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 ~ m i r "

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). The fourth 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

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

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

characterized Pib gene, one of the genes conferring 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

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).

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, 1 pL of 2.5 mM dNTPs, 1 pL 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

Z=X& Kencana Bali genotypes were~~usce~ t ib le to Pyricularla NV grisea race 04 1.

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

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.

Genotype Race 033 Race 04 1 5 dai 9 dai 5 dai 9 dai

Orvza ~nalarnpuzl~aenms 2.5 0.0 1.6 0 6 iIrvzo punclala Jatiluhur Asahan Laur Tdwar Maninjau Kalimutu Way Rare111 Danau Ten~pe Cirata Ke~~cana Ball

dai (day after inoculation)

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

Figure I. SCAR niarker analysis by using Pib primer pair. Lane no 1 to no I I represent Orvza n~alm~p~~zhnen~vis , 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-I11 digest).

Based on resistance responses to race 033 a i d racc 041. and existence of Pib gene in the genome. ~ h c evaluated genotypes can be classified into six groups. First group consisted of Jatiluhur and Asahan, wluch wcre resistant to race 033 and race 041, and canied Pih gene. Second group consisted of O t y o ~ ~ ~ a l a ~ t ~ p u z h o e ~ i . and 0,:vza pui~cfoto, which wcrc resistmt to race 033 and race 041, but did not cany Pib

gem. Third group consisted oCl(alimutu, Maninjau and Laut Tawar. which were resistant to racc 033 and canied Pib gene. Fourth group consisted of Danau Tempe, wlucli rvcre resistant to race 041 and canying Pib gene. Fifth group consisted of Way Rarem. w11icl1 was resistant to race 033 only. Sixth group consisted of Cirata and Kencana Bali, which were susceptible to racc 033 and 041. did not c a n , Pib gene (Table 2).

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

Fluoresen Klorof i l 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

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

yang masih hijau memiliki daya berkecambah yang 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 (LIP 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 Fluoresen Klorofil sebagai Pencir~ Mutu Bmih: Kasus benih berdasarkan fluoresen klorofil ini mampu memilah Benih Tomaf benih yang memiliki perbedaan warna (hijauklorofil) yang sangat kecil yang tidak mampu diamati dengan Kandungan klorofil pada benih tomat berkorelasi

lnata telanjang, seperti pada benih tomat, cabe, kubis, negatif dengan daya berkecambahnya (Gambar I).

wortel dan lain-lain. Benih-benih ini sangat sulit Masak fisiologis yang dicerminkan oleh daya berkecambah mencapai maksimum pada saat kandungan dipisahkan karena pada periode pemasakan memiliki klorofil mencapai minimum. Mutu benih sangat ukuran, bentuk dan berat yang relatif sama. Karena

relatif merupakan parameter baru, fluoresen dari klorofil ditentukan oleh tingkat kemasakan benih tersebut.

benih diharapkan dapat bersinergi dengan parameter sehingga dapat dikatakan juga bahwa kandungan klorofil benih juga menentukan mutu benih tersebut. fisiologis lainnya untuk mengungkap masalah mutu

benih

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 e t 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~npercepat penurunan 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 I 1 (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 mi 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 (2)

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 (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 =oa 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 =oa 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 IS total N uptake of oat growing with N-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 Donor root enrichment method N transfer2' N transferred (mg ~ d f t root3)

(%I N planf I) Oat growing 0.68 1.280 2.51 With Minica Oat growing 0.58 0.927 1.62 With Scirocco HSD(0,OS) ns ns 11s

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 ~nedia of '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

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~-enrichment and 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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