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UNIVERSITI PUTRA MALAYSIA

RICE STRAW COMPOSTING BY LIGNOCELLULYTIC MICROORGANISMS FOR IMPROVED STABILITY AND

BIOEFFICACY IN SCLEROTIAL DISEASE SUPPRESSION

MD. KAUSAR HOSSAIN

ITA 2011 2

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RICE STRAW COMPOSTING BY LIGNOCELLULYTIC MICROORGANISMS FOR IMPROVED STABILITY AND BIOEFFICACY IN SCLEROTIAL

DISEASE SUPPRESSION

By

MD. KAUSAR HOSSAIN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in fulfilment of the Requirement for the Degree of Doctor of Philosophy

July 2011

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Dedicated

to

Departed Parents

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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

RICE STRAW COMPOSTING BY LIGNOCELLULYTIC MICROORGANISMS FOR IMPROVED STABILITY AND BIOEFFICACY IN SCLEROTIAL

DISEASE SUPPRESSION

By

MD. KAUSAR HOSSAIN

July 2011

Chairman: Professor Sariah Meon, PhD

Institute: Institute of Tropical Agriculture

Rice (Oryza sativa L.) is an important cereal crop in the world. Annually, a huge

amount of rice straw (RST) is produced as by-product of rice cultivation. Proper

disposal of RST is a concern across the world. Composting is an alternative for

recycling of RST into a useful product. Composting of RST however is time consuming

as it comprises of lignocellulosic material. Therefore, in the present study, the ability of

a lignocellulolytic microbial consortium (Aspergillus niger and Trichoderma viride) at

different pH conditions as activator and enhancer of maturity and stability of RST

compost was evaluated, and its bioefficacy against sclerotial foot rot of chilli was

determined. A total of 125 microbes were isolated from different in-situ and in-vitro

RST compost sources. Twenty lignocellulolytic microbial isolates (5-bacteria, 5-

actinobacteria and 10-fungi) were selected based on enzymatic degradation of starch,

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cellulose and lignin, followed by screening for adaptability on rice straw powder (RSP)-

amended media. Six isolates (B37, A7, F26, F28, F29 and F44) were selected as

lignocellulolytic agents for in-vitro RST biodegradation based on their optimum growth

rate, biomass production and lignocellulolytic activities on RSP-amended media. Fungal

isolates were found to be more efficient than bacteria and actinobacteria in terms of

decomposing cellulose, hemicellulose, and lignin, and total carbon during RST

biodegradation. Four fungal isolates (F26, F28, F29 and F44) were evaluated for their

in-vitro compatibility. Six different interactions were found between the four interacting

fungal isolates in the form of mutual intermingling, partial mutual intermingling and

inhibition at the contact point. Finally, a consortium of A. niger (F44) and T. viride

(F26) was selected as the best potential lignocellulolytic microbial consortium for rapid

composting of RST. Compost stability and maturity were determined by monitoring the

physical, biochemical and biological changes during composting. The RST compost

produced was termed as microbial infused RST compost was also tested for its

bioefficacy in terms of seed germination and seedling establishment, plant growth and

disease suppression in chilli under plant house condition. Composting of RST amended

with lignocellulolytic microbial consortium (A. niger and T. viride) under natural (pH

6.75) pH condition showed significant changes in terms of physical, biochemical and

biological parameters compared to acidic (pH 5.75) and alkaline (pH 7.75) pH

conditions. After day 21, microbial consortium under natural pH condition was found to

reduce C/N ratio to 17.5 from an initial value of 29.2 and increased germination index to

75.5. The remaining lignin and cellulose contents in microbial infused RST compost

were 9.8 and 12.4%, respectively. Enzymatic activities namely, �-1,4-endo-glucanase,

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�-1,4-exo-glucanase and total dehydrogenase activity were significantly lower than non-

inoculated treatments. The contents of N, P, K, Ca and Mg were 2.3, 1.1, 2.6, 2.5 and

1.4%, respectively. These results suggest that, after day 21, microbial infused RST

compost produced under natural pH could be used as a substitute for inorganic

fertilizers. Under plant house condition, it was found that application of microbial

infused RST compost significantly increased seed germination, plant growth and

suppressed the development of foot rot caused by S. rolfsii in chilli compared to

commercial compost (BFC, CMT Agro Resources Sdn Bhd) or the use of fungicide

Benomyl. Application at a rate of 15 t/ha was optimum for seed germination and

seedling establishment, plant growth and disease suppression suggesting that microbial

infused RST compost could be used as an alternative to chemical fungicide Benomyl for

the control of sclerotial disease in chilli.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PENGKOMPOSAN JERAMI PADI MMENGGUNAKAN MIKROB UNTUK PENAMBAHBAIKAN KESTABILAN DAN EFIKASI BIOLOGI

Oleh

MD. KAUSAR HOSSAIN

Julai 2011

Pengerusi: Profesor Sariah Meon, PhD

Institut: Institut Pertanian Tropika

Padi (Sativa Oryza L.) merupakan tanaman bijirin penting di dunia. Setiap tahun,

sejumlah besar jerami padi (RST) dihasilkan sebagai hasil sampingan penanaman padi.

Cara pelupusan yang betul bagi RST telah menjadi satu kebimbangan merentasi dunia.

Pengkomposan merupakan satu alternatif untuk kitar semula RST menjadi produk yang

bermanfaat. Pengkomposan RST bagaimanapun memakan masa yamg lama kerana

ianya mengandungi bahan lignosellulosa. Oleh itu, kajian ini telah menilai kemampuan

konsortium mikrob lignoselulolitik (Aspergillus niger dan Trichoderma viride) pada pH

yang berbeza sebagai pemangkin dan penambah kematangan dan kestabilan kompos

RST, dan efikasi biologinya terhadap jangkitan Sclerotium pangkal cili telah ditentukan.

Sebanyak 125 mikrob telah diasingkan dari berbagai sumber kompos RST in-situ and

in-vitro. Dua puluh isolat mikrob lignoselulolitik berpotensi (5 bakteria, 5-

aktinobakteria dan 10 kulat) dipilih berdasarkan pendegradan enzim kanji, selulosa dan

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lignin, diikuti oleh penyaringan pada media ubahsuai serbuk jerami padi (RSP). Enam

isolat (B37, A7, F26, F28, F29 dan F44) dipilih sebagai agen lignoselulolitik dalam

biodegradasi RST in vitro berdasarkan kadar pertumbuhan optimum, pengeluaran

biojisim dan aktiviti lignoselulolitik pada media ubahsuai serbuk jerami padi. Isolat

kulat didapati lebih cekap daripada bakteria dan aktinobakteria dari aspek penguraian

selulosa, hemiselulosa, dan lignin, dan jumlah karbon semasa biodegradasi jerami padi.

Empat isolat kulat (F26, F28, F29 and F44) telah dinilai untuk keserasian in-vitro. Enam

interaksi berbeza telah ditemui antara empat interaksi isolat kulat dalam bentuk

penggabungjalinan saling, penggabungjalinan saling separa dan perencatan pada titik

sentuhan. Akhirnya, konsortium terdiri dari pada A. niger (F44) dan T. viride (F26) telah

dipilih sebagai konsortium kulat lignoselulolitik berpotensi untuk pengkomposan RST.

Kematangan dan kestabilan kompos ditentukan dengan memantau perubahan fizikal,

biokimia dan biologi yang berlaku semasa proses pengkomposan. Kompos RST terhasil

dikenali sebagai microbial infused juga diuji untuk keberkesanannya dalam

percambahan biji benih dan kematangan anak benih, pertumbuhan pokok dan

penindasan jangkitan penyakit pada cili di dalam rumah tanaman. Pengkomposan RST

dengan konsortium kulat lignoselulolitik (F44 and F26) pada pH normal (pH 6.75)

menunjukkan perubahan signifikan dalam ciri fizikal, biokimia dan biologi berbanding

dengan pH asid (pH 5.75) dan pH alkali (pH 7.75). Selepas hari ke 21, konsortium kulat

dan pH normal mengurangkan nisbah C/N kepada 17.5 daripada nilai awal 29.2, dan

meningkatkan indeks percambahan (GI) kepada 75.5. Lignin dan selulosa yang tidak

terurai dalam kompos microbial infused adalah 9.8 dan 12.4 %. �-1,4-endo-glucanase,

�-1,4-exo-glucanase dan jumlah aktiviti dehidrogenase adalah lebih rendah secara

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signifikannya dibanding dengan kompos tanpa rawatna mikrob. Kandungan N, P, K, Ca

dan Mg dalam kompos microbial infused adalah 2,3, 1,1, 2,6, 2.5 dan 1.4 %. Keputusan

ini menunjukkan bahawa kompos RST microbial infused yang terhasil pada pH normal

selepas hari ke21 boleh digunakan sebagai pengganti baja bukan organik. Di bawah

rumah tanaman, aplikasi kompos microbial infused meningkatkan secara signifikan

percambahan biji benih, pertumbuhan tanaman dan mengurangkan jangkitan S. rolfsii

pada tanaman cili berbanding kompos komersial (BFC, CMT Agro Resources Sdn Bhd)

atau racun kulat Benomyl. Aplikasi pada tahap 15 t/ha juga adalah optimum untuk

percambahan biji benih dan kematangan anak benih, pertumbuhan pokok serta

pengurangan jangkitan penyakit, mencadangkan kompos RST microbial infused boleh

digunakan sebagai alternatif kepada racun kulat Benomyl untuk mengawal jangkitan

Sclerotium pada tanaman cili.

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ACKNOWLEDGEMENTS

I would like to give thanks to the Almighty Allah for giving me strength and wisdom to

complete my study. May His name be glorified and praised.

I would like to express my gratitude to my supervisor Professor Dr. Sariah Meon for her

keen interest, valuable guidance and contribution provided during the planning and

preparation of this thesis. Her countless patience, dedicated efforts, encouragement,

support and constructive criticisms are admirable. My sincere appreciations are

extended to my supervisory committee members, Professor Dr. Mohd. Razi Ismail,

Assoc. Prof. Dr. Halimi Mohd. Saud and Professor Dr. Md. Zahangir Alam for their

guidance, suggestions and advice throughout the research work.

It is my greatest pleasure to acknowledge Universiti Putra Malaysia for awarding me the

fellowship for the study. I am also grateful to my employer organization Sher-e-Bangla

Agricultural University for granting my leave to pursue my study.

Thanks are also extended to all the staff-members in the Institute of Tropical

Agriculture, Plant Pathology, Microbiology and Nematology Laboratories for their kind

assistance. Special thanks are also extended to Dr. Yasmeen Siddique, Lee Cheu, Aswad

and Yuvarani for their help and moral support towards the completion of this study.

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I wish to express my heartfelt gratitude and appreciation to my wife Sheikh Hasna

Habib and my son A. M. Farhan Ishrak for their endless love, sacrifice, physical and

mental support to make this dream a reality.

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I certify that a Thesis Examination Committee has met on -/-/- to conduct the final examination of Md. Kausar Hossain on his thesis entitled “Microbial composting of rice straw for improved stability and bioefficacy” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee recommends that the student be awarded the Doctor of Philosophy. Members of the Examination Committee are as follows: Name of Chairperson, PhD Title (e.g. Professor/ associate Professor/Ir) Name of Faculty Universiti Putra Malaysia (Chairman) Name of Examinar 1, PhD Title (e.g. Professor/ associate Professor/Ir) Name of Faculty Universiti Putra Malaysia (Internal Examinar) Name of Examinar 2, PhD Title (e.g. Professor/ associate Professor/Ir) Name of Faculty Universiti Putra Malaysia (Internal Examinar) Name of External Examinar, PhD Title (e.g. Professor/ associate Professor/Ir) Name of Department and/ orFaculty Name of Organization (Universiti/Institute) Country (External Examinar)

SHAMSUDDIN SULAIMAN, Ph.D. Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:………………………….

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirements for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Sariah Meon, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Mohd. Razi Ismail, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Member) Halimi Mohd. Saud, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member) Md. Zahangir Alam, PhD Professor Faculty of Engineering International Islamic University Malaysia (Member) HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or any other institution. MD. KAUSAR HOSSAIN

Date: 18 July 2011

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TABLE OF CONTENTS Page DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xxi CHAPTER

1 INTRODUCTION

1

2 LITERATURE REVIEW 7 2.1 Rice plant 7 2.1.1 Production of rice and rice straw (RST) 7 2.1.2 Problems of RST disposal 8 2.2 Properties of RST 9 2.2.1 Cellulose 9 2.2.2 Hemicellulose 10 2.2.3 Lignin 11 2.2.4 Other cell wall components 11 2.3 Potential microorganisms in composting 12 2.3.1 Bacteria 13 2.3.2 Actinobacteria 14 2.3.3 Fungi 14 2.3.4 Trichoderma spp 15 2.3.5 Aspergillus niger 16 2.4 Biodegradation of lignocellulosic materials 18 2.4.1 Biodegradation of cellulose 18 2.4.2 Biodegradation of hemicellulose 19 2.4.3 Biodegradation of lignin 20 2.5 Microbial composting 23 2.5.1 Definition of composting 23 2.5.2 Composting process 24 2.5.3 Biochemical changes in microbial composting 25 2.6 Factors affecting microbial composting 27 2.6.1 Temperature 27 2.6.2 pH 28 2.6.3 C/N ratio 29 2.6.4 Particle size 30 2.6.5 Moisture 31 2.6.6 Aeration 32

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2.7 Compost Maturity and Stability 33 2.8 Bioefficacy of RST compost 35 2.8.1 Soil health enhancement 35 2.8.2 Plant disease suppression 39 2.8.3 Mechanism of disease suppression 40 3 ISOLATION AND SCREENING OF LIGNOCELLULOLYTIC

MICROBES FOR RAPID COMPOSTING OF RST

3.1 Introduction 44 3.2 Materials and Methods 46 3.2.1 Source of materials 46 3.2.2 Isolation of microorganisms associated with composted

RST 47

3.2.3 In-vitro screening for lignocellulolytic activity 48 3.2.4 Lignocellulolytic activity of selected microbes on rice straw

powder (RSP) amended media 50

3.2.5 In-vitro Biodegradation of RST 51 3.2.6 In-vitro Compatibility Evaluation of Mixed Fungal Culture 56 3.2.7 Identification of lignocellulolytic fungi 57 3.2.8 Experimental Design and data analysis 61 3.3 Results 62 3.3.1 Isolation of microorganisms associated with composted

RST 62

3.3.2 In vitro screening for lignocellulolytic activity 62 3.3.3 Lignocellulolytic activity of selected microbial isolates

tested on RSP- amended media 73

3.3.4 In-vitro biodegradation of RST 78 3.3.5 In vitro compatibility evaluation of fungal isolates 82 3.3.6 Identification of the selected lignocellulolytic microbial

consortium 85

3.4 Discussion 87 3.5 Conclusion 96 4 EFFECT OF MICROBIAL INOCULATION AND pH ON

COMPOSTING OF RICE STRAW

4.1 Introduction 97 4.2 Materials and Methods 99 4.2.1 Materials 99 4.2.2 Characteristics of composting substrates 99 4.2.3 Preparation of microbial inoculants 100 4.2.4 Composting procedure 101 4.2.5 Analysis of compost 102 4.2.6 Experimental design and data analysis 113 4.3 Results 114 4.3.1 Electrical conductivity 114 4.3.2 Bulk density 115

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4.3.3 Germination Index 116 4.3.4 C/N ratio 117 4.3.5 Microbial counts 118 4.3.6 Lignin peroxidase 121 4.3.7 Endo-1, 4-�-glucanase 122 4.3.8 Exo-1, 4-�-glucanase 123 4.3.9 Fluorescein diacetate (FDA) hydrolysis 124 4.3.10 Lignin 125 4.3.11 Cellulose 126 4.3.12 Accumulation of macronutrients 127 4.3.13 Relationship among different parameters 128 4.3.14 Characteristics of matured microbial infused RST compost 130 4.4 Discussion 131 4.5 Conclusion 138 5 EVALUATION OF MICROBIAL INFUSED RST COMPOST ON

PLANT GROWTH PERFORMANCE AND INDUCTION OF DISEASE RESISTANCE IN CHILLI

5.1 Introduction 139 5.2 Materials and Methods 141 5.2.1 Experimental site 141 5.2.2 Preparation of inoculum 141 5.2.3 Compost sources 142 5.2.4 Plant materials and growing medium 142 5.2.5 Seed germination and seedling establishment 142 5.2.6 Growth performance and disease incidence 143 5.2.7 Experimental design and data analysis 145 5.3 Results 146 5.3.1 Seed germination and seedling establishment 146 5.3.2 Plant growth 147 5.3.3 Disease incidence 148 5.4 Discussion 153 5.5 Conclusion 156 6 SUMMARY, CONCLUSION AND RECOMMENDATIONS 6.1 Summary 159 6.2 Conclusion and Recommendations

165

REFERENCES 167 APPENDICES 194 BIODATA OF STUDENT 207 LIST OF PUBLICATIONS 208