of
Reading, Reading RG6 2AS, UK, I lp.
Cochran, V. L., S. F. Schlentner. 1985. Intercropped
oat and fababean in Alaska: dry matter production.
dinitrogen fixation, nitrogen transfer and nitrogen
fertilizer response. Agron. J. 87,420-424 .
Danso, S. K. A., F. Zapata, G. Hardarson. 1987.
Nitrogen fixation in faba beans as affected by plant
population density in sole or intercropped system
with barley. Soil Biol. Biochein. 19.41 1-415.
FAO. 2000. Production Year Book F A 0 Rome
http:l/w.FAO.or~statistical databaseslagricul-
turelprimary crops. (September, 2000)
Giller, K. E., I<. J. Wilson. 1991. Nitrogen fixation in
tropical cropping systeins. CAB International.
Wallingford. Oxon. UK. 313p.
Giller, I<. E., J. Ormesher, F. M. Awah. 1991. Nitrogen
.transfer from Phaseolus bean to intercropped
maize measured using lS~-enrichmentand 1 5 ~ -
isotope dilution methods. Soil Biol. Biochem. 23,
339-346.
Jensen, E. S. 1996. Barley uptake of N deposited in the
rhizosphere of associated field pea. Soil Biol.
Biochem. 28 : 159 - 168.
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