UNIVERSITI PUTRA MALAYSIA

EFFECTS OF ROOT EXUDATES ON SPECIFIC DIAZOTROPH-RICE GENOTYPE ASSOCIATION

UMME AMINUN NAHER

FP 2009 20

EFFECTS OF ROOT EXUDATES ON SPECIFIC DIAZOTROPH-RICE GENOTYPE ASSOCIATION

By

UMME AMINUN NAHER

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy

SEPTEMBER 2009

DEDICATED

to

My son Azmaine Iqtidar

my husband Mesbahuddin Ahmed

my parents Syed Ahmed Ali Amina Ahmed

and Family

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in partial fulfilment of the requirement for the degree of Doctor of Philosophy

EFFECTS OF ROOT EXUDATES ON SPECIFIC DIAZOTROPH-RICE GENOTYPE ASSOCIATION

By

UMME AMINUN NAHER

September 2009

Chairman: Associate Professor Radziah Othman, PhD Faculty : Agriculture Diazotrophs are known to utilize root exudate carbon compounds, form natural

associations with rice plants and subsequently fix nitrogen. The specific association can

be influenced by the bacterial strains and rice genotypes. A series of experiments were

conducted in laboratory and glasshouse conditions with the following objectives; (i) to

isolate and characterize the indigenous diazotrophs, (ii) to determine the root exudate

sugars and amino acids of different rice genotypes (iii) to determine the utilization of

root exudates sugars by the diazotrophs (Rhizobium sp. and Corynebacterium sp.) during

colonization and (iv) to determine the effects of specific sugars on plant-diazotrophs

associations, biological nitrogen fixation and growth of different rice genotypes. The

diazotrophs were isolated from Tanjong Karang rice irrigation project area using N-free

semi-solid media and the nitrogenase enzyme activity was determined by Acetylene

Reduction Assay (ARA) technique. The isolated diazotrophs were identified using

Biolog Identification method. Root exudates sugars and amino acids of the three rice

genotypes (Mahsuri, Mayang Segumpal and MR219) were determined using high

iii

performance liquid chromatography (HPLC). The root (rhizosphere and endophytic)

colonization were visually observed by using Scanning Electron Microscope (SEM) and

Transmission Electron Microscope (TEM). The diazotroph–plant association and

biological nitrogen fixation (BNF) was estimated using 15N dilution technique in

glasshouse condition. Results showed that the indigenous diazotrophic populations were

significantly (P<0.01) influenced by soil types, plant age and rice varieties. Bacterial

populations were significantly higher in soil (1.8-2.2 x 106 cfu g-1soil) and rhizosphere

(1.4-4.2 x 107 cfu g-1 root) of rice grown in Organic Clay & Muck, Bakau, Sedu and

Serong soil series. Molecular analysis indicated a diverse group of diazotrophic strains

were present in the different soil types. Ten of the strains were identified by the Biolog

Identification method as Rhizobium, Burkholderia and Corynebacterium spp..

Biochemical tests of 19 isolates showed that these strains were positive for N2 fixation,

capable of degrading cellulose and able to produc high amounts of indoleacetic acid

(IAA) which ranged from 15 to 69 mg L-1. The diazotrophs exhibited differences in the

specific growth rate, generation time, and utilized mono and disaccharide sugars as sole

energy sources. A total of seven sugars and 16 amino acids were determined from rice

root exudates. The concentration of root exudate sugars, amino acids and their release

patterns differed significantly among rice genotypes. Mahsuri released the highest root

exudate sugars (25.73%) followed by MR219 (23.14%) and Mayang Segumpal

(20.85%). Inoculated plants produced different amounts of sugars and amino acids

compared to non-inoculated plants. Mahsuri inoculated with Corynebacterium sp.

(Sb26) released the highest amount of fructose and arabinose, while Mayang Segumpal

inoculated with Rhizobium sp. (Sb16) produced the highest amount of sucrose in the root

exudate. All rice genotypes produced significantly higher amounts of glycine and

iv

isoleucine in root exudates as compared to other amino acids. In general, plants

inoculated with Sb16 produced higher amounts of total sugars and amino acids in their

root exudates compared to those inoculated with Sb26. A significant relationship was

observed between diazotrophic populations and utilization of root exudates sugars and

amino acids in the rice genotypes. The Sb16 strain utilized higher amounts of sugars and

stimulating higher rhizosphere population compared to Sb26 strain. The diazotrophs

were able to colonize and proliferate endophytically in the rice roots. SEM micrographs

showed the occurrence of bacterial colonization on surfaces of primary and lateral roots,

root hair zone, lateral root junction, in crevices and root tips. TEM view of roots

revealed the presence of diazotroph in the intercellular spaces of cortical parenchyma,

within epidermis, inner cortex, and near vascular tissue. The results of invitro and

glasshouse study using 15N dilution studies showed that Mayang Segumpal inoculated

with Sb16 and applied with galactose significantly increased plant-N content and fixed

42 % of atmospheric N (Ndfa). This association increased 147-245 % plant biomass

compared to non-inoculated control and 8-52 % over 60 kg ha-1 N-fertilizer

application.The study proved that diazotroph inoculation enhanced root exudate sugar

production and provides specific sugars for specific diazotroph-rice plant association.

Application of galactose and arabinose as external carbon source enhanced the growth

and N2 fixation activity of the Rhizobium sp. (Sb16) and Corynebacterium sp. (Sb26),

respectively. The association of Mayang Segumpal with Sb16 and MR219 rice with

Sb26 significantly improved nitrogen fixation and subsequently plant growth.

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

KESAN EKSUDAT AKAR KE ATAS GABUNGAN ANTARA DIAZOTROF

KHUSUS-GENOTIP PADI

Oleh

UMME AMINUN NAHER

September 2009

Pengerusi : Professor Madya Radziah Othman, PhD Fakulti : Pertanian Diazotrof diketahui menggunakan kompaun karbon dalam eksudat akar, membentuk

penyatuan semulajadi dengan pokok padi dan seterusnya mengikat nitrogen. Penyatuan

khusus boleh dipengaruhi oleh strain bakteria dan genotip padi. Beberapa siri

eksperimen telah dijalankan di dalam makmal dan di rumah kaca dengan objektif-

objektif berikut; (i) untuk mengasingkan dan mencirikan diazotrof asal, (ii) untuk

menentukan gula eksudat akar dan asid amino pada genotip padi berbeza, (iii) untuk

menentukan penggunaan gula eksudat akar oleh diazotrof (Rhizobium sp. and

Corynebacterium sp.) semasa kolonisasi dan (iv) untuk menentukan kesan gula khusus

ke atas penyatuan tanaman-diazotrof, pengikatan nitrogen biologikal dan pertumbuhan

genotip padi berbeza. Diazotrof telah dipencilkan dari kawasan projek pengairan padi

Tanjong Karang menggunakan media separa pepejal bebas nitrogen dan aktiviti enzim

nitrogenase ditentukan oleh teknik asai penurunan asetilena (ARA). Diazotrof tersebut

telah dikenalpasti menggunakan kaedah pengecaman Biolog. Gula eksudat akar dan asid

amino tiga genotip padi (Mahsuri, Mayang Segumpal dan MR219) telah ditentukan

dengan menggunakan kromatografi cecair berkeupayaan tinggi (HPLC). Pengkolonian

bakteria pada akar (rhizosfera dan endofitik) telah diperhati secara visual menggunakan

vi

Mikroskop Pengimbas Elektron (SEM) dan Mikroskop Pemancar Electron (TEM).

Pengikatan nitrogen biologikal (BNF) pada penyatuan bakteria-tanaman telah ditentukan

dengan menggunakan kajian pencairan 15N dalam rumah kaca. Keputusan kajian

menunjukkan populasi diazotrof asal dipengaruhi dengan signifikan (P<0.01) oleh jenis

tanah, umur tanaman dan varieti padi. Populasi bakteria adalah lebih tinggi secara

signifikan dalam tanah (1.8-2.2 x 106 cfu g-1 tanah) dan rhizosfera (1.4-4.2 x 107 cfu g-1

akar) pertumbuhan padi dalam siri tanah Organic Clay & Muck, Bakau, Sedu dan

Serong. Analisis molekular menunjukkan pelbagai kumpulan strain bakteria hadir dalam

jenis tanah berlainan. Sepuluh strain telah dikenalpasti oleh kaedah Biolog sebagai

Rhizobium, Burkholderia dan Corynebacterium spp. Ujian biokimia ke atas 19 isolat

menunjukkan bahawa strain-strain ini adalah positif pada pengikatan N2, berkebolehan

menghancurkan selulosa dan berupaya menghasilkan jumlah asid indolasetik (IAA) yang

tinggi pada julat antara 15 hingga 69 mg L-1. Diazotrof telah menunjukkan perbezaan

pada kadar pertumbuhan tentu, masa generasi dan penggunaan gula mono dan disakarida

sebagai sumber tenaga utama. Sejumlah tujuh gula dan 16 asid amino telah ditentukan

daripada eksudat akar padi. Kepekatan gula eksudat akar, asid amino dan corak

pembebasannya berbeza dengan ketara antara genotip padi. Mahsuri menghasilkan gula

eksudat tertinggi (25.73%), diikuti dengan MR219 (23.14%) dan Mayang Segumpal

(20.85%). Pokok yang diinokulasi menghasilkan kandungan gula dan asid amino yang

berbeza berbanding pokok yang tidak diinokulasi. Mahsuri yang diinokulasi dengan

Corynebacterium sp. (Sb26) telah membebaskan kandungan fruktosa dan arabinosa

tertinggi, manakala Mayang Segumpal yang diinokulasi dengan Rhizobium sp. (Sb16)

telah menghasilkan kandungan sukrosa yang tertinggi dalam eksudat akar. Semua varieti

padi telah menghasilkan kandungan glisina dan isoleusina yang ketara tinggi dalam

eksudat akar berbanding dengan asid amino yang lain. Pada amnya, pokok yang

diinokulasi dengan Sb16 telah menghasilkan jumlah gula dan asid amino yang lebih

vii

tinggi dalam eksudat akar berbanding dengan pokok yang diinokulasi dengan Sb26.

Interaksi signifikan dapat dilihat di antara populasi diazotrof dengan penggunaan gula

dan asid amino pada tiga jenis varieti padi. Sb16 menggunakan lebih banyak gula dan

menghasilkan lebih tinggi populasi rhizosfera berbanding Sb26. Perbezaan penggunaan

gula oleh diazotrof dapat dilihat untuk kesemua tiga varieti padi. Strain Sb16

menggunakan jumlah gula yang lebih tinggi dan menggalakkan populasi bakteria pada

rhizosfera yang lebih tinggi berbanding strain Sb26. Diazotrof mampu mengkolonisasi

dan bercambah secara endofitik dalam akar padi. SEM menunjukkan pengkolonian

bakteria di atas permukaan akar primer dan akar sisi, zon akar rerambut, persimpangan

pengeluaran akar sisi, di celahan dan hujung akar. TEM menunjukkan kehadiran

diazotrof di ruang interselular pada parenkima kortikal, di antara epidermis, korteks

dalam dan saluran tisu vaskular. Keputusan kajian dalam makmal dan rumah kaca

dengan menggunakan kajian pencairan 15N menunjukkan bahawa Mayang Segumpal

yang diinokulasi dengan Sb16 dan diberi galaktosa meningkatkan dengan signifikan

kandungan N-pokok dan mengikat 42% N atmosfera (Ndfa). Penyatuan ini telah

meningkatkan 147-245% biomas pokok berbanding pokok yang tidak diinokulasi dan 8-

52% berbanding pokok yang diaplikasi dengan 60 kg ha-1 baja N. Kajian ini

membuktikan bahawa inokulasi diazotrof meningkatkan pengeluaran gula eksudat akar

dan membekalkan gula khusus untuk penyatuan khusus diazotrof-pokok padi. Aplikasi

galaktosa dan arabinosa sebagai sumber karbon luaran telah masing-masing

meningkatkan pertumbuhan dan aktiviti pengikatan nitrogen Rhizobium sp. (Sb16) dan

Corynebacterium sp. (Sb26). Penyatuan padi Mayang Segumpal dengan Sb16 dan padi

MR219 dengan Sb26 meningkatkan dengan ketara pengikatan nitrogen dan seterusnya

pertumbuhan pokok.

viii

ACKNOWLEDGEMENTS

‘Glory be to the Almighty’. I owe a debt of gratitude to the Almighty for His blessing to

accomplish this study. Alhamdulillah.

I wish to express my heartfelt gratitude, indebtedness, and deep sense of respect to

Associate Professor Dr. Radziah Othman, the Chairman of the Supervisory Committee

for her sincere support, guidance, constant encouragement, invaluable suggestions and

generous help throughout the study period. I am much indebted and grateful to Professor

Dr. Zulkifli Hj Shamsuddin, Associate Professor Dr. Halimi Mohd Saud and Professor

Dr. Mohd Razi Ismail, members of the supervisory committee, for their constructive

suggestions and guidance in formulation and execution of the research projects and

critical review of the manuscript. I express my deep sense of respect to all of the

lecturers and professors department of land management for their valuable suggestions

and constructive comments.

Thankfully acknowledge the assistance of TWOWS (Third World Organization for

Women in Science) for financial support and facilitating my study in Malaysia, and

University Putra Malaysia for providing research facilities under the project Ministry of

Science, Technology and Innovation (MOSTI) for Science fund project: 02-01-04-

SF0169. Furthermore, I am also grateful to the Bangladesh Rice Research Institute

(BRRI) for providing deputation and help for the accomplishment of my degree.

ix

I am grateful to the administrative staff of the Soil Microbiology laboratory, Department

of Land Management, Department of Crop Science, Crop Protection, Biotech III, and

Institute of Bioscience. Special thanks to Nuclear Malaysia, Bangi, for 15N analyses,

University Science Malaysia for acetylene reduction activity test and Institute of

Veterinary Science, Nilai for amino acids analyses. I respectfully acknowledge the

blessings and good wishes of my parents, brother, sister, late father in law, mother in law

and all relatives. Special gratitude goes to my only son Azmaine Iqtidar, for his patience

during the hard time of my study period.

I am be indebted to my husband Mesbahuddin Ahmed, not only for his sacrifice but also

for bearing responsibilities of taking care of our son and for his encouragement to

pursuing a higher degree. Without his love, great sacrifice, and support this study would

not have been possible.

I would like to extend my heartfelt thanks to Mr. Qurban Ali Panhwar, for helping and

sharing my workload, frustration, anxiety and happy moments during the study period.

My appreciation goes to all of my friends specially Monthar, Noraini, Rizal, Asilah,

Aizat, Ayuni, Haniza, Hidayah, Fadhlil, Rahimi, Shamsol, Hun, Diana, Aishah, Mazrina

and all well wishers.

Finally I would like to express my humble apology to those persons, who had helped me

in some ways but may not find their names in my narration here.

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I certify that an Examination Committee has met on 15th September, 2009 of viva voce to conduct the final examination of Umme Aminun Naher on her Doctor of Philosophy thesis entitled “Effect of Root Exudates on Specific Diazotroph-Rice Genotypes Association” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Examination Committee were as follows: Name of Chairperson, Ph.D. Prof. Dr. Shamsudin Jusop Faculty of Agriculture Universiti Putra Malaysia (Chairman) Name of Examiner 1, Ph.D. Prof. Dr. Mohd. Khanif Yusop Faculty of Agriculture Universiti Putra Malaysia (Internal Examinar) Name of Examiner 2, PhD. Associate Prof. Dr. Kamaruzaman Sijam Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Name of External Examiner, Ph.D. Professor Dr. Asghari Bano Department of Plant Sciences Quaid-I-Azam University Islamabad Pakistan (External Examiner) -------------------------------------------------- BUJANG KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 18th October 2009

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This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement of the degree of Doctor of Philosophy. The members of the Supervisory Committee were follows: Radziah Othman, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Zulkifli Hj Shamsuddin, Ph.D. Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

Halimi Mohd Saud, PhD. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member) Mohd Razi Ismail, Ph.D. Professor Faculty of Agriculture Universiti Putra Malaysia (Member) -------------------------------------------------- HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 16th November 2009

xii

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions. ------------------------------------------ UMME AMINUN NAHER Date:

xiii

TABLE OF CONTENTS Page DEDICATION iiABSTRACT iiiABSTRAK viiACKNOWLEDGEMENTS xiAPPROVAL xiiDECLARATION xvLIST OF TABLES xxiLIST OF FIGURES xxivGLOSSARY OF ABBREVIATIONS xxviii CHAPTER 1 INTRODUCTION 1.1 2 LITERATURE REVIEW 2.0 Rice (Oryza sativa L.) 2.1 2.1 Global nitrogen fertilizer production and uses 2.2 2.2 Nitrogen fertilizer uses in Malaysia for rice production 2.3 2.3 Rice production in sustainable agriculture 2.4 2.4 Diazotroph associations in rice 2.6 2.4.1 Diazotrophic PGPR 2.8 2.4.2 Mechanism of nitrogen fixation 2.10 2.4.3 Mechanism of carbon uptake for biological nitrogen

fixation 2.14

2.5 Mechanism of plant growth promotion 2.16 2.5.1 Growth promotion by Rhizobium spp. 2.17 2.5.2 Growth promotion by Burkholderia sp. 2.19 2.5.3 Growth promotion by Corynebacterium spp. 2.22 2.6 Relationship between phytohormone and nitrogen fixation 2.22 2.7 Factors affecting diazotrophic population and nitrogen fixation 2.24 2.7.1 Soil physico-chemical properties 2.24 2.7.2 Soil microbial communities 2.27 2.7.3 Plant genotypes and growth stages 2.28 2.7.4 Root exudate and root colonization 2.29 2.8 Mechanism of attachment to the root system 2.31 2.9 Influence of root exudation on colonization 2.34 2.10 Evidence of root colonization 2.35

xiv

3 ISOLATION AND CHARACTERIZATION OF INDIGENOUS DIAZOTROPH FROM RICE PLANTS GROWN IN TANJONG KARANG RICE IRRIGATION PROJECT

3.0 Introduction 3.1 3.1 Materials and Methods 3.3 3.1.0 Sampling procedure for isolation of indigenous diazotroph 3.3 3.1.1 Isolation and estimation of diazotroph population from

soil and rice-rhizosphere 3.3

3.1.2 Isolation and estimation of endophytic diazotroph population from rice root and stem

3.4

3.1.3 Soil chemical analysis 3.4 3.1.4 Gram staining and KOH test 3.5 3.1.5 Estimation of acetylene reduction (ARA) 3.5 3.1.6 Determination of indoleacetic acid (IAA) production 3.6 3.1.7 Determination of cellulase activity 3.6 3.1.8 Strain identification using Biolog Identification method 3.7 3.1.9 Strain diversity of the isolates 3.8 3.2 Results 3.9 3.2.1 Physico-chemical properties of the experimental soil 3.9 3.2.2 Population of diazotroph 3.10 3.2.3 Gram staining and KOH test 3.11 3.2.4 Acetylene reduction assay (ARA) 3.11 3.2.5 Cellulase activity 3.12 3.2.6 Indoleacetic acid production (IAA) 3.12 3.2.7 Biolog identification and strain diversity 3.15 3.3 Discussion 3.17 3.4 Conclusion 3.20 4 SPECIFIC GROWTH RATE AND SUGAR CONSUMPTION OF

DIAZOTROPH ISOLATED FROM RICE RHIZOSPHERE

4.0 Introduction 4.1 4.1 Materials and Methods 4.3 4.1.1 Substrate preparation 4.3 4.1.2 Inocula preparation 4.3 4.1.3 Determination of population growth 4.4 4.1.4 Determination of sugar consumption 4.4 4.1.5 Specific growth rate 4.5 4.1.6 Cell doubling time 4.5 4.2 Results 4.5 4.2.1 Sugar consumption by Burkholderia sp. (Sb13) 4.5 4.2.2 Sugar consumption by Rhizobium sp. (Sb16) 4.7 4.2.3 Sugar consumption by Corynebacterium sp. (Sb 26) 4.8 4.2.4 Sugar consumption by Corynebacterium sp. (Sb35) 4.9 4.3 Discussion 4.11 4.4 Conclusion 4.13

xv

5 EFFECT OF ROOT EXUDATE SUGAR AND AMINO ACID OF DIFFERENT RICE GENOTYPES ON DIAZOTROPH COLONIZATION

5.1

5.0 Introduction 5.4 5.1 Materials and Methods 5.4 5.1.1 Seed surface sterilization 5.4 5.1.2 Preparation of inocula and rice seedlings inoculation in

vitro 5.5

5.1.3 In vitro growth of rice seedlings 5.5 5.1.4 Collection of root exudate 5.6 5.1.5 Determination of root exudate sugar concentration 5.7 5.1.6 Determination of root exudate amino acid concentration 5.7 5.1.7 Total sugar and amino acid production in root exudate 5.8 5.1.8 Sugar and amino acid utilization by the diazotroph from

root exudate 5.9

5.1.9 Determination of IAA production in the growth culture medium

5.9

5.1.10 Determination of non-rhizosphere population 5.9 5.1.11 Determination of rhizosphere population 5.9 5.1.12 Determination of root-endosphere population 5.10 5.1.13 Scanning and Transmission Electron Microscopic

observation 5.10

5.2 Results 5.11 5.2.1 Sugar and amino acid in the root exudate of non-

inoculated rice genotypes 5.11

5.2.2 Effect of inoculation on sugar production 5.12 5.2.3 Effect of inoculation on amino acid production 5.15 5.2.4 Production and utilization of sugars in root exudate 5.17 5.2.5 Production and utilization of amino acids in root exudate 5.23 5.2.6 Relationship between root exudate sugar and amino acid

utilization and diazotroph populations 5.25

5.2.7 Relationship between indoleacetic acid production and tryptophan concentration of the growth culture solution

5.31

5.2.8 Root colonization 5.33 5.3 Discussion 5.39 5.4 Conclusion 5.47 6 INFLUENCE OF SUGARS ON DIAZOTROPH ASSOCIATION AND

NITROGEN FIXATION IN RICE

6.0 Introduction 6.1 6.1

6.1.1 Materials and Methods

Growth chamber(in vitro) study 6.4

6.1.1.1 Seed surface sterilization 6.4

xvi

6.1.1.2 Preparation of sand and growing of rice seedlings 6.5 6.1.1.3 Preparation of inocula and inoculation of rice seedlings

in vitro condition 6.6

6.1.1.4 Application of sugar to the inoculated and non-inoculated rice genotypes

6.6

6.1.1.5 Application of nitrogen fertilizer to the non-inoculated rice genotypes

6.6

6.1.1.6 Determination of non rhizosphere population 6.6 6.1.1.7 Determination of rhizosphere population 6.7 6.1.1.8 Determination of root-endosphere population 6.7 6.1.1.9 Study of root morphology 6.7 6.1.1.10 Plant-nitrogen analyses 6.8 6.1.1.11 Determination of residual sugar 6.8 6.1.2 Glasshouse study 6.8 6.1.2.1 Soil preparation 6.8 6.1.2.2 Seedling preparation and transplanting 6.9 6.1.2.3 Preparation of inocula and inoculation of rice seedlings 6.10 6.1.2.4 Application of sugar to the inoculated and non-inoculated

rice genotypes 6.10

6.1.2.5 Application of nitrogen fertilizer to the non-inoculated rice genotypes

6.11

6.1.2.6 Determination of leaf chlorophyll (SPAD value) 6.11 6.1.2.7 Determination of photosynthesis rate 6.11 6.1.2.8 Determination of leaf area index 6.11 6.1.2.9 Plant-tissue nitrogen content analyses 6.12 6.1.2.10 15N isotope dilution analyses and estimation of N2

fixation 6.12

6.1.2.11 Determination of plant biomass 6.13 6.2 Results 6.14 6.2.1 Growth chamber(in vitro) study 6.14 6.2.1.1 Effect of sugar application on diazotroph population 6.14 6.2.1.2 Effect of sugars application on tissue-N content of rice

genotypes 6.15

6.2.1.3 Relationship between plant N-content and diazotroph population

6.15

6.2.1.4 Effect of sugar and nitrogen on root-morphology of rice genotypes

6.19

6.2.2 Glasshouse study 6.24 6.2.2.1 Plant-nitrogen concentration of rice genotypes 6.24 6.2.2.2 Estimation of biological nitrogen fixation and proportion

of nitrogen derived from atmosphere (% Ndfa) (15N isotope technique)

6.25

6.2.2.3 Total amount of nitrogen fixed 6.27 6.2.2.4 Leaf chlorophyll content (SPAD value) and 6.28

xvii

photosynthesis of rice genotypes 6.2.2.5 Nitrogen increment (%) in the plant 6.30 6.2.2.6 Plant height, leaf area index and biomass production of

rice genotypes 6.31

6.3 Discussion 6.36 6.4 Conclusion 6.42 7 General Discussion and Conclusion 7.1

REFERENCES R.1APPENDICES A.1BIODATA OF STUDENT B.1

xviii

LIST OF TABLES Table

Page

2.1 Recent and projected fertilizer use for agriculture in some countries 2.3

2.2 Estimated nitrogen fertilizer uses in rice production of different states in Malaysia

2.4

2.3 Diazotrophic strains able to fix N2 and increase crop production

2.10

2.4 Growth promotion by Burkholderia vietnamiensis in two rice cultivars (Ponni and ADT-43)

2.21

2.5 Grain yield and estimated amounts of fixed N2 by different N2 fixing systems

2.24

3.1 Chemical properties of different soils in Tanjong Karang rice irrigation project

3.10

3.2 Diazotroph population in soil, rhizosphere, root and shoot of different rice varieties grown in Tanjong Karang rice irrigation project

3.11

3.3 Nitrogen fixation of isolated diazotroph and some of the biochemical properties

3.13

3.4 Identification of diazotrophic bacteria isolated from Tanjong Karang rice irrigation project using Biolog Identification method

3.15

4.1 Cell generation time and percent sugar consumption of diazotroph in different carbon substrates

4.11

5.1 Sugar production in the root exudate of non-inoculated three rice genotypes grown in axenic condition

5.12

5.2 Total amino acid production in the root exudate of three rice genotypes

5.12

5.3 Sugar production and utilization (µmol g-1 root dry wt.) of inoculated and non-inoculated Mahsuri rice (Oryza sativa) root exudate during 5, 8, 14 and 20 days of growth period in an axenic condition

5.20

5.4 Sugar production and utilization (µmol g-1 root dry wt.) of inoculated and non-inoculated Mayang Segumpal rice (Oryza sativa) root exudate during 5, 8, 14 and 20 days of growth period in an axenic condition

5.21

xix

5.5 Sugar production and utilization (µmol g-1 root dry wt.) of inoculated and non-inoculated MR219 rice (Oryza sativa) root exudate during 5, 8, 14 and 20 days of growth period in an axenic condition

5.22

5.6 The correlation between root exudate sugar consumption and population growth of Rhizobium and Corynebacterium spp. in Mahsuri, Mayang and MR219 rice varieties

5.28

5.7 The correlation between root exudate amino acids utilization and population growth of Rhizobium and Corynebacterium spp. in Mahsuri, Mayang and MR219 rice varieties

5.29

5.8 Indoleacetic acid production in the growth culture solution of inoculated rice varieties

5.31

5.9 Correlation matrix of root exudate tryptophan concentration and indoleacetic acid production of three rice genotypes

5.32

6.1.0 Chemical properties of Bernam soil series

6.10

6.2.1.1 Diazotroph population at 45 days of inoculation and provided with different sugars in two rice genotypes under in vitro condition

6.17

6.2.1.2 The correlation matrix for diazotroph population and plant-N concentration of rice genotypes with sugar application

6.19

6.2.2.1 Effect of diazotroph inoculation and fertilized-N application on plant-N concentrations of two rice genotypes (60 days after transplanting) in glasshouse

6.24

6.2.2.2 Effect of diazotroph on % N atom excess (a.e.) in rice genotypes after 60 days of growth in 15 N labeled Bernam soil series

6.26

6.2.2.3 Effect of diazotroph inoculation and sugar application on total N2 fixed after 60 days of growth in 15N labeled Bernam soil series

6.28

6.2.2.4 Effect of diazotroph inoculation and fertilizer-N application on leaf chlorophyll (SPAD value), and leaf photosynthesis of two rice genotypes

6.29

6.2.2.5 Influence of diazotroph inoculation and sugar application on Plant-N (%) increased in Mayang Segumpal and MR219 rice genotypes at 60 days of growth

6.31

xx

6.2.2.6 Effect of diazotroph inoculation and sugar application on leaf area of two rice genotypes (60 days after transplanting) in glass house

6.33

6.2.2.7 Influence of diazotroph inoculation and sugar application on biomass yield increment (%) of two rice genotypes at 60 days in glasshouse

6.35

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LIST OF FIGURES

Figure

Page

2.0 Schematic representation of the possible source of nitrogen nutrient to the rice plant and the genotypic factors involved N acquisition efficiency traits (NAE); N fixation stimulation traits (NFS); N utilization efficiency traits (NUE)

2.5

3.1 Cellulose activity assayed after 24 hours incubation in Jensen-CMC plate

3.14

3.2 IAA production of the isolated diazotroph grown in Tanjong Karang rice irrigation project

3.14

3.3 The dendogram of cluster analyses of isolated diazotroph based on REP-PCR marker (Veasalovic et al., 1997)

3.16

4.1 Comparison growth of different strains in glucose, fructose, sucrose and arabinose substrate

4.6

4.2 Specific growth rate of Burkholderia sp. (Sb13) in different substrates

4.7

4.3 Specific growth rate of Rhizobium sp.(Sb16) in different substrates

4.8

4.4 Specific growth rate of Corynebacterium sp.(Sb26) in different substrates

4.9

4.5 Specific growth rate of Corynebacterium sp.(Sb35) in different substrates

4.10

5.1a Total sugar production by inoculated and non-inoculated Mahsuri rice variety

5.13

5.1b Total sugar production by inoculated and non-inoculated Mayang Segumpal rice variety

5.14

5.1.c Total sugar production by inoculated and non-inoculated MR219 rice variety

5.14

5.2a Total amino acids production by inoculated and non-inoculated Mahsuri rice

5.16

5.2b Total amino acids production by inoculated and non-inoculated Mayang Segumpal rice

5.16

xxii

5.2c Total amino acids production by inoculated and non-inoculated MR219 rice variety

5.17

5.3a Root exudate sugar production and utilization by Rhizobium and Corynebacterium spp. in Mahsuri rice during 18 days of inoculation period

5.18

5.3b Root exudate sugar production and utilization by Rhizobium and Corynebacterium spp. in Mayang Segumpal rice during 18 days of inoculation period

5.19

5.3c Root exudate sugar production and utilization by Rhizobium and Corynebacterium spp. in MR219 rice during 18 days of inoculation period

5.19

5.4a Root exudate amino acids production and utilization by Rhizobium and Corynebacterium spp. in Mahsuri rice during 18 days of growth

5.24

5.4b Root exudate amino acids production and utilization by Rhizobium and Corynebacterium spp. in Mayang Segumpal rice during 18 days of growth

5.24

5.4c Root exudate amino acids production and utilization by Rhizobium and Corynebacterium spp. in MR219 rice during 18 days of growth

5.25

5.5 Changes in growth of diazotroph population in the rice varieties inoculated with Rhizobium and Corynebacterium spp.

5.27

5.6 IAA production and tryptophan concentrations of the growth culture solution of inoculated (Sb16, Sb26) Mahsuri, Mayang Segumpal and MR219 rice

5.32

5.7 a-b Diazotroph colonization: a) Root hair zone. b) Junction of lateral root

5.34

5.7 c-d Diazotroph colonization: c) Junction of lateral root meristem, d) Crevices

5.35

5.7 e-f Diazotroph colonization: e) Inside crevices. f) Zone of elongation

5.36

5.7 g-h Diazotroph colonization: g) In between cell wall of root tip. h) Bacterial cell aggregation on the root hair zone covered by mucilage material

5.37

5.8 a-d Diazotroph colonization (Transverse section): a & b) In between cell wall. c & d) Inter cellular spaces and within epidermis

5.38

xxiii

5.8 e -h Diazotroph colonization (Transverse section): (e) In between cell wall. f) Inner cortex. g&h) near vascular bundles

5.39

6.1 Plant-N concentration of inoculated, non-inoculated, and N fertilized treatments of two rice genotypes with different sugar application

6.18

6.2 Root length of inoculated, non-inoculated, and N-fertilized treatments of two rice genotypes with different sugar application

6.21

6.3 Root surface area of inoculated, non-inoculated, and N-fertilized treatments of two rice genotypes with different sugar application

6.22

6.4 Root volume of inoculated, non-inoculated, and N-fertilized treatments of two rice genotypes with different sugar application

6.23

6.5 Percent nitrogen derived from atmosphere and other sources 6.27

6.6 Relationship between leaf photosynthesis and plant-N content of Mayang Segumpal and Mahsuri rice

6.30

6.7 Influence of inoculation and sugar application on root and shoot length of rice genotypes

6.32

6.8 Influence of inoculation and sugar application on biomass production of rice genotypes

6.34

xxiv