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UNIVERSITI PUTRA MALAYSIA INFLUENCE OF ZINC-SOLUBILIZING BACTERIA, ZINC SULFATE AND ORGANIC ACIDS ON GROWTH AND YIELD OF FLOODED RICE NUR MAIZATUL IDAYU BINTI OTHMAN FP 2018 31
UNIVERSITI PUTRA MALAYSIA
INFLUENCE OF ZINC-SOLUBILIZING BACTERIA, ZINC SULFATE AND ORGANIC ACIDS ON GROWTH AND YIELD OF FLOODED RICE
NUR MAIZATUL IDAYU BINTI OTHMAN
FP 2018 31
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INFLUENCE OF ZINC-SOLUBILIZING BACTERIA, ZINC SULFATE AND
ORGANIC ACIDS ON GROWTH AND YIELD OF FLOODED RICE
By
NUR MAIZATUL IDAYU BINTI OTHMAN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfillment of the Requirements for the Degree of Doctor of Philosophy
Dicember 2017
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COPYRIGHT
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photographs, and all other artwork, is copyright material of Universiti Putra Malaysia
unless otherwise stated. Use may be made of any material contained within the thesis
for non-commercial purposes from the copyright holder. Commercial use of material
may only be made with the express, prior, written permission of Universiti Putra
Malaysia.
Copyright © Universiti Putra Malaysia
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the degree of Doctor of Philosophy
INFLUENCE OF ZINC-SOLUBILIZING BACTERIA, ZINC SULFATE AND
ORGANIC ACIDS ON GROWTH AND YIELD OF FLOODED RICE
By
NUR MAIZATUL IDAYU BINTI OTHMAN
Dicember 2017
Chairman : Associate Professor Radziah Binti Othman, PhD
Faculty : Agriculture
Zinc (Zn) is an essential micronutrient required for plant growth. The unavailability
of zinc due to the insoluble form cause deficiency of Zn in rice plant. Zinc-
solubilization bacteria (ZSB) has been known to aid in Zn solubilization in soil making
zinc available for plants. A series of experiments were conducted in laboratory and
glasshouse conditions with the following objectives; i) to isolate and characterize ZSB
from rice roots and soil from Tanjung Karang Irrigation Area, Selangor, Malaysia ii)
to evaluate the effects of different Zn rates on root colonization of rice plant inoculated
with ZSB, iii) to determine the effects of ZSB inoculation, Zn sources and rates on
growth of rice plant, iv) to determine the influence of ZSB inoculation, types and rates
of organic acids and on Zn uptake and growth of rice plant applied with zinc sulfate
and v) to determine the influence of ZSB, zinc sulfate and malic acid on growth and
yield of rice. A total of 88 bacteria were isolated from rice soil and roots. Zinc-
solubilizing bacteria strains were evaluated for in vitro Zn solubilizing activity and
characterized for multiple plant growth-promoting properties. Colonization and
penetration of ZSB strains into rice roots were visualized using Scanning Electron
Microscope (SEM) and Transmission Electron Microscope (TEM). Growth chamber
study was conducted to determine the effects of ZSB, Zn sources and Zn rates on
growth of rice using zinc oxide and zinc sulfate. The effect of ZSB on growth of rice
in the presence of two organic acids (malic acid and citric acid) at four different rates
(0, 0.1, 1.0 and 10 mM) was evaluated on growth of rice. Finally, glasshouse studies
was conducted to determine the influence of ZSB strain Acinetobacter sp. (TM56),
zinc sulfate and malic acid on Zn distribution in rice plant roots, rice grain yield and
Zn fraction in zinc treated soils. Grain yield parameters were recorded after 120 days
of growth. Zinc fraction in soil was referred to Community Bureau of Reference
(BCR) sequential method. Results showed that nine of the isolates were able to
solubilize the insoluble Zn. The highest Zn solubilizing efficiency (611%) was
recorded by isolate TM67 in media supplemented with zinc carbonate. ZSB isolates
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were positive for nitrogen (N) fixation, phosphate solubilization, siderophore, indole-
3-acetic acid and hydrolyzing enzyme production. Two of the ZSB isolates (TM56
and TM23) were identified as Acinetobacter spp. and the other two (TM9 and BM13)
were identified as Serratia sp. Micrographs of SEM and TEM showed the presence of
ZSB strains in the intercellular spaces of rice root cells indicating their ability to
colonise root surface and endosphere. Endosphere population of ZSB was differed
with Zn rates in nutrient solution. Serratia sp. (TM9) showed highest endosphere
population (5.56 CFU mL-1) at 0.2 mg L-1 of Zn rates compared to control. Application
of ZSB, Zn sources and Zn rates increased plant growth. Plants inoculated with
Acinetobacter sp. (TM56) with zinc sulfate at 0.2 mg L-1 showed the highest plant
height (23.57 cm) compared to control. While, ZSB inoculation, organic acids and
rates of organic acids increased plant growth and Zn uptake. In ZSB inoculated
treatment with Acinetobacter sp. (TM56) and malic acid at 0.1 mM showed highest
(777.64 mg 3 plant-1) Zn uptake, (39.57) SPAD reading , (29.57 cm) plant height and
(135.67 mg) plant biomass were recorded compared to control. The EDX analysis
showed that application of ZSB, zinc sulfate and malic acid in combination deposited
highest Zn distribution (2.04 %) around exodermises and sclerenchyma of roots. The
combined treatments showed highest filled spikelet (87.7 %) and harvest index (0.52).
Zinc fractions in zinc treated soils was in the following order: residual > reducible >
oxidisable > exchangeable. The combined treatments of ZSB, zinc sulfate and malic
acid showed highest exchangeable (0.36 mg kg-1), oxidisable (0.26 mg kg-1) and
reducible (0.35 mg kg-1) of Zn fraction compared to control. This study proved that
application of ZSB, zinc sulfate and organic acids in combination was able to improve
growth and Zn uptake of rice plant.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
PENGARUH BAKTERIA PELARUT-ZINK, ZINK SULFAT DAN ASID
ORGANIK PADA PERTUMBUHAN DAN HASIL PADI SAWAH
Oleh
NUR MAIZATUL IDAYU BINTI OTHMAN
Disember 2017
Pengerusi : Profesor Madya Radziah Binti Othman, PhD
Fakulti : Pertanian
Zink (Zn) adalah mikronutrien penting yang diperlukan untuk pertumbuhan tanaman.
Ketidaktersediaan Zn disebabkan bentuk tidak larut menyebabkan kekurangan Zn
dalam pokok padi. Bakteria Pelarut-Zink (ZSB) dikenali dalam melarutkan Zn dalam
tanah menjadikan zinc tersedia untuk tumbuhan. Satu siri kajian dijalankan di makmal
dan rumah kaca dengan tujuan berikut: i) untuk mengasingkan dan mencirikan ZSB
dari akar padi dan tanah dari Kawasan Pengairan Tanjung Karang, Selangor, Malaysia
ii) untuk menilai kesan kadar Zn yang berlainan pada pengkolonian akar padi oleh
ZSB, iii) untuk menentukan kesan inokulasi ZSB dengan sumber dan kadar Zn yang
berbeza pada pertumbuhan padi, iv) untuk menentukan pengaruh inokulasi ZSB, jenis
dan kadar asid organik pada pengambilan Zn dan pertumbuhan padi menggunakan
baja zink sulfat, dan v) untuk menilai pengaruh inokulasi ZSB, zink sulfat dan asid
malik pada pertumbuhan dan hasil padi. Lapan puluh lapan bakteria telah diasingkan
dari tanah dan akar padi. Bakteria pelarut-zink berpotensi diuji dan dicirikan secara in
vitro untuk aktiviti solubilisasi Zn dan pelbagai ciri bermanfaat sebagai bakteria
penggalak-tumbesaran tumbuhan (PGPB). Pengkolonian dan penembusan isolat ZSB
ke dalam akar tanaman padi telah divisualisasikan menggunakan Mikroskop
Pengimbas Elektron (SEM) dan Mikroskop Transmisi Elektron (TEM). Kajian
pertumbuhan dalam kebuk dilakukan untuk menentukan kesan ZSB, sumber Zn dan
kadar Zn pada pertumbuhan tanaman padi menggunakan zink oksida dan zink sulfat.
Kesan ZSB dan zink sulfat dengan kehadiran dua asid organik komersil (asid malik
dan asid sitrik) pada empat kadar berbeza (0, 0.1, 1.0 dan 10 mM) diuji untuk
menentukan kesan pada pertumbuhan tumbuhan padi. Yang terkahir, kajian rumah
kaca dilakukan untuk menyiasat pengaruh inokulasi strain ZSB Acinetobacter sp.
(TM56), zink sulfat dan asid malik terhadap pengagihan Zn dalam akar padi, kajian
hasil padi dan penentuan pemecahan Zn dalam tanah yang dirawat. Parameter hasil
padi telah direkodkan selepas 120 hari tempoh pertumbuhan. Sementara itu,
penentuan pecahan Zn pada tanah yang dirawat telah dirujuk kepada kaedah ekstraksi
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berurutan Community Bureau of References (BCR) . Menurut keputusan kajian,
sembilan strain ZSB berupaya melarutkan Zn. Keupayaan melarutkan Zn yang
tertinggi (611 %) ialah isolat TM67 dalam media yang dimasukkan Zn karbonat.
Seluruh isolat ZSB juga menunjukkan ciri sebagai PGPB secara positif dalam
pengikatan nitrogen (N), pelarutan fosfat, pengeluaran IAA dan pengeluaran enzim
hidrolisis. Dua Isolat ZSB telah dikenal pasti sebagai Acinetobacter sp. (isolat TM56
dan TM23), manakala dua lagi adalah Serratia sp (isolat TM9 dan BM13). Mikrograf
dari SEM dan TEM menunjukkan kehadiran strain ZSB dalam ruang sel-sel akar
menunjukkan keupayaan menembusi akar tanaman padi. Populasi endosfera ZSB
berbeza dengan kadar Zn.padi dalan larutan nutrient. Serratia sp. (TM9) menunjukkan
populasi endosfera tertinggi (5.56 CFU mL-1) pada kadar 0.2 mg L-1 Zn berbanding
kawalan. Aplikasi ZSB, sumber Zn dan kadar Zn mempengaruhi pertumbuhan
tanaman. Tanaman diinokulasi Acinetobacter sp. (TM56), dengan zink sulfat pada 0.2
mg L-1 menunjukkan tinggi tanaman tertinggi (23.57 cm) berbanding kawalan.
Sementara itu, inokulasi ZSB menggunakan Acinetobacter sp. (TM56) dan asid malik
pada 0.1 mM menunjukkan pengambilan Zn (777.64 mg 3 tumbuhan-1), bacaan SPAD
(39.57), ketinggian tanaman (29.57 cm) dan jisim tanaman (135.67 mg) tertinggi
dicatatkan berbanding kawalan. Gabungan ZSB, zink sulfat dan asid malik dapat
mengedar Zn paling tinggi (2.04%) di sekitar eksodermis dan skelerenkima akar.
Gabungan ini juga menunjukkan peratusan tertinggi spikelet dipenuhi (87.7%) dan
indeks tuaian (0.52). Pecahan Zn untuk semua tanah yang dirawat adalah dalam
susunan berikut: sisa> terturun> teroksida> bertukar ganti. Gabungan rawatan ZSB,
zink sulfat dan malic acid menunjukkan pecahan boleh ditukar (0.36 mg kg -1),
teroksida (0.26 mg kg-1), diturunkan (0.35 mg kg-1) yang tertinggi. Kajian ini
membuktikan bahawa penggunaan gabungan ZSB, zink sulfat dan organik asid
berupaya untuk meningkatkan pertumbuhan dan pengambilan Zn tanaman padi.
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ACKNOWLEDGEMENTS
First and formost, I am thankful to Allah SWT for blessing me with wisdom, good
health, strength and support throughout my journey as a PhD student.
I would like to take this opportunity to express my deepest gratitude and sincere
appreciation to Associate Professor Dr. Radziah Binti Othman, the Chairman of
Supervisory Committee for her endless support, motivation and understandings,
invaluable suggestions, precious times, and generous help throughout this study period
and during my thesis write up. My sincere appreciation is also extended to my co-
supervisors, Associate Professor Dr. Halimi Bin Mohd Saud and Dr. Puteri Edaroyati
Binti Megat Wahab for their valuable advice, constructive suggestions and critical
reviews of the manuscript.
Special thanks to the laboratory assistants in the Department of Land Management,
Faculty of Agriculture especially Mr. Dzulkifli Duaji and Mr. Jamil Omar. I really
appreciate help from Mr. Sabri, from Biotechnology Laboratory for his help and guidance
on using HPLC and also Mrs Musliyana for the assistance on molecular identification at
the Institute of Bioscience I would like to thank the Ministry of Higher Education
Malaysia and also the Faculty of Plantation and Agrotechnology, Universiti Teknologi
Mara for giving me t opportunity to pursue my Ph.D degree.
I am greatly indebted to my mother, Hajjah Patimah Mamat, for your care and love.
Thank you for your willingness to look after Iman during my lab works. Thanks to
my father, Haji Othman Bin Sidek for his advices and encouragement. Special thanks
also to my aunt, Mrs. Zaidah Bt Sidek and my parents in law, Mrs. Ramlah Awang
and Mr. Mohd Noor Sidik. Not forgotten, my siblings, Mohd Ziarul Faizal,
Maisyatinil Halimatul and Mohd Ziarul Fadhli, thanks a lot for your support and care
towards me. Thank you too to my best friends, Siti Aishah Binti Mohd Sharip,
Rusyamimi Rosman, Nur Syamimi Abdul Rahman, Nur Hanani Hanis Mohd Nawar
and Mohd Fahmi Feesol for time and valuable friendship. Thanks to my lab mates
especially Nur Hidayah Hashim for her help and knowledge.
Finally, to the one who really cares, supports, and also my pillar of strength, my beloved
husband, Mr. Mohd Firdaus Bin Mohd Noor. Thanks a lot for giving me your time,
advices, patience and support throughout my lab works and thesis preparation. Thanks for
being by my side during hard times and never lose hope to see me success. I am really
grateful to Allah for giving us our son, Muhammad Iman Mohd Firdaus. Their sacrifices
in the course of my study will never be paid. Thank you so much.
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This thesis was submitted to the Senate of the Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The
members of the Supervisory Committee were as follows:
Radziah Othman, PhD
Associate Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Chairman)
Halimi Mohd Saud, PhD
Associate Profesor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
Puteri Edaroyati Megat Wahab, PhD
Senior Lecturer
Faculty of Agricultre
Universiti Putra Malaysia
(Member)
ROBIAH BINTI YUNUS, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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Declaration by graduate student
I hereby confirm that:
this thesis is my original work;
quotations, illustrations and citations have been duly referenced;
this thesis has not been submitted previously or concurrently for any other degree
at any institutions;
intellectual property from the thesis and copyright of thesis are fully-owned by
Universiti Putra Malaysia, as according to the Universiti Putra Malaysia
(Research) Rules 2012;
written permission must be obtained from supervisor and the office of Deputy
Vice-Chancellor (Research and innovation) before thesis is published (in the form
of written, printed or in electronic form) including books, journals, modules,
proceedings, popular writings, seminar papers, manuscripts, posters, reports,
lecture notes, learning modules or any other materials as stated in the Universiti
Putra Malaysia (Research) Rules 2012;
there is no plagiarism or data falsification/fabrication in the thesis, and scholarly
integrity is upheld as according to the Universiti Putra Malaysia (Graduate
Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia
(Research) Rules 2012. The thesis has undergone plagiarism detection software
Signature: Date:
Name and Matric No: Nur Maizatul Idayu binti Othman, GS41223
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Declaration by Members of Supervisory Committee
This is to confirm that:
the research conducted and the writing of this thesis was under our supervision;
supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate
Studies) Rules 2003 (Revision 2012-2013) were adhered to.
Signature:
Name of Chairman
of Supervisory
Committee:
Associate Professor Dr. Radziah Othman
Signature:
Name of Member
of Supervisory
Committee:
Associate Professor Dr. Halimi Mohd Saud
Signature:
Name of Member
of Supervisory
Committee:
Dr. Puteri Edaroyati Megat Wahab
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TABLE OF CONTENTS
Page
ABSTRACT i
ABSTRAK iii
ACKNOWLEDGEMENTS v
APPROVAL vi
DECLARATION viii
LIST OF TABLES xvi
LIST OF FIGURES xviii
LIST OF PLATES xxi
LIST OF ABBREVIATIONS xxii
CHAPTER
1 INTRODUCTION 1 1.1 Introduction 1 1.2 Problem Statement 1 1.3 Significance of Study 2 1.4 Objectives of Study 2
2 LITERATURE REVIEW 3 2.1 Rice and its production in Malaysia 3 2.2 Constraints of rice production in Malaysia 3
2.2.1 Zinc deficiency in rice production 4 2.2.2 Zn depletion in Malaysian rice 5
2.3 Zinc and its function in rice 6 2.4 Zinc in flooded system 6 2.5 Mechanism of zinc solubilization 7 2.6 Source of zinc fertilizers 10 2.7 Zinc solubilization by microorganism 11
2.7.1 Zinc solubilization by chelating agent 12 2.7.2 Fungal zinc solubilization 12
2.8 Factor affecting survival and activity by zinc-solubilizing
bacteria 12 2.9 Low molecular weight organic acid (LMWOA) complexes with
zinc 12 2.10 Zinc distribution using energy dispersive x-ray (EDX) technique
13 2.11 Physiological mechanism of zinc uptake by rice plants 13
2.11.1 Xylem and phloem transport of Zinc 15
2.12 Summary 16
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3 MATERIALS AND GENERAL METHODOLOGY 17 3.1 Enumeration and isolation of indigenous zinc-solubilizing
bacteria strains from soils and rice roots 17 3.2 Preparation of inoculum and rice seedlings inoculation 17 3.3 Seed surface sterilization 17 3.4 Preparation of sand culture and growing rice seedlings 18 3.5 Production of organic acids by zinc-solubilizing bacteria isolates
18 3.6 Determination of leaf area index (LAI) 18 3.7 Determination of relative leaf chlorophyll content 19 3.8 Zinc tissue analysis 19 3.9 Determination of plant biomass 19
3.10 Determination of root morphology 19 3.11 Determination of pH 19
4 CHARACTERIZATION OF ZINC-SOLUBILIZING BACTERIA
ISOLATED FROM RICE ROOTS 20 4.1 Introduction 20 4.2 Materials and methods 21
4.2.1 Sampling of soil and rice roots 21 4.2.2 Screening of Zinc-solubilizing activity by bacteria
isolates 22 4.2.2.1 Isolation of bacteria from rice roots and soil 22
4.2.2.2 Zinc-solubilizing activity using plate assay 22 4.2.3 Determination of nitrogen fixation activity 22 4.2.4 Determination of siderophore production 22 4.2.5 Determination of indole -3-acetic acid production 22 4.2.6 Determination of phosphate solubilisation ability 23
4.2.7 Hydrolysing enzyme production 23 4.2.8 Zinc solubilisation in liquid medium 23
4.2.9 Determination of zinc-solubilizing bacteria population 23 4.2.10 Specific growth rates 24
4.2.11 Bacterial identification using 16S rDNA polymerase
chain reaction (PCR) 24
4.2.12 Data analysis 25
4.3 Results 26
4.3.1 Isolation and characterization of zinc-solubilizing
bacteria 26 4.3.2 Zinc-solubilizing activities on media plates 26 4.3.3 Biochemical properties of indigenous isolated ZSB
strains 27
4.3.4 Zinc solubilizing in liquid medium assay 28 4.3.5 Population of zinc-solubilizing in liquid media
supplemented with insoluble Zn 29
4.3.6 Changes of pH in different insoluble Zn media 31 4.3.7 Kinetics of Zn Solubilisation of ZSB in liquid media
supplemented with insoluble zinc 33
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4.3.8 Bacterial identification 35 4.4 Discussion 36 4.5 Conclusion 37
5 EFFECT OF DIFFERENT ZINC RATES ON ROOT
COLONIZATION OF RICE PLANT INOCULATED WITH
ZINC SOLUBILIZING BACTERIA 38 5.1 Introduction 38 5.2 Materials and methods 39
5.2.1 Plant pathogenicity test 39 5.2.2 Seed surface sterilization 39
5.2.3 Preparation of inoculum and rice seedlings inoculation in
vitro 39 5.2.4 Experimental design 40 5.2.5 In vitro growth of rice seedlings 40 5.2.6 Zinc-solubilizing bacterial population during growth of
rice plants 40 5.2.7 Visual observation of root colonization by zinc
solubilizing bacteria using SEM and TEM 41 5.2.8 Data analysis 41
5.3 Results 41 5.3.1 Plant pathogenicity 41 5.3.2 ZSB population at different rates of zinc fertilizer during
rice growth period 42 5.3.3 Visual observation of root colonization by zinc-
solubilizing bacteria 43 5.4 Discussion 46 5.5 Conclusion 47
6 EFFECTS OF ZINC-SOLUBILIZING BACTERIA, ZINC
SOURCES AND RATES OF ZINC ON GROWTH OF RICE 48
6.1 Introduction 48 6.2 Materials and methods 49
6.2.1 Production of organic acids by zinc-solubilizing bacterial
isolates 49
6.2.2 Experimental design for soil culture in growth chamber 49 6.2.3 Preparation of sand culture and growing of rice seedlings
49 6.2.4 Preparation of inoculums and inoculations of rice
seedlings in vitro condition 49
6.2.5 Seed Surface Sterilization 50 6.2.6 Determination of organic acids in growth medium 50 6.2.7 Determination of organic acids from rice roots 50
6.2.8 Root Morphology measurement 50 6.2.9 Zinc tissue analysis 50 6.2.10 Data analysis 50
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6.3 Results 51 6.3.1 Production of organic acids in rice plant root 51 6.3.2 Production of organic acids in growth medium 53 6.3.3 Effects of ZSB inoculation, Zn sources and Zn rates on
zinc concentration in rice 56 6.3.4 Effect of ZSB inoculation, Zn sources, and Zn rates on
Zn uptake in rice 59 6.3.5 Effect of ZSB inoculation, Zn sources, and Zn rates on
plant biomass 61 6.3.6 Effect of ZSB inoculation, Zn sources, and Zn rates in
rice plant height 63 6.3.7 Effects of ZSB inoculation, Zn sources and Zn rates on
root development of rice 65 6.3.8 Correlation between various organic acids production
with plant biomass and Zn uptake 70 6.4 Discussion 71 6.5 Conclusion 72
7 EFFECT OF ZINC-SOLUBILIZING BACTERIA AND
ORGANIC ACIDS ON ZINC UPTAKE AND GROWTH OF
RICE APPLIED WITH ZINC SULFATE 73 7.1 Introduction 73 7.2 Materials and methods 74
7.2.1 Solubilization of zinc source by different organic acids 74 7.2.2 Growth chamber study of the effects of ZSB inoculation,
organic acids types and rates on rice plant 74 7.2.2.1 Experimental design for sand culture under
controlled condition 74 7.2.2.2 Seed surface sterilization 75 7.2.2.3 Preparation of sand culture 75 7.2.2.4 Preparation of bacterial inoculums and
inoculation of rice seedlings 75 7.2.3 Determination of plant growth parameters 75 7.2.4 Zinc tissue analysis 75 7.2.5 Bacterial population of inoculated treatments 75
7.2.6 Determination of pH of sand culture 75 7.2.7 Data analysis 76
7.3 Results 76
7.3.1 Zn solubilisation by different types of organic acids 76 7.3.2 Effect of ZSB inoculation, types of organic acids and its
rates on growth of rice 77 7.3.3 Effect of ZSB inoculation, organic acid types and rates of
organic acids on relative chlorophyll contents in rice
plant leaves 80 7.3.4 Effects of ZSB inoculation, organic acid types and rates
of organic acids on Zn concentration in rice 82
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7.3.5 Effect of ZSB inoculation, organic acid types and rates of
organic acids on Zn uptake in rice plant 83 7.3.6 Effect of ZSB, organic acid types and rates of organic
acid to root development of rice plant 85 7.3.7 Effects of organic acid types and rates on bacterial
population of inoculated treatment 87 7.3.8 Effects of ZSB inoculation, organic acid types and rates
on pH of sand culture 88 7.4 Discussion 89 7.5 Conclusion 90
8 INFLUENCE OF ZINC-SOLUBILIZING BACTERIA, ZINC
SULFATE AND MALIC ACID ON GROWTH AND YIELD OF
RICE 91 8.1 Introduction 91 8.2 Materials and methods 92
8.2.1 Effect of ZSB inoculation, zinc sulfate and organic acids
on Zn distribution and Zn uptake in rice plant 92 8.2.1.1 Sand culture preparation 92 8.2.1.2 Preparation of inoculum and inoculation of rice
seedlings 92 8.2.1.3 Plant growth parameters and root morphology 92 8.2.1.4 Energy dispersive X-Ray (EDX) analysis of
zinc distribution in roots 93 8.2.1.5 Root and shoot nutrient analysis 93
8.2.2 Effects of ZSB, zinc sulfate and malic acid on grain yield
of rice 93 8.2.2.1 Pot experimental design 93 8.2.2.2 Soil preparation 94 8.2.2.3 Seedlings Preparation and Planting 94 8.2.2.4 Determination of Zn analysis and other
macronutrients 94 8.2.3 Effects of ZSB inoculation, zinc sulfate and malic acid on
fractionation and soil enzyme 95 8.2.3.1 Zinc fraction in rice soil 95
8.2.3.2 Dehydrogenase and protease activities in soil 95 8.2.4 Data analysis 96
8.3 Results 96
8.3.1 SEM/EDX micrograph 96 8.3.2 Effects of ZSB inoculation, zinc sulfate and malic acid on
rice plant biomass 96
8.3.3 Zinc concentration and Zinc uptake in rice 97 8.3.4 Plant height, chlorophyll content, number of tillers, and
LAI 98 8.3.5 Bacteria and ZSB population 99
8.3.6 Root development 100 8.3.7 Nutrients Concentration in Root and Shoot of Rice 102
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8.3.8 Grain yield parameters 102 8.3.9 Nutrient concentration in straw and grain 104 8.3.10 Dehydrogenase and protease enzyme activities in rice
soil 107 8.4 Discussion 108 8.5 Conclusion 110
9 GENERAL DISCUSSION, CONCLUSIONS AND
RECOMMENDATIONS FOR FUTURE RESEARCH 111
REFERENCES 114 APPENDICES 130 BIODATA OF STUDENT 147 LIST OF PUBLICATIONS 148
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LIST OF TABLES
Table Page
1 Zinc status (mg kg-1) of 15 soil series at different depths 5
2 Different Zn source based on their formula, percentage of Zn, water
solubility, soil type and cost
11
3 Chemical properties of sampling area in Tg Karang Rice Irrigation
Project
21
4 PCR Cycling Step 25
5 ZSB population (CFU g-1) in rhizosphere and endosphere of rice from
different location and soil series
26
6 Biochemical properties of zinc-solubilizing bacteria 28
7 Zn solubilized in medium broth supplemented with different
insoluble Zn after 48 h of incubation
29
8 Plant pathogenicity test of ZSB on rice seedlings 41
9 Bacterial population from endosphere population of inoculated
MR219 rice plant at different rates of Zn
42
10 Effects of ZSB inoculation, Zn sources and Zn rates on the Zn
concentration of rice plant
57
11 Effects of ZSB inoculation, Zn sources, and Zn rates on Zn uptake of
rice plant
59
12 Effects of ZSB inoculation, Zn sources, and Zn rates on the biomass
production of rice
61
13 Effects of ZSB, Zn sources, and rates of Zn on plant height in rice 63
14 Effects of zinc source, zinc rate, and ZSB and their interactions with
different root development of rice plant
66
15 Correlation between various organic acids production in nutrient
solution with plant biomass and Zn uptake
70
16 Correlation between various organic acids production in rice plant
root with plant biomass and Zn uptake
70
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17 Effects of ZSB inoculation, organic acids and its rates on plant
biomass
78
18 Effects of ZSB inoculation, organic acids and rates of organic acids
on rice plant height
79
19 Effects of ZSB inoculation, organic acids and rates of organic acids
on SPAD reading in rice plant.
81
20 Effects of ZSB inoculation, organic acids and organic acids rate on 82
21 Effects of ZSB inoculation, organic acids and organic acid rates on
Zn uptake of rice
84
22 Effects of ZSB, organic acid types and rates of organic acids and their
interactions on root development of rice plant
86
23 Effects of organic acid types and rates on bacterial population of
inoculated treatment
88
24 Effects of organic acid types and rates on pH of sand culture 89
25 Chemical properties of Kangkong series soil, from Kedah 94
26 Distribution of Zn and its increment over control in different
treatments
96
27 Effect of ZSB, malic acid, and zinc sulfate on Zn concentration and
Zn uptake in root and shoot of rice
98
28 Effect of ZSB, zinc sulfate and malic acid on populations of total
bacteria and ZSB.
100
29 Effect of ZSB,zinc sulfate, and malic acid on nutrients concentrations
in root and shoot tissue of rice plant
102
30 Effects of ZSB, zinc sulfate and malic acid on grain yield parameters
after rice harvest
103
31 Effect of ZSB, zinc sulfate and malic acid on nutrients in straw and
grain
104
32 Zinc fractions in rice soil at 120 days 106
33 Correlation between different Zn fractions with total Zn and Zn
uptake in straw and grain of rice
107
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LIST OF FIGURES
Figure Page
1 Causes of zinc deficiency in crops 4
2 Effects of root exudates components on nutrient availability by
plants and rhizosphere members
9
3 Major soil physical and chemical properties affecting Zn availability
to roots
10
4 Factors affecting Zn accumulation in plants 14
5 Soil processes affecting Zn bioavailability in soil solution 15
6 Zinc solubilisation strength of nine isolates on media agar consisted
with zinc carbonate, zinc oxide and zinc phosphate
27
7 Bacterial population in liquid media supplemented with zinc oxide
(a), zinc carbonate (b) and zinc phosphate (c) during 48 h incubation
period
30
8 Effects of bacterial strains on culture pH in media supplemented with
zinc oxide (a), zinc carbonate (b) and zinc phosphate (c)
32
9 Kinetics of Zn Solubilisation of isolate; a) BM13 b) TM23 c) TM56
d) TM9 in liquid media supplemented with zinc carbonate
33
10 Kinetics of Zn Solubilisation of isolate: a) BM13 b) TM23 c) TM56
d) TM9 in liquid media supplemented with zinc oxide
34
11 Kinetics of Zn Solubilisation of isolate: a) BM13 b) TM23 c) TM56
d) TM9 in liquid media supplemented with zinc phosphate
35
12 Interactions between inoculation type and zinc rate on bacterial
population in b) endosphere and a) rhizosphere of inoculated MR219
rice plant at different rates of Zn
43
13 Production of organic acids in roots of rice inoculated with ZSB at
different rates of zinc oxide
52
14 Production of organic acids in roots of rice inoculated with ZSB at
different rates of zinc sulfate
53
15 Production of organic acids in growth medium of rice inoculated
with ZSB at different rates of zinc oxide
55
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16 Production of organic acids in growth medium of rice inoculated
with ZSB at different rates of zinc sulfate
56
17 Effect of ZSB, Zn sources, and rates of Zn on Zn concentration of
rice plant; a) non-inoculated b) with Acinetobacter sp. (TM56) and
c) with Serratia sp. (TM9)
58
18 Effect of ZSB, Zn sources, and rates of Zn on Zn uptake of rice plant;
a) non-inoculated b) with Acinetobacter sp. (TM56) and c) with
Serratia sp. (TM9)
60
19 Effect of ZSB, Zn sources, and rates of Zn on plant biomass of rice
plant; a) non-inoculated b) with Acinetobacter sp. (TM56) and c)
with Serratia sp. (TM9)
62
20 Effect of ZSB, Zn sources, and rates of Zn on plant height of rice
plant; a) non-inoculated b) with Acinetobacter sp. (TM56) and c)
with Serratia sp. (TM9)
64
21 Effect of ZSB, Zn sources, and rates of Zn on root volume of rice
plant a) non-inoculated b) with Acinetobacter sp. (TM56) and c) with
Serratia sp. (TM9)
67
22 Effect of ZSB, Zn sources, and rates of Zn on root length of rice
plant; a) non-inoculated b) with Acinetobacter sp. (TM56) and c)
with Serratia sp. (TM9)
68
23 Effect of ZSB, Zn sources, and rates of Zn on root area of rice plant;
a) non-inoculated b) with Acinetobacter sp. (TM56) and c) with
Serratia sp. (TM9)
69
24 Zn solubilisation using organic acids in liquid media. a) malic acid
b) citric acid
77
25 Effects of ZSB Inoculation, organic acid types and its rates on plant
biomass of rice plant: a) inoculated and b) non-inoculated
78
26 Effects of ZSB Inoculation, organic acid types and its rates on plant
height of rice plant: a) inoculated and b) non-inoculated
80
27 Effects of ZSB Inoculation, organic acid types and rates on relative
chlorophyll content reading of rice plant: a) inoculated and b) non-
inoculated
81
28 Effects of ZSB Inoculation, organic acid types and its rates on Zn
concentration of rice plant: a) inoculated and b) non-inoculated
83
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29 Effects of ZSB Inoculation, organic acid types and rates on Zn uptake
of rice plant: a) inoculated and b) non-inoculated
84
30 Effects of ZSB inoculation, organic acid types and its rates on root
length of rice plant: a) inoculated and b) non-inoculated
87
31 Effects of ZSB inoculation, organic acids and its rates on root volume
of rice plant: a) inoculated and b) non-inoculated
87
32 Effect of ZSB, zinc sulfate and malic acid on plant biomass, root
biomass, and shoot biomass
97
33 Effect of ZSB, zinc sulfate, and malic acid on a) plant height, b)
chlorophyll content, c) number of tillers and d) leaf area index (LAI)
in rice
99
34 Effect of ZSB, zinc sulfate, and malic acid on a) root area, b) root
volume and c) root tips in rice
101
35 Effect of ZSB, malic acid, and zinc sulfate on a) Zn concentration in
grain and straw and b) Zn uptake in grain and plant tissue of rice at
120 days of plant growth
105
36 Effects of ZSB, zinc sulfate, and malic acid on the percentage of Zn
fraction in rice soil after 120 days of plant growth
106
37 Effect of ZSB, zinc sulfate and malic acid on dehydrogenase activity
and protease activity in soils at 120 of plant growth.
108
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LIST OF PLATES
Plate Page
1 Pseudomonas aeruginosa that can solubilize zinc oxide and zinc
phosphate
8
2 Zinc solubilisation by bacteria isolates as indicated by halazone
formation mineral salts agar medium
27
3 SEM micrograph shows the location of ZSB colonization inoculated
on MR219 rice plant roots
44
4 TEM micrographs shows the ZSB colonization inoculated on
MR219 rice roots of inner spaces
45
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LIST OF ABBREVIATIONS
ANOVA Analysis of Variance
CAS Chrome Azurol Sulphonate
CFU Colony Forming Unit
CRD Completele Randomized Design
DAN National Agrofood Policy
DW Dry Weight
EDTA Ethylenediaminetetraacetic Acid
EDX Energy Dispersive X-Ray
HDTMA Hexadecyltrimethylammonium Bromide
HPLC High Performance Liquid Chromatography
IAA Indole-3-Acetic Acid
IRRI International Rice Research Institute
L-try L-tryptophan
LMWOA Low Molecular Weight Organic Acid
MA Malic Acid
NBRIP National Botanical Research Institute’s phosphate growth
medium
NA Nutrient Agar
NB Nutrient Broth
OA Organic Acids
OD Optical Density
PBS Phosphate Buffer Saline
PGPB Plant Growth-Promoting Bacteria
RCBD Randomized Complete Block Design
SAS Statistical Analysis Software
SEM Scanning Electron Microscopy
Sp. Species (singular)
TEM Transmission Electron Microscopy
SPAD Soil Plant Analysis Division: Chlorophyll Content
ZSB Zinc-Solubilizing Bacteria
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CHAPTER 1
1 INTRODUCTION
1.1 Introduction
In Malaysia, widespread zinc (Zn) deficiency has been found responsible for yield
reduction in rice due to its unavailability to plants (Hafeez et al., 2013). Zinc is an
essential micronutrient for microorganisms and plants. The role of Zn in the nutrition
and physiology of both eukaryotic and prokaryotic organisms, especially its
importance for activity of many enzymes is widely studied. Exogenous application of
soluble Zn sources, similar to fertilizer application, has been advocated to various
crops to overcome Zn. Apart from that, phytosiderophores and organic acids are two
classes of Zn chelators secreted from roots that have been linked to the release of Zn
from soil-bound forms and its subsequent uptake by plants (Gao et al., 2011). Zn made
unavailable can be reverted back to available form by inoculating bacterial strains
capable of solubilizing it (Saravanan et al, 2003). Rhizosphere acidification from
activities of bacteria and fungi can enhance the utilization of not only P but also Zn.
The excretion of organic acids or phytosiderophores capable of increasing the
bioavailability of Zn in the rhizosphere is a potential mechanism of reducing Zn
deficiency in rice. In general bacteria can contribute to metal immobilization by
several processes such as precipitation and adsorption (Bapiri et al., 2012). As for
example phosphate solubilizing bacteria also can produce organic acids which
transform immobile phosphorus into available form for plant growth (Sundra et al.,
2002). In addition to these traits, the plant growth promoting bacteria (PGPB) must be
very competitive, able to survive and colonize the rhizosphere system (Cattelan et al.,
1999). According to Ahmad et al. (2008), the interactions between beneficial bacteria
and plants can be affected by various environmental factors that will influence to
them and their effects on plants. For good interaction between bacteria and plants it
is necessary to understand how the rhizobacteria exerts their effects on plant and the
effects of the inoculation on the uptake of nutrient including Zn.
1.2 Problem Statement
In recent years, Zn deficiency problem in important crops like rice has received
increasing attention. Because the concentration of Zn in most crops is inherently very
low, growing crops on potentially Zn-deficient soils can further decrease grain Zn
concentration. Applications of zinc sulfate in the form of fertilizers could ameliorate
Zn deficiency and improve plant yields. However, the applied Zn gets transformed
into insoluble forms depending on the soil types and totally become unavailable
within 7 days of application (Saravanan et al., 2007). The major reason for the
widespread occurrence of Zn deficiency problem in plants is the low solubility of Zn
in soils rather than the low total amount (Cakmak, 2007). In Malaysia, the application
of solely NPK fertilizers without Zn have caused continuous removal of Zn after
harvesting, as well as losses due to leaching or surface runoffs (Liew et al., 2010).
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Flooding and submergence also bring about a decline in available Zn due to pH
changes and the formation of insoluble Zn compounds. Zn deficiency in rice
production is widespread globally and studies of Zn deficiency in rice production in
Malaysia is limited (Hafeez et al., 2016).
1.3 Significance of Study
Zinc is an essential plant micro-nutrient for several biochemical processes in the rice
plant, including chlorophyll production and cell membrane integrity (Goteti et al.,
2013). Rice yield reduction occurs due to disturbance in biochemical processes for
enzyme activities and chlorophyll production. As an alternative to Zn fertilizer
application , Zn-solubilizing microorganisms can be used to solubilize Zn from
inorganic and organic pools of total soil Zn for increased Zn availability to plants
(Sushil et al., 2012). An ecologically sound, efficient, and cost-effective alternative
is important to improve Zn availability for enhance yield and income to rice farmers
in Malaysia. It is possible that exploitation of Zn solubilizing bacteria may aid in
overcoming Zn deficiency in flooded rice (Liew et al., 2010). Application of beneficial
bacteria can be an effective method for enhancing the growth of plant and maintaining
nutrient quality for a sustainable rice production.
1.4 Objectives of Study
The following studies were conducted with the objectives;
1) To isolate and characterize zinc-solubilizing bacteria (ZSB) from rice roots and soil
in Tanjung Karang, Selangor, Malaysia.
2) To evaluate the effects of different zinc rates on root colonization of rice plant
inoculated with ZSB.
3) To determine the effects of ZSB inoculation, Zn sources and rates on growth of rice
plant.
4) To determine the influence of ZSB, types and rates of organic acids on zinc uptake
and growth of rice plant applied with zinc sulfate.
5) To determine the influence of ZSB, zinc sulfate and malic acid on growth and yield
of rice.
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13 LIST OF PUBLICATIONS
Journal papers:
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri
Edaroyati Megat Wahab 2016. Effects of root colonization by zinc-
solubilizing bacteria on growth of rice plant (Oryza sativa MR219). Agr. Nat.
Resour .50(x): xx– xx. (Manuscripts in Revision for Acceptance)
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri
Edaroyati Megat Wahab 2017. Efficiency of zinc-solubilizng bacteria for in
vitro zinc solubilization and its effects on IAA rice production. Bangladesh J.
Bot. 46(1): 511-516. (Published)
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri
Edaroyati Megat Wahab 2017. Inoculation of zinc-solubilizing bacteria with
different zinc sources and rates for improved growth and zinc uptake in rice.
Accepted. Int. J. Agric. Biol., 00: 000-000. (Article in press)
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri
Edaroyati Megat Wahab 2017. Influence of Zn-solubilizing bacteria , Zn
sources and Malic acid on growth and yield in rice. (Manuscript in preparation
for submission)
Conference/Congress:
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri
Edaroyati Megat Wahab 2015. Evaluation of zinc solubilization potential by
bacteria associated with rice plants. In: International Conference on Crop
Improvement, 29- 30 December 2015. Faculty of Engineering, Universiti Putra
Malaysia (Poster Presentation)
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri Edaroyati
Megat Wahab 2016. Efficiency of bacteria isolated from lowland rice soil for In
Vitro zinc solubilization. In: International Conference on Sustainable Soil
Management. Tabung Haji Hotel , Kuala Terengganu. (Best Poster Award)
Nur Maizatul Idayu Othman, Radziah Othman, Halimi Mohd Saud and Puteri
Edaroyati Megat Wahab 2016. Influence of Zinc-Solubilizing Bacteria (ZSB),
zinc fertilizer and different rates of zinc on lowland rice growth. International
Agriculture congress 2016.4-6 Oktober 2016. Hotel Bangi-Putrajaya,
Selangor. (Oral presentation)
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