UNIVERSITI PUTRA MALAYSIA

IN VITRO AND IN SILICO INVESTIGATION OF THE PLANT GROWTH-PROMOTING RHIZOBACTERIA FROM PEPPER

(Piper nigrum L.)

ZAKRY FITRI BIN AB. AZIZ

FP 2013 8

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IN VITRO AND IN SILICO INVESTIGATION OF

THE PLANT GROWTH-PROMOTING

RHIZOBACTERIA FROM PEPPER

(Piper nigrum L.)

ZAKRY FITRI BIN AB. AZIZ

DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2013

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“IN THE NAME OF ALLAH THE MOST GRACIOUS AND THE MOST MERCIFUL”

3) “And it is He Who spread out the earth, and placed therein firm mountains and

rivers and of every kind of fruits He made Zawjain Ithnaîn (two in pairs - may mean

two kinds or it may mean: of two sorts, e.g. black and white, sweet and sour, small

and big, etc.) He brings the night as a cover over the day. Verily, in these things,

there are Ayât (proofs, evidences, lessons, signs, etc.) for people who reflect.”

4) “And in the earth are neighbouring tracts, and gardens of vines, and green crops

(fields etc.), and date-palms, growing out two or three from a single stem root, or

otherwise (one stem root for every palm), watered with the same water, yet some of

them We make more excellent than others to eat. Verily, in these things, there are

Ayât (proofs, evidences, lessons, signs) for the people who understand.”

(Al-Qur’an: Ar-Rad: 3-4)

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IN VITRO AND IN SILICO INVESTIGATION OF THE PLANT GROWTH-

PROMOTING RHIZOBACTERIA FROM PEPPER (Piper nigrum L.)

By

ZAKRY FITRI BIN AB. AZIZ

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

September 2013

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All material contained within the thesis, including without limitation text, logos, icons, 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 fulfillment of the requirements for the degree of Doctor of Philosophy

IN VITRO AND IN SILICO INVESTIGATION OF THE PLANT GROWTH-

PROMOTING RHIZOBACTERIA FROM PEPPER (Piper nigrum L.)

By

ZAKRY FITRI BIN AB. AZIZ

September 2013

Chairman: Associate Professor Halimi Mohd. Saud, PhD

Faculty: Agriculture

The increase in awareness of the public to use inexpensive, safe, organic and

environmentally friendly agricultural inputs makes plant growth-promoting

rhizobacteria as one of the more attractive biological applications used to reduce or

complement the use of synthetic agricultural inputs. In the present study, diligent

investigation has been conducted in search of the most promising plant growth-

promoting rhizobacteria from pepper (Piper nigrum L.) rhizosphere. As a result of

the search, 14 rhizobacteria were isolated from the rhizosphere of the pepper plant

(Piper nigrum L.). In vitro analyses showed that the isolates possessed many traits

beneficial to agronomy, viz. biological nitrogen fixation, phosphate solubilisation and

indole acetic acid (IAA) production. Analyses by 16S rRNA sequencing indicated

that the isolates belonged to Acinetobacter radioresistens (UPMLH19), Bacillus

cereus (UPMLH1, UPMLH13, UPMLH24, UPMLH41 and UPMLH42), Bacillus

megaterium (UPMLH3 and UPMLH22), Bacillus subtilis (UPMLH5), Bacillus spp.

(UPMLH8, UPMLH23, UPMLH34 and UPMLH43) and Leclercia sp. (UPMLH2).

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Phylogenetic analyses indicated that the newly isolated rhizobacteria from P. nigrum

were from Firmicutes and Proteobacteria, with the former being more common. In

the present study, computational methods were used to predict information within the

small subunit 16S rRNA gene from B. cereus strain UPMLH24. The computational

methods employed revealed that the small subunit 16S rRNA gene sequence from B.

cereus strain UPMLH24 contained several open reading frames that encoded

gamma-polyglutamic acid protein, lipoprotein, cytoplasmic protein and ribosomal

protein S10. Computational predictions based on protein-protein identity classified

seven phyla of organisms associated with small open reading frames of novel B.

cereus strain UPMLH24. Plant bioassay technique was used to select the most

promising isolates. Three vegetable crop species were used as test plants, namely

mustard (Brassica juncea), mung bean (Vigna radiata) and shallot (Allium

ascalinocum). Of the 14 inocula, Bacillus spp. strains (UPML23, UPMLH34 and

UPMLH43) and B. cereus UPMLH24 improved early plant growth of mustard

significantly. Early development of mung bean seedlings showed significant

improvement after inoculation with Bacillus sp. strain UPMLH8, B. megaterium

strains UPMLH3 and UPMLH22, and B. cereus strain UPMLH24. Only B. cereus

strain UPMLH1, significantly promoted root number and shoot height of shallot

plants. B. cereus strains UPMLH1 and UPMLH24 inoculum were selected and used

in further investigation after showing to be the most promising in enhancing the early

development of mustard, bean and shallot plants. The selected strains of B. cereus

were then used to evaluate the effect of inoculation on their original host, P. nigrum

stem cuttings of Kuching variety, studied for root formation under nursery

conditions. Results showed that B. cereus UPMLH24 inoculation on fresh pepper

stem cuttings stimulated root number (55% increase over control), length of longest

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root (25% increase over control), total root length (87% increase over control), root

fresh weight (28% increase over control) and root dry weight (112% increase over

control). Present study recommends Bacillus cereus UPMLH24 as a potential

candidate in a formulation of a biostimulant for organic and sustainable nursery for

pepper production.

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

PENYIASATAN IN VITRO DAN IN SILICO RIZOBAKTERIA

PENGGALAK-TUMBESARAN TUMBUHAN DARI LADA (Piper nigrum L.)

Oleh

ZAKRY FITRI BIN AB. AZIZ

September 2013

Pengerusi: Profesor Madya Halimi Mohd. Saud, PhD

Fakulti: Pertanian

Peningkatan kesedaran awam ke atas penggunaan input pertanian yang murah,

selamat, organik dan mesra alam telah menjadikan rizobakteria penggalak-

tumbesaran satu daripada aplikasi biologi yang menarik untuk mengurangkan atau

mengganti input pertanian sintetik. Satu kajian terperinci telah dijalankan untuk

mencari rizobakteria penggalak-tumbesaran dari rizosfera lada (Piper nigrum L.)

yang berpotensi tinggi. Hasil dari pencarian ini, 14 rizobakteria berjaya dipencilkan

dari rizosfera tanaman lada (Piper nigrum L.). Analisis in vitro telah menunjukkan

asingan itu mempunyai beberapa ciri berfaedah dari segi agronomi, seperti

pengikatan nitrogen biologi, penguraian fosfat dan penghasilan asid asetik indol.

Analisis penjujukan 16S rRNA menunjukkan isolat terdiri dari Acinetobacter

radioresistens (UPMLH19), Bacillus cereus (UPMLH1, UPMLH13, UPMLH24,

UPMLH41 dan UPMLH42), Bacillus megaterium (UPMLH3 dan UPMLH22),

Bacillus subtilis (UPMLH5), Bacillus spp. (UPMLH8, UPMLH23, UPMLH34 dan

UPMLH43) dan Leclercia sp. (UPMLH2). Analisis filogenetik menunjukkan

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rizobakteria dari P. nigrum yang baru dipencilkan ini adalah terdiri dari Firmicutes

dan Proteobacteria, dengan Firmicutes mempunyai bilangan terbanyak. Dalam kajian

ini, kaedah-kaedah pengkomputeran digunakan bagi meramal maklumat di dalam

subunit kecil gen 16S rRNA dari Bacillus cereus UPMLH24. Kaedah-kaedah

pengkomputeran yang dijalankan membuktikan subunit kecil gen 16S rRNA dari B.

cereus UPMLH24 mengandungi beberapa “open reading frames” yang mengekod

protein asid poliglutamik, lipoprotein, protein sitoplasmik dan protein ribosom S10.

Teknik bioasai tumbuhan telah digunakan untuk membuat pilihan isolat yang paling

berpotensi. Tiga tanaman sayuran telah dijadikan tumbuhan ujian, iaitu sawi

(Brassica juncea), kacang hijau (Vigna radiata) dan bawang merah (Allium

ascalinocum). Dari 14 inokula, Bacillus spp. (UPML23, UPMLH34 dan UPMLH43)

dan B. cereus UPMLH24 telah meningkatkan pertumbuhan awal tanaman sawi

secara bererti. Perkembangan awal tanaman kacang hijau menunjukkan

perkembangan yang bererti setelah inokulasi dengan Bacillus sp. UPMLH8, B.

megaterium (UPMLH3 dan UPMLH22) dan B. cereus UPMLH24. Hanya B. cereus

UPMLH1, merangsang secara bererti ke atas bilangan akar dan tinggi pucuk

pertumbuhan tanaman bawang merah. Inokulum B. cereus UPMLH1 dan UPMLH24

telah dipilih dan digunakan dalam kajian lanjutan setelah menunjukkan peningkatan

ke atas perkembangan awal tanaman sawi, kacang hijau dan bawang merah. Isolat B.

cereus yang terpilih kemudiannya dinilai kesan inokulasinya ke atas hos asalnya,

iaitu keratan batang P. nigrum varieti Kuching, untuk mengkaji pembentukan akar di

nurseri. Keputusan kajian menunjukkan inokulasi B. cereus UPMLH24 ke atas

keratan-keratan batang lada merangsang bilangan akar (peningkatan 55% berbanding

kawalan), panjang akar terpanjang (peningkatan 25% berbanding kawalan), jumlah

panjang akar (peningkatan 87% berbanding kawalan), berat basah akar (peningkatan

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28% berbanding kawalan) dan berat kering akar (peningkatan 112% berbanding

kawalan). Kajian ini mencadangkan Bacillus cereus UPMLH24 adalah calon yang

berpotensi di dalam formulasi biopenggalak bagi pengeluaran nurseri tanaman lada

organik dan lestari.

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ACKNOWLEDGEMENTS

First of all, I thank Allah S.W.T. with His compassion and mercifulness, for giving

the strength and endurance to carry out and complete this thesis. I wish to extend my

deep appreciation and sincere thanks to my main supervisor, Associate Professor Dr.

Halimi Mohd Saud for his guidance throughout the thesis project. I also extend my

sincere appreciation to Dr. Khairuddin Abdul Rahim and Associate Professor Dr.

Osumanu H. Ahmed for their suggestions and critical comments as members of my

supervisory committee. My deep appreciation also goes to Prof. Dr. Zulkifli Hj.

Shamsuddin for his advice and motivation and Dato’ Prof. Dr. Nik Muhammad Ab.

Majid for his help and advice on research grant. Special thanks to my laboratory co-

workers, Ms. Piriya L. Veerasamy, Ms. Tavamani Balan, Mrs. Menakumarry

Shantrakumarru, Ms. Nursyahda Zakaria, Ms. Lisna Nurul Afza Zulkifli and Mr. Sze

Hua Kiong. I also thank the staff members of the Faculty of Agriculture and Food

Sciences of Universiti Putra Malaysia Bintulu Sarawak Campus for their

congeniality, especially Prof. Dr. Japar Sidik Bujang, Associate Prof. Dr. Mutaharah

Zakaria, Dr. Stephen Leong Chan Teck, Dr. Wong Sing King, Mr. Make Jiwan, Mr.

Franklin Ragai Kundat and Mrs. Siti Fatimah Razali. Finally, I am grateful for the

personal support, understanding and encouragement I received from my parents Hj.

Ab. Aziz Hj. Zainal and Hjh Rokiah Hj. Ibrahim, my in laws Hj. Malahubban Hj.

Mohammad and Hjh Saburayah Husin and also to my wife Mrs. Masnindah Hj.

Malahubban, daughter Zunah Farwizah and son Hadiff Fathullah throughout this

years. Finally, I would like to acknowledge the Ministry of Education Malaysia,

Malaysian Pepper Board and Universiti Putra Malaysia for financial and overall

support.

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I certify that a Thesis Examination Committee has met on 6th September 2013 to conduct the final examination of Zakry Fitri bin Ab. Aziz on his thesis entitled “In

vitro and In silico Investigation of the Plant Growth-Promoting Rhizobacteria from Pepper (Piper nigrum L.)” 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 Thesis Examination Committee were as follows:

Ahmad Husni b. Mohd. Haniff, PhD

Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Mohd. Razi b. Ismail, PhD

Professor Institute of Tropical Agriculture Universiti Putra Malaysia (Internal Examiner)

Radziah bt. Othman, PhD

Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

Syed Dilnawaz Ahmad Gardezi, PhD

Professor University of Azad Jammu & Kashmir Pakistan (External Examiner)

________________________________ NORITAH OMAR, PhD

Associate Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 21 January 2014

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This thesis was submitted to the Senate of 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:

Halimi bin Mohd. Saud, PhD

Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Osumanu Haruna Ahmed, PhD

Associate Professor Faculty of Agriculture and Food Sciences Universiti Putra Malaysia Bintulu Campus Sarawak (Member)

Khairuddin bin Abdul Rahim, PhD

Director Division of Agrotechnology and Biosciences Malaysian Nuclear Agency (Member)

________________________________ BUJANG BIN KIM HUAT, 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 other 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.: __________________________________________________

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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) are adhered to.

Signature: _____________________ Signature: ____________________ Name of Name of Chairman of Member of Supervisory Supervisory Committee: _____________________ Committee: ____________________

Signature: _____________________ Signature: ____________________ Name of Name of Member of Member of Supervisory Supervisory Committee: _____________________ Committee: ____________________

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TABLE OF CONTENTS

Page

ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL ix DECLARATION xi LIST OF TABLES xv LIST OF FIGURES xvi LIST OF ABBREVIATIONS xviii

CHAPTER

1 GENERAL INTRODUCTION 1

2 LITERATURE REVIEW 4 2.1 The Importance of Black Pepper (Piper nigrum Linn.) 4 2.2 Rhizosphere 9 2.3 Plant Growth-Promoting Rhizobacteria (PGPR) 10 2.3.1 N2 Fixation 11 2.3.2 P Solubilisation 12 2.3.3 Phytohormone IAA Production 13 2.4 PGPR Research Progress on Pepper (Piper nigrum L.) 14 2.5 Screening and Selecting the Most Promising Plant Growth-Promoting Rhizobacteria

16

2.6 The 16S ribosomal RNA Gene 17 2.7 The Era of Big Molecular Data 22 2.8 Use of Bioinformatics for Information and Knowledge Generation in Microbiology

24

2.9 Summary of Literature Review 27

3 BIOCHEMICAL AND MOLECULAR ANALYSIS OF

RHIZOBACTERIA ISOLATED FROM PEPPER (Piper

nigrum L.)

28

3.1 Introduction 28 3.2 Materials and Methods 28 3.2.1 Isolation of Bacteria from Pepper (Piper nigrum L.) Rhizosphere

28

3.2.2 Biochemical Characterisation and Measurement 30 3.2.3 16S rRNA Molecular Identification and Phylogenetic Analysis

31

3.2.4 16S rRNA Bioinformatics Analysis 32 3.3 Results 35 3.3.1 Isolation, Biochemical and Molecular Characterisation

35

3.3.2 16S rRNA Phylogenetic Relationships of the Isolates

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3.3.3 16S rRNA Bioinformatics Prediction 413.4 Discussion 53 3.5 Conclusion 61

4 PLANT BIOASSAY SCREENING AND SELECTION

FOR THE MOST PROMISING PLANT GROWTH-

PROMOTING RHIZOBACTERIA FROM

PEPPER PLANTS (Piper nigrum L.)

63

4.1 Introduction 63 4.2 Materials and Methods 63 4.3 Results 65 4.3.1 Mustard Plant Inoculation 65 4.3.2 Mung Bean Plant Inoculation 66 4.3.3 Shallot Plant Inoculation 66 4.3.4 Selection for the Most Promising PGPR Isolates 67 4.4 Discussion 68 4.5 Conclusion 69

5 EFFECTS OF INOCULATION OF PEPPER

(Piper nigrum L.) STEM CUTTINGS WITH TWO

STRAINS OF PLANT GROWTH-PROMOTING

RHIZOBACTERIUM, Bacillus cereus, ON ROOT

FORMATION

71

5.1 Introduction 71 5.2 Materials and Methods 71 5.2.1 The Rhizobacteria 71 5.2.2 PGPR Bacillus cereus Inoculation on Root Formation of Pepper Cuttings

71

5.2.3 Bacterial Population of Inoculated Pepper Stem Cuttings

73

5.3 Results 73 5.4 Discussion 76 5.5 Conclusion 78

6 GENERAL DISCUSSION, CONCLUSIONS AND

RECOMMENDATIONS FOR FUTURE RESEARCH

79

REFERENCES 83 APPENDICES 100 BIODATA OF STUDENT 109 LIST OF PUBLICATIONS 110