Doctor of Philosophy

231 Pages·2002·8.77 MB·English

Isolation and characterization of methylotrophic
bacteria from wetland paddy ecosystem and their
plant growth promoting rhizobacterial (PGPR) traits

A
THESIS
SUBMITTED TO
ANAND AGRICULTURAL UNIVERSITY
FOR PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF DEGREE

OF

Doctor of Philosophy

IN

AGRICULTURAL MICROBIOLOGY

BY

JHALA YOGESHVARI KISHORSINH
M. Sc. (AGRICULTURAL MICROBIOLOGY)

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
B. A. COLLEGE OF AGRICULTURE
ANAND AGRICULTURAL UNIVERSITY
ANAND-388 110

2013

Registration No. 04-1255-2010
“Isolation and characterization of methylotrophic bacteria
from wetland paddy ecosystem and their plant growth
promoting rhizobacterial (PGPR) traits”

Name of Student Major Advisor
Jhala Yogeshvari Kishorsinh Dr. R. V. Vyas

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
B. A. COLLEGE OF AGRICULTURE
ANAND AGRICULTURAL UNIVERSITY
ANAND –388 110, GUJARAT (INDIA)

ABSTRACT

Present investigation was carried out to isolate efficient
methylotrophic cultures from wetland paddy fields of Gujarat. Out of 74
cultures obtained, 5 were found proficient utilizer of methane and
methanol. Morphological, biochemical and 16S rDNA sequencing
identified isolate M 8 as Bacillus aerius AAU M 8; M 10 as Rhizobium sp.
AAU M 10; M 14 as B. subtilis AAU M 14; M 17 as Paenibacillus illinoisensis
AAU M 17 and M 29 as B. megaterium AAU M 29. Among all the tested
isolates, P. illinoisensis, B. aerius, Rhizobium sp. and B. subtilis showed
presence of pmoA gene encoding α subunit particulate methane
monooxygenase (pMMO) cluster and B. megaterium, P. illinoisensis,
Rhizobium sp. and M. extrorquens showed presence of mmoX gene
encoding α subunit of the hydroxylase component of the soluble methane
monooxygenase (sMMO) cluster. P. illinoisensis and Rhizobium sp. showed
presence mxaF gene encoding α subunit region of methanol
dehydrogenase gene cluster showing that both bacterium are efficient
utilizers of methane.
All the isolates were capable of producing IAA. All the isolates were
capable of solubilizing phosphate, among which B. megaterium showed
the highest activity (305.6 and 309.9 μg /ml P release at 3 and 5 DAI,
respectively). B. aerius and Rhizobium sp. also showed presence of nifH
gene. Whereas, B. aerius and Rhizobium sp. proved capacity to produce
ACC deaminase. Moreover, Rhizobium sp. showed potash solubilization
activity. All the isolates showed more or less antagonistic activity against
soil borne pathogenic fungi.
All the isolates showed plant growth promotion of rice cultivars
Gurjari and IR 64 in pots with possible reduction of 25 % NP chemical
fertilizers, wherein, B. megaterium proved to be the best followed by P.
illinoisensis. All the results clearly signify their multiple utility as methane
degrader and PGPR cum bioagent to be explored in rice fields.
ii
Dr. R. V. Vyas
Professor and Head
Department of Agricultural Microbiology
B. A. College of Agriculture
Anand Agricultural University
Anand- 388 110
Gujarat, India.

CCEERRTTIIFFIICCAATTEE

This is to certify that the thesis entitled “Isolation
and characterization of methylotrophic bacteria from
wetland paddy ecosystem and their plant growth
promoting rhizobacterial (PGPR) traits” submitted by
JHALA YOGESHVARI KISHORSINH in partial fulfillment of the
requirements for the award of the degree of DOCTOR OF
PHILOSOPHY in AGRICULTURAL MICROBIOLOGY of the
Anand Agricultural University is a record of bonafide research
work carried out by her under my personal guidance and
supervision. The thesis has not previously formed the basis for
the award of any degree, diploma or other similar title.

Place: Anand (R. V. VYAS)
Date: / / Major Advisor
DDEECCLLAARRAATTIIOONN

This is to declare that the whole of the research
work reported here in the thesis entitled “Isolation and
characterization of methylotrophic bacteria from wetland
paddy ecosystem and their plant growth promoting
rhizobacterial (PGPR) traits” for the partial fulfillment of the
requirements for the degree of Doctor of Philosophy in
Agricultural Microbiology by the undersigned is the results of
investigationdone by me under the direct guidance and
supervision of Dr. R. V. Vyas, Professor and Head, Department
of Microbiology, Anand Agricultural University, Anand and no
part of the work has been submitted for any other degree so
far.

Place: Anand (Jhala Yogeshvari Kishorsinh)
Date: / /2013

Counter signed by

((RR.. VV.. VVyyaass))
Professor and Head
Department of Agricultural Microbiology,
B. A. College of Agriculture
Anand Agricultural University,
Anand - 388 110.
Acknowledgement

„A journey is easier when you travel together‟. Interdependence is certainly
more valuable than independence. This thesis is the result of three years of work
whereby I have been accompanied and supported by many peoples. It is a pleasant
aspect that I have now got the opportunity to express my gratitude for all of them.
I take this opportunity to extend my deep sense of gratitude and words of
appreciation towards those who helped me during the pursuit of my present study.
At this inexplicable moment, words are not enough to express my whole
hearted sense of gratitude to my major advisor, Dr. R. V. Vyas, Professor & Head,
Department of Microbiology, B.A. College of Agriculture, Anand Agricultural
University, Anand for his invaluable, judicious, constantly inspiring and thorough
guidance with inspiring encouragement, active persuasion and supervision,
illuminating suggestions and diligent efforts throughout the course of my study.
His caring attitude, faith, integral view on research and his mission for “only high
quality work and not less” had made a deep impression on me. I confess that I am
very fortunate to get chance of working with him as a student.
It‟s my privilege to accord my sincere thanks to Mrs H. N. Shelat,
Associate research Scientist, Department of Microbiology for her humane attitude,
sustained encouragement, critical suggestions, everwilling help and valuable
advice.
I am indeed thankful to my Minor Advisor Dr Y. M. Shukla, Associate
Professor, Department of Biochemistry and the members of my advisory committee Dr J.
S. Patel, Professor, Department of Agricultural statistics and Dr Vyas Pandey, Professor
& Head, Department of Meteorology for their kind counsel and valuable suggestions
during the tenure of my study and research work.
I also wish to express my thanks to Dr. K. P. Patel, Principal, B.A. College
of Agriculture, AAU, Anand and University authorities for providing me
opportunity to pursue higher studies from such a prestigious institute.
I express my deep sense of gratitude for College of Food Processing Technology
and Bioenergy, AAU, Anand for providing methane gas as and when required
I limit myself to express my love to every staff members of the department
(Ashish, Jiya, Nishal, Niketa, Dilip, Bharat, Jayantibhai, Ambaben, Chhotubhai,
Jashvant, Pankaj and Raju) for providing lively, friendly and encouraging
atmosphere throughout the period of my study.
I extend my sincere thanks to my friends Deepmala and Keyur for their
unreserved help, everwilling support and memorable company during course of
study.
This work would not have seen the light of the day, if I had not been
constantly inspired and supported by Harit and Hirenbhai especially during
molecular analysis.
Friends are the integral part of life and without them life is vague. I
cannot forget the love and affections received from Armi, Archna, Bhumika,
Chetna, Ekta, Hetal , Shraddha, Sneha, Leena, Aashish, Ankit, Arpan, Deepak,
Kuldeep,Naushad and Ronak I acknowledge all of them from the depth of my
heart, their help and company caused me through this study.
Without the support of nears and dears, this work would not have been a
success. I want to express my heartful accolade to my revered parents (Kishorsinh
and Hemaba) who brought me to this stage. I am also grateful for their kind
blessings, love, patience, overwhelming support and inspiration. I fall short of
words for the moral and unrelenting support extended by my loving brother Dr.
Shivraj during my all ups and down.
Last but not least, I thank almighty, for guiding me through my life,
giving me opportunities and fulfilling my dreams.

Place: ANAND
Date: / /2013
(Jhala Yogeshvari K.)
CONTENTS
Chapter Title Page
No.
I INTRODUCTION 1-7
II REVIEW OF LITERATURE 8-31
2.1 Natural balance sheet of methane emission 9
from rice fields
2.2 Isolation and characterization of 11
methylotrophic bacteria
2.3 Biochemistry of methylotrophic bacteria 18
2.4 PGPR traits of methylotrophic bacteria 24
2.5 Plant growth promoting effects of 27
methylotrophs on growth and yield of
crops
III MATERIAL AND METHODS 32-61
3.1 General Materials 32

3.1.1 Standard strains used in the study 32
3.1.2 Glass-wares and chemicals 32

3.1.3 Media, Stains and Reagents 33
3.2 Methodology 33
3.2.1 Isolation of methylotrophic bacteria 33-34
from wetland paddy of Gujarat
3.2.1.1 Source of isolation 33

3.2.1.2 Isolation of methylotrophic bacteria 33
following enrichment culture technique
3.2.1.3 Screening of isolates 34
3.2.2 Characterization of isolates and 34-47
assessment of in vitro enzyme activity
for methane degradation
3.2.2.1 Evaluation of isolates for methane gas and 34
methanol utilization

3.2.2.2 Characterization of the potential 34
methylotrophic isolates
3.2.2.2.1 Morphological characterization 35
3.2.2.2.2 Physiological characterization 35
3.2.2.2.2.1 pH tolerance 35
3.2.2.2.2.2 Salt tolerance 35
3.2.2.2.2.3 Antibiotics resistance profile 36
3.2.2.2.3 Biochemical characterization 37
3.2.2.2.4 Utilization of C compounds 39
1
3.2.2.2.5 Molecular characterization. 39
3.2.2.2.5.1 DNA extraction 39
3.2.2.2.5.2 Purity and quantification test of DNA by 40
spectrophotometric Analysis (Nanodrop)
3.2.2.2.5.3 16S rRNA amplification 41
3.2.2.2.5.3.1 Oligonucleotide primers used 41
3.2.2.2.5.3.2 Analysis of the PCR Products 42
3.2.2.2.5.4 Amplified Ribosomal DNA Restriction 42
Analysis (ARDRA) of promising isolates
3.2.2.2.5.5 Bacterial 16S rRNA gene sequencing and 44
identification of promising methylotrophic
isolates
3.2.2.2.5.5.1 PCR product purification 44
3.2.2.2.5.5.2 DNA sequencing 44
3.2.2.3 In vitro enzyme activities for methane 45
degradation
3.2.2.3.1 Methane monooxygenase activity 45
3.2.2.3.2 Methanol dehydrogenase assay 46
3.2.2.3.3 Detection of bacterial methane metabolism 46
genes
3.2.3 Evaluation of PGPR traits of 48-55
methylotrophic isolates in laboratory
3.2.3.1 Detectioin of nifH gene 48
3.2.3.2 Phosphate solubilization capacity 49

3.2.3.3 Phytase activity 50

3.2.4.3 Organic acid production 50

3.2.4.3.1 Detection of organic acid production on 50
solid media

3.2.4.3.2 Qualitative and quantitative analysis of 50
Organic acids production in liquid media
by HPLC

3.2.4.3.2.1 Sample preparation for HPLC analysis 50
3.2.4.3.2.2 Equipment and operating conditions 51
3.2.4.3.2.3 Calibration and method validation 51

3.2.4.3 Indole acetic acid (IAA) production 52
3.2.4.4 ACC-deaminase activity 52
3.2.4.5 Potash solubilization efficiency 53
3.2.4.6 Biocontrol potential of native potential 53
methylotrophic isolates
3.2.4.6.1 Bioassay against plant pathogenic fungi 53
3.2.4.6.2 Siderophore production 54
3.2.4.6.3 Production of cell wall degrading enzymes 55

3.2.4.6.3.1 Determination of lipase 55